BERG | BioEngineering Research Group · BERG aims at excellence in research and advanced education...

2009

Annual Report

BERG | BioEngineering Research Group

Contents

04

About BERG

06 Executive Summary

07 The Research Group

08 Main Indicators

10

Research Activities

12 Bioprocess Engineering and

Biocatalysis Laboratory

14 Nucleic Acid Bioengineering

Laboratory

16 Stem Cell Bioengineering

Laboratory

20 3 09 Annual Report

18

Research Highlights

20 Miniaturization in Biocatalysis

22 Bio-inspired Affinity Polymer Systems

for Antibody Recognition

24 Affinity-enhanced Extraction of Human

Antibodies

26 Designing Safer DNA Vaccines

28 Membrane Chromatography for DNA

Vaccine Purification

30 Ex-vivo Expansion of Mesenchymal

Stem Cells for Cellular Therapies: from

Lab to Clinic

32 Ex-vivo Expansion of mouse Embry-

onic Stem Cell-derived Neural Stem Cells

Under Low Oxygen

34

Scientific Output

36 Articles in Peer-reviewed Journals

38 Articles in Conference Proceedings

39 Book Chapters

39 Patents

39 Dissertations

40 Oral Communications

42 Poster Communications

46 Prizes

About BERG

20092009

BERG Annual ReportBERG Annual Report


Executive Summary

General Description

Main Indicators

The annual report of the BioEngineering Research Group (BERG) of Centre for Biological and Chemi-cal Engineering at IST highlights the activities of its three laboratories, within the Associated Labo-ratory Institute for Biotechnology and Bioengineer-ing (IBB). The major achievements in Bioprocess Engineer-ing include a novel separation process for the puri-fication of monoclonal antibodies based on multi-stage liquid-liquid extraction in aqueous two-phase systems, which led to a joint world patent with Bayer Technology Services (Leverkusen, DE). At the interface between Bioprocess Engineering and Nucleic Acid Bioengineering, unit operations (e.g. precipitation, tangential flow filtration, and aqueous two phase extraction) were evaluated, as alterna-tives in the intermediate recovery of plasmid DNA from E. coli lysates; and phenyl-boronate chroma-tography and hydrophobic interaction chromatog-raphy were successfully used for the purification of plasmid DNA directly from alkaline lysates. In Biocatalysis and Biotransformations, a new strategy for penicillin G acylase immobilization in sol-gel was successfully developed, allowing a remarkable improvement of enzyme activity in comparison with previously reported sol-gel tech-niques; and the production of androstenedione from sitosterol was scaled-up from multi-well plates to bench-scale stirred bioreactor using suit-able engineering parameters. The Nanobiotechnology research within the three BERG Laboratories include: (i) the development of portable magneto-resistive biochips for pathogen microorganism with femtomolar DNA limit detec-tion using specific antibodies immobilized on nano-magneto particles (in collaboration with INESC-MN); (ii) the use of electric field pulses to increase the speed of DNA immobilization and hybridization on chemically functionalized SiO2 thin-film chips by several orders of magnitude, with reaction time scaled down from 1 to 2 h to a range between 100 ns and 1 ms (in collaboration with INESC-MN); and (iii) the previously established 3-D cellular

microarray platform demonstrated to be a powerful tool for investigating cellular mechanisms underly-ing mouse ESC self-renewal versus differentiation, while providing the basis for rapid identification of signals and conditions that can be used to direct cell fate. With impact on the Healthcare sector, the major achievements on Stem Cell Bioengineering in-clude: (i) the consolidation of the consortium IBB-IST, Instituto Português de Oncologia de Lisboa Francisco Gentil and Centro de Histocompatibili-dade do Sul, focusing the isolation and ex-vivo expansion of Mesenchymal Stem Cells (MSC) under GMP conditions for Cellular Therapies, with three more patients being successfully infused with expanded MSC; (ii) the improvement of both human MSC and mouse Neural Stem Cells ex-vivo expansion under a low oxygen environment (i.e. hypoxia); and (iii) the successful establish-ment of a non-viral gene delivery platform using cationic liposomes for Bone Marrow MSC, allow-ing the expression of a target gene in a safe and transient way for potential use in Cell and Gene Therapy settings. A research network on Stem Cells, Tissue Engi-neering and Regenerative Medicine, “Stem Cell Engineering & Clinical Research net – StemCell-net” was launched in November, within the MIT-Portugal Program, under the coordination of BERG, to promote excellent quality research and advanced education programmes, ensuring an international competitiveness in the area of Re-generative Medicine.

Joaquim M.S. Cabral

BERG Head and

Director of IBB

Executive Summary


BEBL NABL SCBL

The Research Group

BERG

The BioEngineering Research Group (BERG) is a research unit in engineering and

life sciences at the Centre for Biological and Chemical Engineering (CEBQ). CEBQ is

the leading Centre of the Associated Laboratory Institute for Biotechnology and Bio-

engineering (IBB), a network of research centres across Portugal. IBB has been

identified by the Portuguese Ministry of Science, Technology and Higher Education

as a strategic infrastructure for the development of the Portuguese R&D and innova-

tion policies in the areas of Biotechnology, Bioengineering, Biomaterials and Life,

Biomedical and Agricultural Sciences. BERG activities within the Associated Labora-

tory IBB are focused on the Thematic Areas of Industrial Biotechnology and Health

Biotechnology.

BERG aims at excellence in research and advanced education in biotechnology and

bioengineering. The overall goal is to contribute for a better understanding of the

mechanisms that occur at the molecular and cellular levels, in order to translate them

into rational applications of biological systems relevant to the Industrial and Health

care sectors. BERG research priorities have special emphasis on Bioprocessing and

Biomolecular Engineering, Nucleic Acid Bioengineering, Nanobiotechnology and

Stem Cell Engineering, featuring an integrated cross-disciplinary approach through

three laboratories:

Bioprocess Engineering and Biocatalysis Laboratory (BEBL)

Nucleic Acid Bioengineering Laboratory (NABL)

Stem Cell Bioengineering Laboratory (SCBL)

BERG was established in 1991 as one of the initial three research groups of the Centre for Biological and

Chemical Engineering at IST, under the coordination of Prof. Joaquim Cabral. The research carried out

throughout the years has been considered of excellent level by the international committees, which regularly

evaluate the research units funded by the Portuguese Ministry of Science, Technology and Higher Educa-

tion. The main output of the activities performed by BERG members in 2009 are summarized in the following

tables.

Human Resources

With the creation of the Institute for Biotechnology and Bioengineering (IBB), a scientific policy has been

implemented to integrate complementary expertises in the group through the inclusion of new staff members

and Postdoctoral researchers in strategic scientific areas for the development of advanced biochemical engi-

neering research programs. In 2009, BERG had 66 researchers in three established programs, of which 21

have a PhD degree, 25 a master degree, 12 a five-year diploma degree, and 8 a bachelor degree.

Main Indicators

Human Resources Number

Faculty staff 10

Research scientists 5

Post-doctoral researchers 6

PhD students 28

Master students 9

Research assistants 7

Technicians 1

Male

Female

PhD

MSc

Diploma (5 years)

BSc


Publications

In 2009, the output of BERG‟s activities includes the publication of a patent in collaboration with Bayer Tech-

nology Services (Leverkusen, Germany), 2 book chapters and 50 scientific articles in peer-reviewed jour-

nals.

Scientific events

In 2009, the members of BERG have participate in organizing and scientific committees of national and in-

ternational conferences, research networks, working groups and sections of the European Federation of

Biotechnology.

Type of Event Name of Event Comittee

Conference 4th International Meeting of the Portuguese Society for Stem Cells and Cell Therapies (SPCE-TC), Lisbon, Portugal

Joaquim Cabral and Cláudia Lobato da Silva

Conference International Conference on Biopartitioning and Purification (BPP2009), Brunel, UK

Joaquim Cabral and Raquel Aires-Barros

Forum European Forum for Industrial Biotechnology (EFIB 2009), Lis-bon, Portugal

Joaquim Cabral

Network StemCellnet | Stem Cell Engineering and Research Network, Lisbon, Portugal

Joaquim Cabral

Workshop Workshop on Biosensors, Satellite event on 14th ECB - Barce-lona, Spain

Luís Fonseca

Workshop Global Network on Bioenergy/Biobased Economy Science (GNBES), Lisbon, Portugal

Joaquim Cabral

Workshop Nano09 Workshop “Grand challenges & New Trends in Nanosciences and Nanotecnologies”, Braga, Portugal

Joaquim Cabral

Type of Publication Number

Articles in international peer-reviewed journals 50

Proceedings in international peer-reviewed journals 13

Patents 1

Book chapters 2

Oral communications in international conferences 22

Poster communications in international conferences 44

Oral communications in national conferences 13

Poster communications in national conferences 12

PhD Thesis 5

MSc Thesis 3

Research Activities

20092009



Bioprocess Engineering and Biocatalysis

Laboratory BEBL

Nucleic Acid Bioengineering Laboratory NABL

Stem Cell Bioengineering Laboratory SCBL

Bioprocess Engineering and Biocatalysis

BEB

L

Objectives

Bioprocess Engineering and Biocatalysis aims to

design and develop value-added bioproducts with

potential application in the key areas of food and

feed, aroma, pharmaceutical industry and biofuels.

The current projects are focused on the develop-

ment of technological platforms for biocatalysis and

biomolecules purification organized in four major

areas: i) Bioseparation, ii) Biocatalysis and Biocon-

versions, iii) Biosensors and Miniaturization, and iv)

Bioenergy.

Research Topics

1. Bioseparations - Novel purification processes are

being developed to optimize and intensify the down-

stream processing of proteins and biopharmaceuti-

cals, with special emphasis on monoclonal antibod-

ies, including aqueous two-phase extraction (ATPE)

and affinity precipitation. Extraction separation units

using affinity materials and nano-magnetic particles

are being evaluated, characterized and validated by

modelling. Processes combining ATPE with chro-

matographic steps and using new biospecific

ligands are being exploited. Stimuli-responsive

beads based on PNIPAM are also being developed

as a strategy to produce novel bio-inspired affinity

polymer systems for antibody recognition.

2. Biocatalysis and Bioconversions - Rational strate-

gies for enzyme and whole cell bioconversions are

being developed and implemented, based on hy-

drolases (cutinase, penicillin acylase and inulinase)

and whole bacterial cells, where biocatalyst per-

formance is evaluated through a representative ar-

ray of reactions and operational conditions. Ester

biosyntheses (flavors, biodiesel and macrocyclic

esters) by cutinase and engineered mutants are

assessed in an enzymatic platform. The effect of

alternative media composition in biocatalyst per-

formance and cell wall integrity is being evaluated

using Rhodococcus and Mycobacterium strains as

model systems.

3. Biosensors and Miniaturization - Nano/micro-

biocatalysts (biocomposites) are being developed

based on hydrogels, protein/cell assemblies and

nano-magnetic particles. New protein-ionic-

conducting-based biocompatible materials (Ion

Jelly) with tailor-made properties have been de-

signed to build new planar amperometric biosen-

sors. Portable magneto-resistive biochip is in devel-

opment for pathogen microorganism detection (in

collaboration with INESC-MN). High throughput

systems are being used to assess the effects of

toxic compounds in cellular adaptation mecha-

nisms, to speed the optimization of biotransforma-

tion/fermentation systems and to establish scaling

strategies.

4. Bioenergy - Lipase and cutinase are being used

in non-conventional media for the sustainable pro-

duction of regular and jet biofuel. Oxido-reductases

are used in combination with an innovative material,

Ion Jelly, for structuring enzymatic biofuel cells.

Rhodococcus spp. are being used for the desulfuri-

zation of crude oil, mitigating acid rain and air pollu-

tion, as well as for the production of electricity using

physicochemical cell surface properties.

Main Achievements

A joint world patent with Bayer Technology Ser-

vices (Leverkusen, DE) was published describ-

ing a novel separation process for the purifica-

tion of monoclonal antibodies based on multi-

stage liquid-liquid extraction in aqueous two-

phase systems (ATPS).

Glutaric acid was identified as the most effective

ligand for the purification of human antibodies

from a CHO cell supernatant using PEG/dextran

ATPE. This extraction step was successfully

integrated with cation exchange chromatography

yielding a final product with 100% protein purity.


B

ioprocess E

ngin

eerin

g a

nd B

iocata

lysis

A new strategy for penicillin G acylase immobili-

zation in sol-gel was successfully developed

allowing a remarkable improvement of enzyme

activity in comparison with previously reported

sol-gel techniques.

Production of androstenedione from sitosterol

was scaled-up from multi-well plates to bench-

scale stirred bioreactor using suitable engineer-

ing parameters, namely volumetric power con-

sumption for resting cells and volumetric oxygen

transfer coefficient for growing cells.

Portable magneto-resistive biochip has been

developed for pathogen microorganism with

femtomolar DNA limit detection using specific

antibodies immobilized on nano-magneto parti-

cles.

High throughput systems were developed to

correlate the effect of toxic compounds with cel-

lular adaptation using Rhodococcus erythropolis

as model cells, to improve biotransformation

systems.

Selected Publications

Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., de

Vries, J., Visser, T.J., Aires-Barros, M.R., J. Chro-

matogr. B, 887: 50-58

Carvalho, F., Marques, M.P.C., de Carvalho,

C.C.C.R., Cabral, J.M.S., Fernandes, P., Biore-

source Technol., 100: 4050-4053

Bernardino, S.M.S.A., Fernandes P., Fonseca,

L.P., Biotechnol. J., 4: 695-702

Martins, V.C., Cardoso, F.A., Germano, J., Car-

doso, S., Sousa, L., Piedade, M., Freitas, P.P.,

Fonseca, L.P., Biosens. Bioelectron., 24: 2690-

2695

de Carvalho, C.C.C.R., Wick, L.Y., Heipieper, H.J.,

Appl. Microbiol. Biotechnol., 82: 311–320

Patent

Baecker, W., Sommerfeld, S., Mutter, M., Rosa,

P.A.J., Aires-Barros, M.R., Azevedo, A.M., World

Patent WO/2009/112149

NAB

L

Nucleic Acid Bioengineering

Objectives

Nucleic Acid Bioengineering is focused on: i) plas-

mid DNA vectors and their application in gene ther-

apy or DNA vaccination; and ii) microchips for DNA,

protein and cell detection. The specific objectives

are to address the scientific/technological chal-

lenges associated with plasmid biopharmaceuticals,

by combining biomolecular engineering studies with

bioprocess engineering, and to co-develop (with

INESC-MN) thin-film microchip and microfluidic plat-

forms for the manipulation/detection of DNA, anti-

bodies and cells.

Research Topics

In the case of plasmids, the following research top-

ics are pursued:

1. Structural stability of plasmids - Studies on the

nuclease barriers to gene expression during plas-

mid trafficking through the cytosol of mammalian

cells are performed with the goal of constructing

plasmid vectors with an increased resistance to

nucleases and less prone to recombination, and

thus with a higher transfection activity. Additionally,

the structure of plasmid vectors is probed for the

presence of sequences like direct and inverted re-

peats that promote recombination and other insta-

bility events.

2. Manufacturing of plasmid vectors - Processes for

the production of plasmids are conceptually de-

signed, developed, optimised and compared. The

impact of specific plasmid structural elements in the

performance of upstream and downstream proc-

esses (microfluidization, tangential flow filtration,

membrane and fixed-bed chromatography, aqueous

two-phase extraction) and in the quality of the final

product is evaluated. Analytical procedures to moni-

tor manufacturing and control product quality are

also developed.

3. DNA vaccine prototyping - DNA vaccine candi-

dates are constructed by cloning antigenic proteins

of the causing parasite of sleeping sickness disease

(Trypanosoma brucei brucei) and tested in mice

models for their ability to generate cellular and hu-

moral responses, and to provide protection against

infection.

In the case of microchips for DNA detection the fol-

lowing topics are addressed:

1. Immobilization and handling of DNA and cells -

Thin film technologies, chemical modification, mi-

crofluidics and electronic addressing are used to

develop microchips for the molecular recognition of

specific analytes via hybridization or cell process-

ing. The core of the chips is a flat surface with im-

mobilized probe molecules or entrapped cells.

Other features include the presence of micro-

electrodes to generate electric fields that accelerate

the kinetics of binding/recognition and promote di-

electrophoresis.

2. Photodetectors - Amorphous silicon photodetec-

tors are developed for the optoelectronic detection

of coloured, chemiluminescent and fluorescent

molecules in thin film chips. The presence of these

molecules ultimately reports specific biorecognition

events such as DNA hybridization or metabolic cell

activity.

Main Achievements

Mice immunized intramuscularly with a DNA

vaccine coding for trans-sialidase were able to

survive after a challenge with the infective form

of T. brucei brucei parasites.

The use of increased temperatures (42ºC) im-

proves pCIneo amplification (up to 4-fold) in E.


N

ucle

ic A

cid

Bio

engin

eerin

g

coli and decreases the ratio of plasmid recombi-

nation. The characteristic heterodimeric plasmid

recombinants are differently selected according

to the concentration of kanamycin used.

Unit operations (e.g. alcohol and salt precipita-

tion, tangential flow filtration and aqueous two

phase extraction) were evaluated in terms of

technical performance, economic viability and

environmental impact, as alternatives in the in-

termediate recovery of plasmid DNA from E. coli

lysates. The use of phenyl-boronate chromatog-

raphy and hydrophobic interaction membrane

chromatography for the purification of plasmid

DNA directly from alkaline lysates was demon-

strated.

The examination of the role of probe-to probe

interactions on the hybridization of double

stranded DNA in magnetic microparticles indi-

cated that the intensity of the hybridization sig-

nal is proportional to differences between the

average area available per probe and the pro-

jected surface area of each probe.

The use of electric field pulses was shown to

increases the speed of DNA immobilization and

hybridization on chemically functionalized SiO2

thin-film chips by several orders of magnitude,

with reaction time scaled down from 1 to 2 h to

a range between 100 ns and 1 ms (in collabora-

tion with INESC-MN).


Silva, M.S., Prazeres, D.M.F., Lança, A., Atouguia,

J., Monteiro, G.A., Parasitol. Res., 105: 1223-1229

Oliveira, P.H., Prazeres D.M.F., Monteiro, G.A., J.

Biotechnol., 143: 231-238

Freitas, S.S., Canário, S., Santos, J.A.L., Prazeres,

D.M.F., Biotechnol. J., 4: 265-278

Martins, S., Prazeres, D.M.F., Fonseca. L.P., Mon-

teiro, G.A., Anal. Bioanal. Chem., 394: 1711-1716

Cabeça, R., Rodrigues, M., Prazeres, D.M.F., Chu,

V., Conde, J.P., Nanotechnology, 20: 015503

SCB

L

Stem Cell Bioengineering

Objectives

The Stem Cell Bioengineering Laboratory aims at

the development of highly controlled culture sys-

tems (e.g. bioreactors) for the ex-vivo expansion of

stem cells and their controlled differentiation into

specific cell types. As stem cells (SC) are rare, their

isolation and expansion/differentiation in vitro sig-

nificantly increases the cell population available for

cellular and gene therapy settings, high-throughput

drug screening, tissue engineering and stem cell

research. Human hematopoietic stem cells (HSC),

human mesenchymal stem cells (MSC), as well as

human and mouse embryonic stem cells (ESC) and

neural stem cells (NSC) are used as model sys-

tems.

Research Topics

1. Ex-vivo expansion of HSC in co-culture with MSC

under serum-free conditions - Current research is

focused on: (i) the definition of optimal culture con-

ditions namely concerning cytokine combinations,

enrichment procedures and initial cell concentra-

tions used to provide an amplification of HSC, espe-

cially those obtained from the umbilical cord blood

(UCB); and (ii) the understanding of the mecha-

nisms underlying the hematopoietic supportive ca-

pacity of MSC. These will have implications in terms

of bioreactor design towards the maximization of

human HSC expansion in vitro.

2. Clinical-scale production of MSC for Cellular

Therapies - Culture protocols are being optimized

for the isolation and expansion of MSC under se-

rum-/xeno-free conditions, while maintaining their

multilineage differentiation and immunosuppressive

capacities, as well as their karyotypic stability. MSC

are isolated from adult bone marrow (BM), adipose

tissue (AT) and UCB. Culture of MSC under condi-

tions more closely related to the in vivo environment

(i.e. hypoxia (2%O2), dynamic systems allowing for

3-D cultivation) is currently being exploited to maxi-

mize MSC yield.

3. Multi-scale strategies for the expansion and neu-

ral differentiation of ESC and NSC - The expansion

of ESC and ESC-derived NSC is studied towards

the definition of highly controlled bioreactor systems

to establish an efficient, reproducible and cost-

effective bioprocess to obtain the starting material

to generate mature cells (i.e. neurons) for potential

use in the treatment of neurological disorders, as

well as for drug screening. High-throughput microar-

ray systems are also being developed to elucidate

important microenvironmental factors (i.e. chemical,

physical) affecting cell self-renewal and differentia-

tion, while providing the basis for rapid identification

of signals and conditions that can be used to direct

cellular responses.

4. Gene delivery strategies to stem cells - The main

goal is to develop efficient and safe methodologies

to genetically-engineer SC, using non-viral vectors,

enhancing their therapeutic efficacy in different Re-

generative Medicine applications. Therapeutic

genes or specific genes involved in self-renewal/

lineage commitment of SC, are being targeted for

their transient and safe overexpression, allowing for

the direct use of gene-modified SC in Cellular and

Gene Therapy settings, or the maximization of the

SC yield or their progeny for application in Cellular

Therapies and Tissue Engineering, respectively.

5. Recombinant protein production for stem cell

research - A versatile platform for the efficient, cost-

effective and reproducible production and purifica-

tion of stable and biologically active recombinant

proteins has been developed based on mammalian

bioreactor systems. The main goal is to produce

cytokines/growth factors such as Leukemia Inhibi-

tory Factor (LIF) and Bone Morphogenetic Protein 4

(BMP-4) which can be used to supplement stem cell

cultures, but also as potential therapeutics

(e.g. BMP-4).


Ste

m C

ell B

ioengin

eerin

g

Main Achievements

The consortium established between IBB-IST,

Instituto Português de Oncologia de Lisboa

Francisco Gentil and Centro de Histocompatibili-

dade do Sul, focusing the isolation and ex-vivo

expansion of MSC under GMP conditions for

Cellular Therapies was consolidated with three

more patients being successfully infused with

expanded MSC.

A divergent division kinetics was found between

BM and UCB HSC, elucidating the heterogeneity

underlying phenotypically identical HSC from

different sources. We also showed that the

beneficial effects on HSC expansion provided by

the MSC feeder layer used in our co-culture sys-

tem were mainly contact-mediated, rather than

based on soluble factor trafficking.

A low oxygen environment (i.e. hypoxia) was

demonstrated to improve both human MSC and

mouse NSC ex-vivo expansion. Hypoxia can

thus be considered a powerful tool towards the

maximization of cell yield from a reduced initial

population for potential application in clinical

settings.

Our previously established 3-D cellular microar-

ray platform demonstrated to be a powerful tool

for investigating cellular mechanisms underlying

mouse ESC self-renewal versus differentiation,

while providing the basis for rapid identification

of signals and conditions that can be used to

direct cell fate.

A non-viral gene delivery platform using cationic

liposomes was successfully established for BM

MSC, allowing the expression of a target gene in

a safe and transient way for potential use in Cell

and Gene Therapy settings.


Fernandes, T.G., Diogo, M.M., Clark, D.S., Dordick,

J.S., Cabral, J.M.S., Trends Biotechnol., 27: 342-

349

Madeira, C., Estrela, N., Ferreira, J.A.B., Andrade,

S.M., Costa S.M.B., Melo E.P., J. Biomed. Optics,

14: 044035

Serro, A.P., Completo, C., Colaço, R., dos Santos,

F., da Silva, C.L., Cabral, J.M.S., Araújo, H., Pires,

E., Saramago, B., Surface & Coatings Technol.,

203: 3701-3707

da Silva, C.L., Gonçalves, R., Porada, C.D., Ascen-

são, J.L., Zanjani, E.D., Cabral, J.M.S., Almeida-

Porada, G., J. Cellular Physiol., 220: 102-111

Temtem, M., Silva, L.M.C., Andrade, P.Z., dos San-

tos, F., da Silva, C.L., Cabral, J.M.S., Abecasis,

M.M., Aguiar-Ricardo, A., J. Supercritical Fluids,

48: 269-277

Research Highlights

20092009



Designing Safer DNA Vaccines

Membrane Chromatography for DNA Vaccine Purification

Miniaturization in Biocatalysis

Bio-inspired Affinity Polymer Systems for Antibody Recognition

Affinity-enhanced Extraction of Human Antibodies

BEBL

NABL

Designing Ex-vivo Expansion of Mesenchymal Stem Cells for

Cellular Therapies: from Lab to Clinic

Ex-vivo Expansion of Mouse Embryonic Stem Cell-derived

Neural Stem Cells Under Low Oxygen

SCBL

Pedro Fernandes, Carla C.C.R. Carvalho, Marco Marques and Joaquim M.S. Cabral

The use of miniaturized devices in biocatalysis

has been gaining momentum in recent years and

the trend for a wider dissemination of this ap-

proach to be well established. Based in the use of

vessels with volumes ranging from some mL

down to tenths of microL, with a wide level of par-

allelization (viz. 24- to 384-multiwell plates) and

prone to automation, this concept provides the

high throughput capability required for speeding

up process development with significant cost re-

ductions, considering both chemicals and biologic

agents and manpower. Within the scope of this

methodology, substantial research efforts have

been made in order to establish the conditions

that allow for reproducibility and scalability of the

data generated. These efforts are anchored in a

focused engineering approach, which had been

missing up to roughly the dawn of the new millen-

nium, and had somehow hampered the success in

the implementation of bio-based processes,

namely due to faulty scale-up.

Bioconversion of steroids

Among bioconversion systems with easily fore-

seen or already established industrial application,

several require a cascade of enzyme reactions

and/or a non-conventional environment, given the

sparingly water solubility of plenty of relevant

compounds. The large number of variables in-

volved when the characterization and optimization

of one such system is aimed at, such as the mi-

crobial side-chain cleavage of (phyto)sterols to

androstenedione (AD), makes it a perfect fit for

the miniaturization approach. This particular bio-

conversion was thus selected as model system.

Throughout the research work performed, 24-well

plates were established as suitable platforms for

the detailed study of the model system. The rele-

vance of operational parameters (viz. chemical

compatibility, evaporation rate, filling volume, na-

ture of the substrate/product carrier, hydrody-

namic conditions) in product yield and productivity

BEB

L

Miniaturization in Biocatalysis

Figure 1: Effect of chain length and ramification of aliphatic on the side-chain cleavage activity of Mycobacterium sp. NRRL B-

3805 performed in covered (open bars) and rubber sealed (closed bars) multiwell plates. Activity data referred to the activity

obtained in the presence of methanol, given in the inset.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

meta

no

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eta

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pro

panol

buta

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pen

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ol

hexan

ol

hep

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octa

nol

no

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decan

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un

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do

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nol

2-p

ropanol

2-b

uta

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l

2-p

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2-h

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ol

2-h

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2-o

cta

nol

2-n

onanol

2-d

ecan

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2-u

nd

ecanol

2-d

odecanol

3-n

onanol

Solvent

Rela

tive A

cti

vit

y

0.0

0.5

1.0

1.5

2.0

2.5

Co

nvert

ed

Sit

oste

rol(

mM

)

Activity (mMAD/h) Specific activity (mmol AD/g h)

Open 1,78E-02 7,13E-04

Closed 9,49E-03 3,80E-04

Activity data for methanol


Figure 2: AD production at different oxygen mass transfer

coefficient (kLa) measured in 24-well OxoDish® microtiter

plate. Filling volumes of 500 L were used in tape-sealed

wells. The kLa at 300rpm was 0.05±0.01 s-1 in unsealed micro-

titer plate with a filling volume of 500 l.

was ascertained (Fig. 1) [1]. The behaviour and

performance of the steroid bioconversion systems

anchored in either growing or resting cells were

addressed, and engineering criteria for scale-up

were identified, based in AD yield (Fig. 2). The

use of multiwell plates equipped with sensor spots

for pH and dissolved oxygen monitoring allowed

for gaining further insight on the time course of the

bioconversion, to compare with the pattern ob-

served in stirred bioreactor, and to further validate

scaling criteria [2].

Bacterial adaptation studies

High throughput systems were developed for test-

ing simultaneously a high number of reactions and

conditions during the study of cell resistance and

adaptation mechanisms towards toxic compounds

(Fig. 3). The inhibition concentrations could be

determined and generational adaptation to in-

creasing concentrations of toxic compounds was

studied. The results obtained allowed the im-

provement of biocatalytic and bioremediation sys-

tems, in particular the biotransformation of ter-

penes and the degradation of extremely high con-

centrations of toluene [3]. It also showed the effect

of antineoplastic agents on bacterial cells and the

response mechanisms activated by the cells.

Microfluidic reactors

Aiming for further miniaturization, research has

recently expanded towards the use of microfluidic

reactors, which allows for continuous operation as

well as for easier implementation of production

scale by scaling out rather than scaling-up. The

enzymatic oxidation of cholesterol to choleste-

none, one of the reactions of the pathway leading

to the production of AD from sterols, was effec-

tively performed in an organic-aqueous two-liquid

phase system. Further insight in the system is

aimed at, in order to contribute for implementing

the whole cell bioconversion system leading to

AD.

The work produced so far has not only contributed

to a better knowledge of the model system used,

but has also provided considerable insight to the

requirements of multiphase biocatalytic whole cell

systems. It is also contributing to define the key

issues to be addressed for the design of represen-

tative biotransformation systems based in minia-

ture scale platforms [4].

References

[1] Marques, M.P.C., Carvalho, F., Magalhães, S.,

Cabral, J.M.S., Fernandes, P., Process Biochem.,

44: 556-561 (2009)

[2] Marques, M.P.C., Magalhães, S., Cabral, J. M.

S., Fernandes, P., J. Biotechnol., 141: 174-180

(2009)

[3] de Carvalho, C.C.C.R., Wick, L.Y., Heipieper,

H.J., Appl. Microbiol. Biotechnol. 82: 311-320

(2009).

[4] Carvalho, F., Marques, M.P.C., de Carvalho,

C.C.C.R., Cabral, J.M.S., Fernandes, P., Biore-

source Technol., 100: 4050-4053 (2009)

R

esearch H

ighlights

0

0.002

0.004

0.006

0.008

0.010

0 0.2 0.4 0.6 0.8 1.0

Relative k La

g A

D .(

g b

iom

as

s)-1

0 164 216 246 267 300

rpm

Figure 3: Bacterial cells growing in the presence of

different concentrations of toxic compounds in a micro-titre plate.

BEB

L

Cláudia Silva, Jaqueline Garcia and M. Ângela Taipa

Affinity-based strategies offer high-resolution

separation and are the most commonly used in

the purification of immunoglobulins and other high

-value proteins of pharmaceutical importance [1].

The affinity ligands utilised range from biological

to synthetic designed molecules with enhanced

chemical resistance and stability.

Recent progresses in polymer chemistry science

open a number of possibilities for new materials

by allowing the synthesis of bio-inspired (or biomi-

metic) polymeric materials with highly controlled

structures. These materials can combine advanta-

geous chemical properties like thermal and pH

sensitivity, magnetic or optical behavior, with the

specificity and selectivity characteristic of biologi-

cal systems. “Smart” polymers, such as thermo-

sensitive poly(N-isopropylacrylamide) (PNIPAM),

are particularly suitable for biological applications

[2] and constitute an interesting alternative for

developing novel affinity materials.

The main goal of this project is the synthesis and

application of bio-inspired affinity polymer systems

based on a “smart” thermosensitive polymeric

material (poly (N-isopropylacrylamide) (PNIPAM)),

conjugated to synthetic (Protein L mimic) affinity

ligands with high selectivity for immunoglobulins

or antibodies. The systems are designed and

studied in order to implement mild and cost-

effective affinity methodologies for the molecular

recognition/purification of antibodies and geneti-

cally engineered related molecules (Figure 1).

Cationic PNIPAM microgel particles containing

primary amino groups were obtained via precipita-

tion polymerization under surfactant-free condi-

t ions by co-po lymer isat ion with 2-

aminoethylmethacrylate (AEMH) or N-(3-

aminopropyl)methacrylamide (APMH). Both co-

monomers improve colloidal stability leading to

the formation of smaller and higher number of

particles, being this effect more pronounced for

AEMH (Figure 2).

Figure 1: Schematic representation of an affinity precipitation process of antibodies with a

biomimetic thermosensitive polymer: (A) capture of the target in solution; (B) precipitation of the affinity complex and recovery of the precipitate; (C) dissociation of the target molecule from the affinity complex (D) recovery of the target molecule; and (E) recovery of the macroli-

gand for reuse (adapted from [1]).

Bio-inspired Affinity Polymer Systems for Anti-

body Recognition

Pure Antibody

Soluble-Phase

Affinity Binding

Reversible

Precipitation

Dissociation

Crude Sample

Product

Recovery

T > LCST

T < LCST

BA

CD

E

Pure Antibody

Soluble-Phase

Affinity Binding

Reversible

Precipitation

Dissociation

Crude Sample

Product

Recovery

T > LCST

T < LCST

BA

CD

E


Figure 2: Reversible swelling/shrinking transition of microgels triggered by small changes in temperature within the physiologi-

cal range. The occurrence of this macroscopic event is due to the existence of a lower critical solution temperature (LCST) at ~32ºC for PNIPAM linear homopolymer, corresponding to a volume phase transition temperature (VPTT) for cross-linked parti-cles. The variation of particle size with temperature was evaluated by dynamic light scattering (DLS) for particles synthesis ed

without co-monomer (A) and in the presence of different concentrations of co-monomer either in batch (1% mol) or by shot-

addition (2% mol) (B).

Figure 3: General structure of biomimetic polymer systems obtained by conjugation of synthetic affinity ligands to microgel

particles (A). Adsorption studies of human IgG with lead ligand/polymer systems at 20ºC and in PBS (pH 7.0) (B). Controls

with non-conjugated particles.

The hydrophilic character of co-monomers affects

the thermo-sensitive profile of the particles: with

AEMH (more hydrophilic) the transition occurs

within a broader range and at higher tempera-

tures, while with APMH (more hydrophobic) the

transition is more defined and occurs at slightly

lower temperatures (Figure 2B).

Triazine-scaffolded synthetic affinity ligands with

high affinity and selectivity for immunoglobulins

were covalently grafted to amino-functionalised

PNIPAM microgel particles (Figure 3A). Lead bio-

inspired/biomimetic polymer systems were

screened for the adsorption of human immu-

noglobulin G (hIgG) as a model-protein. Selected

biomimetic particles were shown to be

„biologically‟ active, and specifically recognize and

capture hIgG (Figure 3B).

The project Biomimpolymers is developed in col-

laboration with the Centro de Química-Física Mo-

lecular (CQFM) of Instituto Superior Técnico and

the Laboratoire de Catalyse, Chimie, Polymères

et Procédés-C2P2/CNRS/CPE/UCBLyon1

(France). As a main output this work aims to con-

tribute to the generation of new bio-inspired mate-

rials with controlled structures, projected towards

the molecular recognition of antibody molecules.

Such materials are expected to have impact in

applications regarding separation science and

technology, diagnostics and biomedicine.

References

[1] Roque, A.C.A., Silva, C.S.O., Taipa, M.A., J.

Chromatogr. A, 1160: 44-55 (2007)

[2] Silva, C.S.O., Baptista, R.P., Santos, A.M.,

Martinho, J.M.G., Cabral, J.M.S., Taipa, M.A.,

Biotechnol. Lett., 28: 2019-2025 (2006)

R

esearch H

ighlights

T<VPTT T>VPTT

T

T<VPTT T>VPTT

TT

PNIPAM

100

300

500

700

900

1100

1300

1500

1700

1900

15 20 25 30 35 40 45 50 55

T (°C)

Hyd

rod

yn

am

ic d

iam

ete

r (n

m)

Co-polymers of PNIPAM

100

200

300

400

500

600

15 20 25 30 35 40 45 50 55

T (°C)

Hyd

rod

yn

am

ic d

iam

ete

r (n

m)

2%APMHsa

1%APMH

2%AEMHsa

1%AEMH

A B

A B

-0.3

0.2

0.7

1.2

1.7

2.2

conjugated non conjugated conjugated non conjugated

Ad

so

rbe

d Ig

G (

mg

/g a

ds

orb

en

t)

1mg/g 3mg/g 5mg/g 7mg/g 10mg/g

PNIPAM-AEMH PNIPAM-APMH

Initial IgG:

N

N

N

R1

R2

NH

CH3O

O

N

N

N

R1

R2

NH

CH3O

O

T<VPTT T>VPTT

T

T<VPTT T>VPTT

TT

PNIPAM

100

300

500

700

900

1100

1300

1500

1700

1900

15 20 25 30 35 40 45 50 55

T (°C)

Hyd

rod

yn

am

ic d

iam

ete

r (n

m)

Co-polymers of PNIPAM

100

200

300

400

500

600

15 20 25 30 35 40 45 50 55

T (°C)

Hyd

rod

yn

am

ic d

iam

ete

r (n

m)

2%APMHsa

1%APMH

2%AEMHsa

1%AEMH

A B

BEB

L

Ana Azevedo, Paula Rosa, Filipa Ferreira and Raquel Aires-Barros

Monoclonal antibodies are becoming a major

source of biopharmaceuticals for the treatment of

different type of diseases including cancer. As a

consequence, the design and production of anti-

bodies for therapeutic purposes has been consid-

erably improved in the last decade, with titres sur-

passing 10 g/l. Hence, with capacity bottlenecks

moving towards downstream purification areas,

the need for a broader strategic approach for the

purification of monoclonal antibodies is being in-

creasingly recognized in order to improve the

overall process performance.

The evidence supporting the success and effec-

tiveness of aqueous two-phase extraction as a

unit operation in the downstream processing of

antibodies is compelling [1]. The possibility of

modifying the affinity of a protein for one of the

phases by functionalization of the phase forming

component constitutes a major advance in this

area, by allowing an increase in both binding ca-

pacity and selectivity.

Single-stage extraction

PEG molecules with different molecular weights

have been modified with different type of ligands,

selected on the basis of their expected interaction

with antibodies through electrostatic, hydrophobic

and affinity effects (Fig. 1). The ability of these

ligands to bind to human IgG was assessed either

as phase-forming compounds or as free ligands

(attached to triethylene glycol - TEG molecules). It

was observed that systems containing polymer

molecules modified with glutaric acid, an amino-

acid mimetic ligand, showed the highest affinity for

human antibodies [2].

The selectivity of each ligand was also evaluated,

for the purification of human IgG from a Chinese

Hamster Ovary (CHO) cell supernatant and again

higher recovery yields in the top PEG-rich phase

were observed using glutaric acid ligands. Table 1

summarizes the performance parameters of each

ligand. The highest partition coefficient was ob-

tained in PEG/dextran systems with added

triethylene glycol glutaric acid (TEG-COOH).

The effect of pH, TEG-COOH concentration and

volume ratio on the partitioning of IgG and on the

different components of the CHO supernatant was

studied and it was further observed that the condi-

tions that favoured the selective extraction of IgG

were lower pH values and high TEG-COOH con-

centrations. The extraction of IgG could be en-

hanced using higher volume ratios, however with

a significant decrease in both purity and percent-

age of contaminants removal [3]. After optimiza-

tion, it was possible to recover 96% of IgG in the

Affinity-enhanced Extraction of Human Anti-

bodies

OH

O O

NH2

SONaO

O

N

N

H

N

NN

N

N

N

N

NH

NH

OH

OH

N

N

O

S

Ligands attached to PEG or TEG molecules

Glutaric acid (COOH)

Amino

Benzyl

Pyrimidine (Pym)

Sulfonate Modified Triazine (A2P)

Aminoquinuclidine (AQ1)

Mercaptoethylpyridine (MEP)

Figure 1: Ligands attached to PEG or TEG molecules.


top phase (TP) outlet, with a final concentration of

0.22 g/l (Fig. 2A).

Multi-stage extraction

In order to enhance simultaneously both recovery

yield and purity, a four stage cross-current opera-

tion was simulated and the corresponding liquid-

liquid equilibrium (LLE) data determined allowing

the prediction of an optimised counter current ex-

traction based on McCabe-Thiele diagrams [3].

Accordingly, IgG can be purified in the PEG-rich

top phase with a final recovery yield of 95%, a

final concentration of 1.04 g/l and a protein purity

of 93%, if a PEG/dextran ATPS containing 1.3%

TEG-COOH, 5 stages and volume ratio of 0.4 are

used (Fig. 2B). This represents a significant im-

provement specially in terms of purity in compari-

son to the single-stage extraction.

This project has been developed together with

Bayer Technology Services (Leverkussen, Ger-

many) and Syncom BV (Gronigen, NL).

References

[1] Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F.,

Aires-Barros, M.R., Trends Biotecnhol., 27: 240-

247 (2009)

[2] Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F.,

Pisco, A.M.M.O., de Vries, J., Korporaal, R., Vis-

ser, T.J., Aires-Barros, M.R., Sep. Purif. Tecnhol.,

65: 31-39 (2009)

[3] Rosa, P.A.J., Azevedo, A.M., Ferreira, I.F.,

Sommerfeld, S., Bäcker, W., Aires-Barros, M.R.,

J. Chromatogr. A, 1216: 8741-8749 (2009)

R

esearch H

ighlights

1

YTop = 96%

TP Outlet

[IgG] = 0.22 g/l

PProtein = 87%

PTotal = 43%

BP Inlet

[IgG] = 0.424 g/l

PProtein = 65%

PTotal = 29%

BP Outlet

[IgG] = 0.02 g/l

TP Inlet

[IgG] = 0 g/l

System log KP Yield PProtein PHPLC

PEG/dextran -0.46 23% 39% 29%

PEG-COOH/dextran 0.65 97% 94% 41%

PEG-NH2/dextran 0.09 76% 82% 32%

PEG-Triazine/dextran -1.00 8% - -

PEG-Benzyl/dextran 1.32 88% 87% 34%

PEG-MEP/dextran -0.24 58% 72% 25%

PEG-Pym/dextran -0.16 59% 82% 21%

PEG/dextran+TEG-COOH 1.32 96% 95% 41%

PEG/dextran+TEG-MEP -0.21 45% 61% 13%

PEG/dextran+TEG-SO3 -0.28 37% 52% 33%

PEG/dextran+TEG-AQ1 -0.29 15% - -

Table 1: Performance of PEG/dextran systems modified with different types of ligands for the purification of human

antibodies from CHO cell culture supernatant, including the partition coefficient (KP), recovery yield in the top phase,

protein purity (determined as the IgG/protein ratio - PProtein) and total purity determined by size-exclusion HPLC (PHPLC).

Figure 2. Optimized scheme of: (A) single-stage ATPE of IgG

using a PEG/dextran ATPS containing 1.3% TEG-COOH with a

volume ratio of 2.2 and (B) predicted counter-current multi-stage

ATPE of IgG using a PEG/dextran ATPS containing 1.3% TEG-

COOH, 5 stages and a volume ratio of 0.4; BP: dextran-rich

bottom phase (14.19% dextran and 2.60% PEG 3350); TP:

PEG-rich top phase (9.74% PEG 3350 and 0.004% dextran);

Y: IgG recovery yield in the top phase.

1

Y = 5%

2

Y = 11%

3

Y = 21%

4

Y = 42%

5

Y = 95%

BP Inlet

[IgG] = 0.44 g/l

PProtein = 65%

PTotal = 30%

BP Outlet

[IgG] = 0.02 g/l

TP Outlet

[IgG] = 1.04 g/l

PProtein = 93%

PTotal = 85%

TP Inlet

[IgG] = 0 g/l

[IgG] = 0.06 g/l [IgG] = 0.23 g/l

[IgG] = 0.12 g/l [IgG] = 0.46 g/l

A

B

NAB

L

Pedro H. Oliveira, Sofia C. Ribeiro, Geisa Gonçalves, Duarte Miguel F. Prazeres,

Gabriel A. Monteiro

Gene therapy and DNA vaccination have

emerged in the last two decades as promising

alternatives for the treatment and prevention of

genetic disorders and acquired diseases. Non-

viral vectors, such as naked plasmids, constitute a

safer gene delivery alternative to viral vectors due

to their lower toxicity and larger gene capacity.

Plasmid DNA (pDNA) vaccines also offer a credi-

ble alternative for the prevention and treatment of

infectious and acquired diseases. In order for a

DNA vaccine to be successfully developed, sev-

eral scientific and technological challenges asso-

ciated with the design of pDNA must be ad-

dressed [1]. In fact, one of the main obstacles

found during the developmental stages of a DNA

molecule involves tackling several instability

events, therefore assuring its structural integrity

[1]. The research program on “Nucleic Acid Bioen-

gineering” addresses some of these challenges.

The type and overall organization of the genetic

elements present in pDNA vaccines will directly

impact not only its bulk production, but also shelf-

stability, efficacy and ultimately its clinical ap-

proval. The mammalian expression vector and

DNA vaccine backbone pCIneo was used

throughout our studies as a model plasmid to gain

further insight into genetic instability. During

Figure 2: Plasmid pCIneo::IS2.

shown is the hybrid promoter

generated by the IS2 −35 and

pCIneo −10 hexamer sequen-

ces. Capital letters match the E.

coli promoter consensus

sequence TTGACA N16–18

TATAAT. The boxed region

represents the 5 bp duplicated

sequence generated upon IS2

insertion. The arrow indicates

the junction site between the

right inverted repeat (RIR) of

IS2 and pCIneo.

Designing Safer DNA Vaccines

3788 atcggaaT tcaAg cacctaaacggggatatAaAGgTctgt 3748

-35 hexamer-10 hexamer 17 bp

IS2neor

Ampr

SV40 enhancer and early promoter

Phage f1 region

CMV immediate early enhancer and promoter

Figure 1: A) Recombination between directly repeated sequences (blue arrows) in a parental (P) plasmid can

give rise to monomeric (M) and heterodimeric (1+2; 1+3) deletion products.

P M 1+2 1+3

pCIneo::IS2

6805 bp


1.E-10

1.E-08

1.E-06

1.E-04

1.E-02

0 1000 2000 3000 4000

FR

LS (bp)

18

50

352

800

LR (bp)

Figure 3: A) Recombination frequency (FR) as a function of spacer length (LS) for fix values of repeat length (LR) in recA+

strains. Lines correspond to model predictions while symbols correspond to experimental data. B) Distribution of direct repeats in plasmid vectors. Repeat distribution analysis shows that direct repeats are frequently found within antibiotic resistance genes, non-coding regions and regulator sequences in bacterial expression vectors while in mammalian expression vectors

they are predominantly found within eukaryotic promoters, non coding and regulator sequences.

pCIneo amplification in Escherichia coli cells, we

were faced with a recurrent contamination consist-

ing in aberrant monomeric and heterodimeric side

products (Fig. 1). The latter were the result of dele-

tion-formation events between two 28-bp direct

repeats of pCIneo, ultimately leading to the expres-

sion of a distant neomycin resistance (neoR) gene

[2]. Expression of this neoR gene was explained by

RNAII transcription beyond ori as a result of non-

hybridized RNAII-DNA molecules [3]. Interestingly,

we were able to lower the ratio of deletion forma-

tion products to total plasmid content by exploiting

the runaway properties of this vector at higher tem-

peratures (42ºC) and/or using a minimal growth

medium such as MM9 [4].

Besides deletion-formation, our group also ob-

served the occurrence of IS2-mediated transposi-

tion events in pCIneo. IS2 was found to transpose

upstream the neoR gene and was able to potenti-

ate the transcription of this gene through the gen-

eration of a hybrid promoter in the IS2-neoR junc-

tion (Fig. 2).

Aiming at a broader picture of both frequency and

abundance of instability events mediated by re-

peated sequences, our group has also performed i)

a meta analysis on plasmid recombination fre-

quency in order to develop mathematical functions

able to correlate this variable with repeat and

spacer lengths [5] (Fig. 3A); ii) an in silico search

for direct and inverted repeats using Repseek in a

large number of bacterial and mammalian expres-

sion vectors (Fig. 3B). Some of these sequences

are currently under evaluation for its removal or

modification.

DNA vaccination and gene therapy have matured

to the point where a number of products should be

hitting the market in the wake of the first veterinary

DNA vaccines. Furthermore, new developments

are likely to surface within the coming years be-

cause of increased investments from academia

and industry in the area. Our work is a contribution

to these efforts.

References

[1] Oliveira, P.H., Prather, K.J., Prazeres, D.M.F.,

Monteiro, G.A., Trends Biotechnol., 27: 503-511

(2009)

[2] Ribeiro, S.C., Oliveira, P.H., Prazeres, D.M.F.,

Monteiro, G.A., Mol. Biotechnol., 40: 252-262

(2008)

[3] Ribeiro, S.C., Prazeres, D.M.F., Monteiro, G.A.,

Appl. Microbiol. Biotechnol., doi: 10.1007/s00253-

009-2339-3 (2010)

[4] Oliveira, P.H., Prazeres, D.M.F., Monteiro, G.A.,

J. Biotechnol., 143: 231-238 (2009)

R

esearch H

ighlights

A B

NAB

L

Luis Raiado, Duarte Miguel F. Prazeres, Marília Mateus

The BioEngineering Research Group has for long

been recognized for its relevant experience in

downstream processing, namely, with membrane

technologies. In the framework of the research pro-

gram on “Nucleic Acid Bioengineering”, the appli-

cation of consolidated and design of new mem-

brane technologies, associated with fundamental

studies in areas of biomolecular engineering and

bioprocessing, have been supporting the develop-

ment of DNA vaccine prototypes.

Chromatography is one of the key operations in the

downstream processing of plasmid DNA (pDNA).

However, the increased demand for highly purified

pDNA experienced in recent years has made clear

the need for alternative processes capable of re-

taining the advantages of conventional chromatog-

raphy, such as selectivity, while providing in-

creased throughput at a lower cost. Recent

achievements have proven that alkyl-like chains in

functionalized membrane supports have the ability

to separate and resolve the model plasmid pVAX1-

LacZ (6050bp) from impurities in clarified Es-

cherichia coli cell lysates (specifically RNA) [1].

Characterization studies on derivatized mem-

branes evidenced the influence of reaction media

(Fig. 1) on reepoxydation yields. Membrane A was

at first suggested as the best candidate for the HIC

membrane process due to its higher epoxy surface

density (43% epoxy yield). Membrane C had the

lowest epoxy surface density (only 32% epoxy

yield), had the highest density of residual free

amines and was, therefore, figured as the worst

candidate for HIC. The presence of these residual

amines is undesirable since these moieties can

mediate non-specific interactions with pDNA, and

thus deteriorate HIC resolution [2].

Implementation of chromatography with these

membranes (Figs. 2 & 3) has shown that mem-

brane C presented the highest selectivity among

those tested (gel electrophoresis analysis showed

less contaminated pDNA peaks by RNA). Struc-

tural differences due to cross-linking in the mem-

brane matrix are probably conditioning the selectiv-

ity results. Optimizing elution profiles with the best

performing modified membrane (Fig. 4), led to a

4.7 purification factor with 73% plasmid yield, com-

petitive with its bead process counterpart [2].

Membrane Chromatography for DNA Vaccine

Purification

Figure 1: Derivatization of epoxy membrane by reaction in 3-steps: (I) amination,

(II) re-epoxidation, (III) blocking.

Figure 2: Modified 25 mm membrane

discs in an in-line membrane holder linked to a chromatographic system (ÄKTA Puri-

fier, GE Healthcare).


This project is in its early stages; however the on-

going work aims to perform the essential research

to fasten the development of membrane chroma-

tography for purification of plasmid-mediated DNA

vectors based on hydrophobic and affinity interac-

tions. The main strategy consists of three ap-

proaches: (i) rational design of membrane material

properties, based on chemical composition of func-

tional groups for bio-interaction with nucleic acids

and topography and internal structure of mem-

brane chromatographic matrices, to render high

resolution and high hydrodynamic capacity chro-

matographic media; (ii) the design of rapid, sensi-

tive and specific biological assays to monitor effec-

tiveness of biological interactions between model

plasmid DNA bio-therapeutics and functional

groups built-up on the surfaces of matrices and; (iii)

the improvement of atomic force microscopy proto-

cols for scanning interaction forces between de-

signed chromatographic membranes surfaces and

in-house developed plasmid-DNA probes.

The approach is innovative for tailoring membrane

materials properties and has the potential of be-

coming a high throughput screening technology to

expand membrane chromatography of DNA bio-

therapeutics. For a successful undertaking, the

“Nucleic Acid Bioengineering” research team is

working in close collaboration with the “Materials

Physics” group of the Institute of Materials and Sur-

faces Sciences and Engineering of Instituto Supe-

rior Técnico which has profound knowledge on the

methodological development of AFM applications.

References

[1] Raiado-Pereira, L., Prazeres, D.M.F., Mateus,

M., J. Sep. Sci. (2010) (accepted)

[2] Freitas, S.S., Santos, J.A.L., Prazeres, D.M.F.,

Sep. Purif. Technol., 65: 95-104 (2009).

R

esearch H

ighlights

0

20

40

60

80

100

120

140

160

180

200

220

0

200

400

600

800

1000

1200

1400

0 5 10 15 20 25 30 35

Co

nd

ucti

vit

y (m

S/c

m)

Ab

so

rban

ce @

260n

m (

mA

U)

Time (min)

UV Absorbance Lysate with pDNA

UV Absorbance Lysate without pDNA

Conductivity

(1)

(2,3) (4)

(5-10)

0

20

40

60

80

100

120

140

160

180

200

220

0

200

400

600

800

1000

1200

1400

0 5 10 15 20 25 30 35 40 45 50 55 60

Co

nd

ucti

vit

y (m

S/c

m)

Ab

so

rban

ce @

260n

m (

mA

U)

Time (min)

UV Absorbance injection1

UV Absorbance injection2

Conductivity

Figure 3: Chromatographic performance for feeds of 100 μL

diluted clarified lysate solutions of DH5α E. coli cells bearing

no plasmid (dashed lines) and bearing the pVAX1-LacZ

plasmid model (solid grey lines), over one membrane C disc,

at room temperature. Chromatography performed with 1.5 M

(NH4)2SO4 in 10 mM Tris pH 8.0 as binding buffer and 10mM

Tris pH 8.0 as elution buffer using isocratic elution at 0.5 mL/

min.

Fractions of the chromatographic runs of lysates bearing

pDNA were analysed by agarose gel electrophoresis. Only

those fractions numbered as (2,3) contained significant

amounts of pDNA with a slight contamination by RNA impu-

rities, while increasingly more hydrophobic RNA fractions

were separated (fraction 4 and higher numbered).

Figure 4: Superimposed chromatograms of

repeated chromatographies performed over 2

staked membrane-C specimens at room tempe-

rature using each time 400 μL of lysate volume

to be separated.

Buffers: 1.8 M of (NH4)2SO4 in 10mM Tris pH

8.0 used for equilibration and binding and 10

mM Tris pH 8.0 used in elution.

Elution profile: 20 min long gradient elution from

0% up to 40% elution buffer followed by a step

up to 100% of elution buffer for 20 min; at

2.5MV/min of flowrate.

SCB

L

Stem Cell Bioengineering Science aims to contrib-

ute for a better knowledge of the ex-vivo expan-

sion of stem cells and/or their controlled differen-

tiation into specific cell types in bioreactor sys-

tems. In particular, human mesenchymal stem

cells (MSC) have become one of the most promis-

ing candidates for Tissue Engineering and Regen-

erative Medicine applications, mostly due to the

differentiative potential and immunologic proper-

ties of these multipotent cells. By combining a

cross-disciplinary approach of Stem Cell Bioengi-

neering and Experimental Hematology, our goal at

BERG-SCBL is the optimization of MSC ex-vivo

expansion for application in Cellular Therapy,

namely for supplementation in hematopoietic stem

cell transplantation settings.

A low oxygen environment - Hypoxia

In their bone marrow (BM) niche, self-renewal

and/or differentiation of MSC are governed by a

complex microenvironment signaling that involves

cell-to-cell interactions, soluble factors, mechani-

cal forces and oxygen tension.

A 2% O2 hypoxic environment was found to im-

prove BM MSC expansion levels by inducing an

early start of the exponential growth phase, with

cells starting cell division earlier in culture com-

pared to normoxia. In addition, we observed an

increase of cellular metabolism efficiency, associ-

ated to the adaptation of BM MSC to the low oxy-

gen tension environment. These results gave im-

portant insights on how hypoxia culture favors

human BM MSC ex-vivo expansion, being advan-

tageous towards the maximization of cell yield in a

clinical-scale MSC expansion process (Fig. 1) [1].

Culture under stirred conditions

MSC cultures have been performed in traditional

culture flasks under static conditions, which are

limited in terms of cell productivity, their non-

homogeneous nature, resulting in concentration

gradients (pH, nutrients, metabolites,...), difficulty

of monitoring and an extensive handling require-

ment for feeding/harvesting procedures and lim-

ited productivity. Therefore the development of a

reproducible and robust process is required for

the production of clinical relevant human MSC

numbers.

We demonstrated the feasibility of using a micro-

carrier-based stirred culture system for the effi-

cient expansion of both BM and adipose tissue

(AT)-derived MSC in low serum conditions (Fig.

2).

Francisco dos Santos, Pedro Z. Andrade, Cláudia Lobato da Silva and Joaquim MS

Cabral

Figure 1: Ex-vivo expansion of human BM MSC under hypoxia (2% O2) and normoxia (20% O2).Total cell numbers (A) were

determined throughout the culture for both hypoxia (black circles) and normoxia (white squares) and the fold increase values in total cell number (B) obtained for hypoxia (black bars) and normoxia (gray bars) are presented as mean ± SEM (*p < 0.05)

[1].

Ex-vivo Expansion of Mesenchymal Stem Cells

for Cellular Therapies: from Lab to Clinic

A B


Clinical-scale production of MSC for Cellular

Therapies

The consortium between IBB-IST, Instituto

Português de Oncologia de Lisboa Francisco

Gentil and Centro de Histocompatibilidade do Sul,

focusing the isolation and ex-vivo expansion of

MSC under GMP conditions for Cellular Therapies

was established in 2007. Since then, 8 patients

have already benefited from this pioneer treat-

ment. MSC were used in the treatment or preven-

tion of graft-versus-host disease (GVHD) and also

to facilitate allogeneic hematopoietic stem cell

engraftment and decrease regimen-related toxic-

ity. The clinical cases under this project for the

last 2 years include:

Acute GVHD

Extensive chronic GVHD

Hurler‟s syndrome

Familial hemophagocytic lymphohistiocytosis

Aplastic anemia

References

[1] dos Santos, F., Andrade, P.Z., Boura, J.S.,

Abecasis, M.M., da Silva, C.L., Cabral, J.M.S., J.

Cell Physiol., 223: 27-35 (2010)

Figure 2: Expansion of BM (A) and AT (B) MSC in spinner flasks using low serum (2% Fetal Bovine Serum) media. We com-pared Cultispher S gelatin-microcarriers (blue line) with the animal-free plastic microcarriers (red line) as support for MSC

adhesion and proliferation. Results are presented as mean ± SEM.

Figure 3: Images of an ex-vivo expansion of human MSC for cellular therapy under GMP-conditions in a clean room at Centro

de Histocompatibilidade do Sul, Lusotransplante, Lisboa.

R

esearch H

ighlights

0.0E+00

5.0E+06

1.0E+07

1.5E+07

2.0E+07

2.5E+07

3.0E+07

3.5E+07

0 1 2 3 4 5 6 7 8 9

Ce

ll n

um

be

r

Time (days)

0.E+00

1.E+07

2.E+07

3.E+07

4.E+07

5.E+07

6.E+07

1 3 5 7 9

Ce

ll n

um

ber

Time (days)

Cell n

um

ber

Time (days) Time (days)

Cell n

um

ber

A B

SCB

L

Neural stem (NS) cells can provide a source of

material with potential applications in different

settings. The Stem Cell Bioengineering Labora-

tory at BERG-SCBL aims at the large-scale ex-

vivo expansion of NS cells, as starting material to

generate mature cells (e.g. neurons) for potential

use in regenerative medicine (e.g. treatment of

neurological disorders), as well as for neural drug

testing or developmental studies. The ultimate

goal is to define an efficient, reproducible and cost

-effective process for the production of NS cells in

large scale under highly controlled conditions,

while maintaining their multipotency.

The in vitro derivation of cultures of NS cells grow-

ing under adherent conditions, with no significant

decline in potential over time, was recently dem-

onstrated, from several sources, such as mouse

ES cells. These cells show long-term self renewal

capacity and maintain the capacity to generate

neurons, astrocytes and oligodendrocytes. Under

the presence of epidermal growth factor (EGF)

and basic fibroblast growth factor (bFGF), these

cells expand under adherent conditions, which

may be advantageous compared to the usual NS

cell culture as floating aggregates (neurospheres).

In neurospheres, besides oxygen and nutrients

diffusion limitations to the cluster centre, NS cells

are not directly identifiable and co-exist with com-

mitted progenitors and differentiated cells. Conse-

quently, this heterogeneity can lead to non-

reproducible results amongst different laborato-

ries. In this work, the continuous ex-vivo expan-

sion of the model mES cell-derived NS cell line

CGR8-NS was characterized and optimized in

serum-free conditions [1].

Oxygen and NS cells

Oxygen is an important energy source for cell me-

tabolism and its concentration is tightly regulated

in the central nervous system, where the pO2 val-

ues are similar among different mammalian spe-

cies and range from 2-5% in the cortex. Lower

concentrations of oxygen have been demon-

strated to have a positive effect on the prolifera-

tion of NS cells from different origins, both in

terms of species or tissue and had influence in

their fate, when induced to differentiate. The effect

of low oxygen tension on one mES cell-derived

NS cell line was studied.

mES cell-derived NS cell growth under differ-

ent oxygen tensions

The influence of oxygen on the optimization of

mouse ES cell-derived NS cell culture was evalu-

ated by testing different O2 tensions: 20%, 10%,

Carlos Rodrigues, Margarida Diogo, Cláudia Lobato da Silva and Joaquim MS Cabral

Ex-vivo Expansion of mouse Embryonic Stem Cell-

derived Neural Stem Cells Under Low Oxygen

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

0 3 6 9 12

Cu

mu

lati

ve C

ell

Nu

mb

er

Time (days)

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

0 5 10 15 20

Sp

ecif

ic G

row

th R

ate

(d

ay

-1)

O2 Tension (%)

Figure 1: Effect of O2 on CGR8-NS cell expansion. (A) Cumulative number of cells during successive passaging under 20%

(), 10% (), 5% () and 2% (▲) O2. Cells were passaged every 3 days during a maximum of 12 days. (B) Specific growth

rate determined from the slopes of the cumulative cell number curves plotted in Figure 1A [1].

A B


5% and 2% (Fig. 1). Cells were plated with an

optimal initial cell density and passaged succes-

sively under an atmosphere with the different O2

concentrations mentioned above. It is clear that,

when compared to the standard 20% atmospheric

O2 tension, low oxygen tension strongly enhances

the expansion of CGR8-NS cells. This positive

effect was observed after the first passage and

was consistently maintained throughout the fol-

lowing passages. These results indicate that, to

obtain the best performance in terms of cell prolif-

eration, CGR8-NS cells must be cultured under an

optimum O2 tension range between 2-5%, ap-

proximately the physiological concentration of

oxygen in the brain (Fig. 2). These conditions led

to fold increase values in total cell number about

twice higher than observed under 20% oxygen

being this effect more pronounced when cells

were plated at low density.

Hypoxia does not affect mES-derived NS cell

multipotency

Before selecting 2% as the optimum oxygen ten-

sion value for the expansion of CGR8-NS cells,

we verified that the expression of Nestin, a typical

marker of neural stem/progenitor cells was not

impaired by this condition. The differentiative po-

tential of NS cells expanded under 2% O2 was

also studied in order to assess the multipotency of

these cells. In the case of astrocyte differentiation,

after 3 days, the typical “star-like” cell morphology

was observed and cells were positive for the ex-

pression of astroglial markers. In terms of neu-

ronal differentiation, cells were harvested after 6

days, when expression of early neuronal markers

is expected, and analyzed by flow cytometry. The

results suggest similar efficiency to undergo neu-

ronal differentiation for the cells previously ex-

panded under both conditions. Further studies

were then performed under 2% O2.

Hypoxia leads to increased proliferation of

mES-derived NS cells

The positive effect of low oxygen in the expansion

of CGR8-NS cells may be the result of: (i) en-

hanced proliferation of the cells under 2% O2; (ii)

lower levels of cell death/apoptosis under low oxy-

gen; or (iii) a combined effect of both factors. To

test these hypotheses, cell divisional history, cell

cycle and apoptosis/necrosis studies were per-

formed for both culture conditions. We observed

that although the number of apoptotic and necrotic

cells is comparable under both oxygen tensions,

cells divide faster under hypoxic conditions. A

larger population in a quiescent G0 phase was

observed in normoxic conditions when compared

to cells cultured under 2% O2.

References

[1] Rodrigues, C.A.V., Diogo, M.M., Lobato da

Silva, C., Cabral, J.M.S., Biotechnol. Bioeng., doi:

10.1002/bit.22648 (2010)

Figure 2: Growth kinetics of CGR8-NS cell expansion. (A) Number of viable cells experimentally determined under 2% O2 (♦)

and 20% O2 (■) and determined from a model for hypoxia (green line) and normoxia (blue line). (B) Specific growth rate val-

ues (day-1) during expansion of CGR8-NS cells under hypoxic (green line) and normoxic conditions (blue line). Data pre-

sented are means ± standard error of the mean for 2 determinations from 4 independent experiments.

*, p< 0.05

R

esearch H

ighlights

0.E+00

2.E+05

4.E+05

6.E+05

8.E+05

1.E+06

1.E+06

1.E+06

0 1 2 3 4

Nu

mb

er

of

Cell

s

Time (days)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 0.5 1 1.5 2 2.5 3 3.5 4

Sp

ecif

ic G

row

th R

ate

(d

ay

-1)

Time (days)

*

*

*

A B

Scientific Output

20092009



Articles in Peer-reviewed Journals

Articles in Conference Proceedings

Patents

Book Chapters

Dissertations

Oral Communications

Poster Communications

Prizes

Articles in Peer-reviewed Journals

Azevedo, A.M., Gomes, A.G., Rosa, P.A.J.,

Ferreira, I.F., Pisco, A.M.M.O., Aires-Barros, M.R.,

“Partitioning of human antibodies in polyethylene

glycol - sodium citrate aqueous two-phase sys-

tems”, Sep. Purif. Technol., 65, 14-21

Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., Aires-

Barros, M.R., “Chromatography-free recovery of

biopharmaceuticals through aqueous two-phase

processing”, Trends Biotechnol., 27, 240-247

Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., Pisco,

A.M.M.O., de Vries, J., Korporaal, R., Visser, T. J.,

Aires-Barros, M.R., “Affinity-enhanced purification of

human antibodies by aqueous two-phase extrac-

tion”, Sep. Purif. Technol., 65, 31-39

Azevedo, A.M., Rosa, P.A.J., Ferreira, I.F., de

Vries, J., Visser, T.J., Aires-Barros, M.R.,

“Downstream processing of human antibodies inte-

grating an extraction capture step and cation ex-

change chromatography”, J. Chromatogr. B, 887,

50-58

Bernardino, S.M.S.A., Fernandes, P., Fonseca,

L.P., "A new biocatalyst: Penicillin G acylase immo-

bilized in sol-gel micro-particles with magnetic prop-

erties", Biotechnol. J., 4, 695-702

de Barros, D.P.C., Fonseca, L.P., Cabral, J.M.S.,

Weiss, C.K., Landfester, K., “Synthesis of alkyl es-

ters by cutinase in miniemulsion and organic sol-

vent media”, Biotechnol. J., 4, 674-683

de Barros, D.P.C., Fonseca, L.P., Fernandes, P.,

Cabral, J.M.S., Mojovic L., “Biosynthesis of ethyl

caproate and other short ethyl esters catalyzed by

cutinase in organic solvent”, J. Mol. Catal. B: En-

zym., 60, 178-185

Cabeça, R., Rodrigues, M., Prazeres, D.M.F., Chu,

V., Conde, J.P., “The effect of the shape of single,

sub-ms voltage pulses on the rates of surface im-

mobilization and hybridization of DNA”, Nanotech-

nology, 20: 015503

Calado, C.R.C., Brandão, M., Biscaia, J., Cabral,

J.M.S., Fonseca, L.P., “Effect of Tween-80 on sta-

bility and secretion of hydrophobic tagged-

cutinases”, Chem. Biochem. Eng. Q., 23, 411-417

Cardoso, M.A.T., Antunes, S., Van Keulen, F.,

Ferreira, B.S., Geraldes, A., Cabral, J.M.S., Pa-

lavra, A.M.F., “SAS micronization of synthetic all

trans-beta-carotene with tetrahydrofuran as solvent

and carbon dioxide as antisolvent”, J. Chem. Tech-

nol. Biotechnol., 84, 215-222

Carvalho, F., Marques, M.P.C., de Carvalho

C.C.C.R., Cabral, J.M.S., Fernandes, P., “Sitosterol

bioconversion with resting cells in liquid polymer

based systems”, Bioresource Technol., 100, 4050

-4053

Carvalho, J., Prazeres, D.M.F., Monteiro, G.A.,

“Bringing DNA vaccines closer to commercial use”,

iDrugs, 12, 642-647

de Carvalho, C.C.C.R., Wick, L.Y., Heipieper, H.J.,

“Cell wall adaptations of planktonic and biofilm

Rhodococcus erythropolis cells to growth on C5 to

C16 n-alkane hydrocarbons”, Appl. Microbiol. Bio-

technol., 82, 311-320

Cattorini, S., Marques, M.P.C., Carvalho, F.,

Chheub, V., Cabral, J.M.S., Fernandes, P.,

“Lentikat®-based Biocatalysts: Effective Tools for

Inulin Hydrolysis”, Chem. Biochem. Eng. Q., 23,

429-434

Costa, L., Brissos, V., Lemos, F., Ribeiro, F.R.,

Cabral, J.M.S., “Enhancing the thermal stability of

lipases through mutagenesis and immobilization on

zeolites”, Bioprocess Biosyst. Eng., 32, 53-61

Costa, L., Coelho, A., Lemos, F., Ribeiro, F.R.,

Cabral, J.M.S., “Modulating the acid strength of

zeolite H-ZSM-5 to increase the selectivity in the

racemization of 1-phenylethanol”, Appl. Catal. A:

Gen., 354, 33-37

Fernandes, T.G., Diogo, M.M., Clark, D.S., Dordick,

J.S., Cabral, J.M.S., “High-throughput cellular mi-

croarray platforms: applications in drug discovery,

toxicology and stem cell research”, Trends Bio-

technol., 27, 342-349

Fernandes, P., Marques, M.P.C., Carvalho, F.,

Cabral, J.M.S., “A simple method for biocatalyst

immobilization using PVA-based hydrogel particles”,

J. Chem. Technol. Biotechnol., 84, 561-564

Publications


Freitas, S.S., Canário, S., Santos, J.A.L., Prazeres,

D.M.F., “Alternatives for the intermediate recovery

of plasmid DNA: Performance, economic viability

and environmental impact”, Biotechnol. J., 4, 265-

278

Freitas, S.S., Santos, J.A.L., Prazeres, D.M.F.,

“Plasmid purification by hydrophobic interaction

chromatography using sodium citrate in the mobile

phase”, Sep. Purif. Technol., 65, 95-104

Gomes, A.G., Azevedo, A.M., Aires-Barros, M.R.,

Prazeres, D.M.F., “Purification of plasmid DNA with

aqueous two phase systems of PEG 600 and so-

dium citrate/ammonium sulphate”, Sep. Purif.

Technol., 65, 22-30

Guerrero-Germán, P., Prazeres, D.M.F., Guzmán,

R., Montesinos-Cisneros, R.M., Tejeda-Mansir, A.,

“Purification of plasmid DNA using tangential flow

filtration and tandem anion-exchange membrane

chromatography”, Bioprocess Biosyst. Eng., 32,

615-623

Henriques, A.M., Madeira, C., Fevereiro, M., Praz-

eres, D.M.F., Aires-Barros, M.R., Monteiro, G.A.,

“Effect of cationic liposomes/DNA charge ratio on

gene expression and antibody response of a candi-

date DNA vaccine against Maedi-Visna virus”, Int.

J. Pharm., 377, 92-98

Madeira, C., Estrela, N., Ferreira, J.A.B., Andrade,

S.M., Costa S.M.B., Melo, E.P., “Fluorescence life-

time imaging microscopy and fluorescence reso-

nance energy transfer from cyan to yellow fluores-

cent protein validates a novel method to cluster pro-

teins on solid surfaces”, J. Biomed. Opt., 14:

044035

Marques, M.P.C., Cabral, J.M.S., Fernandes, P.,

“High throughput in biotechnology: from shake-

flasks to fully instrumented microfermentors”, Re-

cent Pat. Biotechnol., 3, 124-140

Marques, M.P.C., Carvalho, F., Magalhães, S.,

Cabral, J.M.S., Fernandes, P., “Screening for suit-

able solvents as substrate carriers for the microbial

side-chain cleavage of sitosterol using microtitre

plates”, Process Biochem., 44, 174-180

Marques, M.P.C., Magalhães, S., Cabral, J.M.S.,

Fernandes, P., “Characterization of 24-well micro-

titer plate reactors for a complex multi-step biocon-

version: from sitosterol to androstenedione”, J. Bio-

technol., 141, 174-180

Martins, S.A.M., Prazeres, D.M.F., Fonseca, L.P.,

Monteiro, G.A., “The role of probe-probe interac-

tions on the hybridization of double stranded DNA

targets onto DNA-modified magnetic microparti-

cles”, Anal. Bioanal. Chem., 394, 1711-1716

Martins, S.A.M., Prazeres, D.M.F., Fonseca, L.P.,

Monteiro, G.A., “Application of central composite

design for DNA hybridization onto magnetic mi-

croparticles”, Anal. Biochem., 391, 17-23

Martins, V.C., Cardoso, F.A., Germano, J., Cardo-

so, S., Sousa, L., Piedade, M., Freitas, P.P., Fon-

seca, L.P. “Femtomolar limit of detection with a

magnetoresistive biochip”, Biosens. Bioelectron.,

24, 2690-2695

Oliveira, P.H., Prather, K.J., Prazeres, D.M.F., Mon-

teiro, G.A., “Structural instabilities of plasmid bio-

pharmaceuticals: Challenges and implications”,

Trends Biotechnol., 27, 503-509

Oliveira, P.H., Prazeres, D.M.F., Monteiro, G.A.,

“Deletion-formation mutations in plasmid expression

vectors are unfavoured by runaway amplification

conditions and differentially selected under kanamy-

cin stress”, J. Biotechnol., 143, 231-238

Paixão, L., Rodrigues, L., Martins, M., Couto, I.,

Fernandes P., de Carvalho, C.C.C.R., Monteiro, G.,

Sansonetty, F., Amaral, L., Viveiros, M.,

“Fluorometric determination of ethidium bromide

efflux kinetics in Escherichia coli”, J. Biol. Eng., 3:

18

Pereira, A.T., Pimentel, A.C, Chu, V., Prazeres,

D.M.F., Conde, J.P., “Chemiluminescence detection

of horseradish peroxidase using an integrated using

an amorphous silicon thin film photosensor”, IEEE

Sensors, 9, 1282-1290

Pimentel, A.C, Pereira, A.T., Prazeres, D.M.F., Chu,

V., Conde, J.P., “Detection of fluorescently labeled

biomolecules immobilized on a detachable sub-

strate using and integrated amorphous silicon

photodetector”, Appl. Phys. Lett., 94: 164106

Pinotti, L.M., Fonseca, L.P., Prazeres, D.M.F., Rod-

rigues, D.S., Nuccia, E.R., Giordano, R.L.C.,

“Recovery and partial purification of penicillin G acy-

lase from E. coli homogenate and B. megaterium

culture medium using an expanded bed adsorption

column”, Biochem. Eng. J., 44, 111-118

Ribeiro, S.C., Monteiro, G.A., Prazeres, D.M.F.,

“Evaluation of the effect of non-B DNA structures on

plasmid integrity via accelerated stability studies”, J.

Pharm. Sci., 98, 1400-1408

Rosa, P.A.J., Azevedo, A.M., Ferreira, I.F., Som-

merfeld, S., Bäcker, W., Aires-Barros, M.R.,

“Downstream processing of antibodies: Single-

stage versus multi-stage aqueous two-phase ex-

traction”, J. Chromatogr. A, 1216, 8741-8749

Rosa, P.A.J., Azevedo A.M., Sommerfeld, S., Mut-

ter, M., Aires-Barros, M.R., Bäcker, W., “Application

of aqueous two-phase systems to antibody purifica-

tion: A multi-stage approach”, J. Biotechnol., 139,

306-313

Serro, A.P., Completo, C., Colaço, R., dos Santos,

F., da Silva, C.L., Cabral, J.M.S., Araújo, H., Pires,

E., Saramago, B., “A comparative study of titanium

nitrides, TiN, TiNbN and TiCN, as coatings for bio-

medical applications”, Surf. Coat. Technol., 203,

3701-3707

Silva, A.I., Mateus, M., “Development of a polysul-

fone hollow fiber vascular bio-artificial pancreas

device for in vitro studies”, J. Biotechnol., 139, 236

-249

da Silva, C.L., Gonçalves, R., Porada, C.D., Ascen-

são, J.L., Zanjani, E.D., Cabral, J.M.S., Almeida-

Porada, G., “Differences amid bone marrow and

cord blood hematopoietic stem/progenitor cell divi-

sion kinetics”, J. Cell. Physiol., 220, 102-111

Silva, M.S., Prazeres, D.M.F., Lança, A., Atouguia,

J., Monteiro, G.A., “Trans-sialidase from Trypano-

soma brucei as a potential target for DNA vaccine

development against African Trypanosomiasis”,

Parasitol. Res., 105, 1223-1229

Silva, T., Lima, P., Hageman, S., Fonseca, L.P.,

Calado, C.R.C., “Prediction of dynamic plasmid pro-

duction by recombinant Escherichia coli fed-batch

cultivations with a generalized regression neural

network”, Chem. Biochem. Eng. Q., 23, 419-427

Sousa, A., Sousa, F., Prazeres, D.M.F., Queiroz,

J.A., “Histidine affinity chromatography of homo-

oligonucleotides. Role of multiple interactions on

retention”, Biomedical Chromatogr., 23, 745-753

Sousa, F., Prazeres, D.M.F., Queiroz, J.A.,

“Improvement of transfection efficiency by using

supercoiled plasmid DNA purified from a clarified

lysate with arginine affinity chromatography”, J.

Gene Med., 11, 79-88

Sousa, F., Prazeres, D.M.F., Queiroz, J.A., “Binding

and elution strategy for improved performance of

arginine affinity chromatography in supercoiled

plasmid DNA purification”, Biomedical Chroma-

togr., 23, 160-165

Sousa, I.T., Ruiu, L., Lowe, C.R., Taipa, M.A.,

“Synthetic affinity ligands as a novel tool to improve

protein stability”, J. Mol. Recognit., 22, 83-90

Temtem, M., Silva, L.M.C., Andrade, P.Z., dos San-

tos, F., da Silva, C.L., Cabral, J.M.S., Abecasis,

M.M., Aguiar-Ricardo, A., “Supercritical CO2 gener-

ating chitosan devices with controlled morphology.

Potential application for drug delivery and mesen-

chymal stem cell culture”, J. Supercritical Fluids,

48, 269-277

Wu, M.L., Freitas, S.S., Monteiro, G.A., Prazeres,

D.M.F., Santos, J.A.L., “Stabilization of naked and

condensed plasmid DNA against degradation in-

duced by ultrasounds and high shear vortices”, Bio-

technol. Appl. Biochem., 53, 237-246

Articles in Conference Proceedings

Andrade, P.Z., dos Santos, F., Almeida-Porada, G.,

da Silva, C.L., Cabral, J.M.S., “Non-viral gene deli-

very to human mesenchymal stem Synergistic cy-

tokine effects modulate ex-vivo expansion of he-

matopoietic stem/progenitor cells in co-culture

with mesenchymal stem cells ” Exp. Hematology,

37, S97


Weiss, C.K., Landfester, K., “Biosynthesis of fatty

acids alkyl esters in miniemulsion as a reaction me-

dia”, New Biotechnol., 25, S116-S117

Bernardino, S., Fernandes, P., Fonseca, L.,

“Penicillin G acylase stabilization by immobilization

in sol-gel micro-particles. Increase in hydrolase and

synthetase activity using Tris buffer”, New Biotech-

nol., 25, S120-S121


Cordas, C.M., Lourenço, N.M.T., Vidinha, P.,

Afonso, C.A.M., Barreiros, S., Fonseca, L.P.,

Cabral, J.M.S., “New conducting biomaterial based

on Ion Jelly technology for development of a new

generation of biosensors”, New Biotechnol., 25,

S138-S139

Madeira, C., Ribeiro, S., Boura, J., da Silva, C.L.,

Cabral, J.M.S., “Microporation of human mesenchy-

mal stem cells promotes high cellular recoveries

and efficient plasmid gene delivery”, Hum. Gene

Ther., 20, 1490-1491

Madeira, C.P., Ribeiro, S.C., dos Santos, F., Mon-

teiro, G.A, da Silva, C.L, Cabral, J.M.S.

“Comparision of polyadenylation signals for use in

transient genetic modification of human mesenchy-

mal stem cells”. Exp. Hematology, 37, S97.

Madeira, C.P., Mendes, R.D., Ribeiro, S.C., Aires-

Barros, M.R., dos Santos, F., da Silva, C.L., Cabral,

J.M.S. “Non-viral gene delivery to human mesen-

chymal stem cells using cationic liposomes”, Exp.

Hematology, 37, S97

Martins, S.A., Prazeres, D.M.F., Fonseca, L.P.,

Monteiro, G., “DNA biosensors: towards a micropar-

ticle based-platform for genomic DNA detection”,

New Biotechnol., 25, S19-S20

Martins, V., Cardoso, F., Freitas, P., Germano, J.,

Cardoso, S., Souse, L., Piedade, M., Fonseca, L.,

“Magneto resistive biochip-based portable platform

for biomolecular recognition detection”, New Bio-

technol., 25, S358-S359

Pereira, A.T., Chu, V., Prazeres, D.M.F., Conde,

J.P., "Enzymatic biosensors with integrated thin film

a-Si:H photodiodes", Mat. Res. Soc. Symp. Proc.,

1153, A08-04


J.P., "Miniaturization of immunoassays using optical

detection with integrated amorphous silicon photo-

diodes", Mat. Res. Soc. Symp. Proc., 1191, OO08-

04

Toledo, M.A.S., Monteiro, G.A., Prazeres, D.M.F.,

Souza, A.P., Azzoni, A.R., “Development of recom-

binant proteins for non viral gene delivery taking

advantage of molecular motor proteins”, Hum.

Gene Ther., 20, 1438-1438


Souza, A.P., Azzoni, A.R., “The use of recombinant

dynein light chains to mediate enhanced plasmidial

DNA transfection proteins for non viral gene deli-

very taking advantage of molecular motor proteins”,

Hum. Gene Ther.,20, 1441

Book chapters

de Carvalho, C.C.C.R., “Biotransformation of ter-

penes by Fungi”, in Advances in Fungal Biotech-

nology (Mahendra Rai, Editor), I.K. International

Publishing House Pvt. Ltd, New Delhi, pp. 528-542

Fernandes, P., “Enzymes in sugar industries”, in

Enzymes in Food Processing: Fundamentals

and Potential Applications (Parmjit S. Panesar,

Satwinder S. Marwaha, Harish Kumar, Editors), I.K.

International Publishing House Pvt. Ltd, New Delhi,

pp. 165-198

Patents

Baecker, W., Sommerfeld, S., Mutter, M., Rosa,

P.A.J., Aires-Barros, M.R., Azevedo, A.M., “Method

for purifying therapeutic proteins” World Patent

WO/2009/112149

Dissertations

Ph.D. Thesis

Ana Isabel Quinta dos Santos Silva, “Study of a

membrane bioreactor as a model of bioartificial pan-

creas”, PhD in Chemical Engineering, IST

(Supervisor: M. Mateus)

Pedro Nuno de Sousa Sampaio, “Processos Inte-

grados para produção e recuperação in situ da

ciprosina por estirpes transformadas de Saccharo-

myces cerevisiae”, PhD in Biotechnology, IST

(Supervisors: L.P. Fonseca - IST, M.S. Soares Pais

- FCUL)

Ricardo Manuel Marques Caleiro Cabeça,

“Electric-Field assisted DNA surface immobilization

and hybridization”, PhD in Biotechnology, IST

(Supervisors: J.P. Conde, D.M.F. Prazeres)

Tiago Paulo Gonçalves Fernandes, "Cell-based

microarray systems for stem cell expansion and

differentiation", PhD in Biotechnology, IST

(Supervisor: J.M.S. Cabral)

Verónica Cristina Baião Martins Romão, “A mag-

netoresistive biochip platform: detection of DNA

hybridization and antibody/cell recognition”, PhD in

Biotechnology, IST (Supervisor: L.P. Fonseca)

M.Sc. Thesis

Laura Coutinho de Almeida Santos, MSc in bio-

logical engineering, “Utilização de Quitosano em

Aplicações Biomédicas” (Supervisors: M.R. Aires

Barros and I.A. Pires)

Meritxell Zurita Turk, MSc in Biotechnology,

“Evaluation of inducible vs. constitutive protein ex-

pression in HEK293Tcells” (Supervisors: J.M.S.

Cabral, T.C.P. Madeira)

Rui Daniel Mendes, MSc in Human Molecular Biol-

ogy from Faculdade de Ciências da Universidade

de Lisboa, “Optimization of differentiation protocolos

for human mesenchymal stem cells (hMSC)”,

(Supervisors: C.L. da Silva - IST, M.G.G.F. Rodri-

gues - FCUL).

Oral Communications

International Conferences

Botelho-Cunha, V., Mateus, M., Petrus, J.C.C., de

Pinho, M.N, "Nanofiltration process in fractionation

of galacto-oligosaccharides continuously produced

in bioreactors", EUROMEMBRANE 2009 Confer-

ence, Montpellier, France, September

de Carvalho, C.C.C.R., “Adaptation of bacterial cells

to antineoplastic agents may increase the risk of

post-chemotherapy infections”, BIT's 2nd World

Cancer Congress, Beijing, P.R. of China, June

(Invited speaker)

de Carvalho, C.C.C.R., “The remarkable adaptabil-

ity of Rhodococcus erythropolis cells”, III Interna-

tional Conference on Environmental, Industrial and

Applied Microbiology | BioMicroWorld2009, Lisbon,

Portugal, December

Carvalho, J., Monteiro, G.A., Atouguia, J., Prazeres,

D.M.F., Rodgers, J., “The challenges of developing

a DNA vaccine for African Trypanosomiasis”, Joint

Malaria and Spring Meeting, British Society for

Parasitology, Edinburgh, UK, April

Ferreira, I.F., de Carvalho, C.C.C.R., Wang, D.I.C.,

Aires-Barros, M.R., “Desulfurization of crude oil by

Rhodococcus erythropolis cells”, III International

Conference on Environmental, Industrial and Ap-

plied Microbiology | BioMicroWorld2009, Lisbon,

Portugal, December

Fernandes, P., Marques, M.P.C., Carvalho, F.,

Cabral, J.M.S., “Carbohydrate hydrolysis with com-

mercial inulinase entrapped in PVA-based beads”,

29th International Exhibition-Congress on Chemical

Engineering, Environmental Protection and Biotechnol-

ogy | ACHEMA 2009, Frankfurt, Germany, May

(Invited speaker)

Fonseca, L.P., “Modern methods for assessment of

enzyme activity in microcapsules”, Spring Work-

shop on Bioencapsulation, Luxembourg, April

Fonseca, L.P., “From conventional biosensors to

new analytical devices for a better quality of life:

Magneto-Resistive Biosensors”, Biosensor Work-

shop, University of Ljubljana, Ljubljana, Slovenia,

April

Fonseca, L.P., “New conducting biomaterial based

on Ion Jelly® technology for development of a new

generation of Biosensors”, Biosensors Workshop,

14th European Congress on Biotechnology, Barce-

lona, Spain, September


Prazeres, D.M.F., "Exploring the interaction of nu-

cleic acids with phenyl boronate ligands as an alter-

native for the purification of pharmaceutical grade

pDNA", 15th International Conference Biopartitioning

and Purification | BPP2009, Uxbridge, UK, June


Marques, M.P.C., Carvalho, F., Fernandes, P.,

Cabral, J.M.S., “Steroids bioconversion: towards

green processes”, 29th International Exhibition-

Congress on Chemical Engineering, Environmental

Protection and Biotechnology | ACHEMA 2009,

Frankfurt, Germany, May


teiro, G.A., “Structural instability in plasmid vectors

for DNA vaccination”, III International Conference

on Environmental, Industrial and Applied Microbiol-

ogy | BioMicroWorld2009, Lisbon, Portugal, Decem-

ber


J.P., "Enzymatic biosensors with integrated thin film

a-Si:H photodiodes", Spring Meeting of the Material

Research Society | MRS, San Francisco, USA, April


J.P., "Miniaturization of immunoassays using optical

detection with integrated amorphous silicon photo-

diodes", Spring Meeting of the Material Research

Society | MRS, San Francisco, USA, April

Raiado-Pereira, L., Prazeres, D.M.F. Mateus, M.,

"Effect of ligand type on membrane hydrophobic

interaction chromatography for the purification of

plasmid DNA vaccines", 15th International Confer-

ence on Biopartitioning and Purification | BPP2009,

Uxbridge, UK, June

Raiado-Pereira, L., Prazeres, D.M.F. Mateus, M.,

"Preparation, characterization and testing of ad-

sorption membranes for chromatographic separa-

tion of therapeutic plasmids", EUROMEMBRANE

2009 Conference, Montpellier, France, September

Rosa, P.A.J., Ferreira, I.F., Azevedo, A.M., Aires-

Barros, M.R., "Affinity partition and purification of

therapeutic proteins, namely antibodies", 15th Inter-

national Conference on Biopartitioning and Purifica-

tion | BPP2009, Uxbridge, UK, June (invited key-

note lecture)

Rosa, P.A.J., Azevedo, A.M., Sommerfeld, S.,

Bäcker, W., Aires-Barros, M.R., "Continuous aque-

ous two-phase extraction process for the purifica-

tion of antibodies: Performance and economical

feasibility", 15th International Conference on Biopar-

titioning and Purification | BPP2009, Uxbridge, UK,

June

Santa, G.L.M., Bernardino, S.M.S.A., Chheub V,

Fonseca, L.P., Fernandes, P., “From inulin to fruc-

tose syrups: evaluation of the sol-gel approach for

inulinase immobilization”, 8th International Meeting

of the Portuguese Carbohydrate Group | Glupor 8,

Braga, Portugal, September

Santos, R., Marques, M., Oliveira, P., Carvalho, F.,

de Carvalho, C.C.C.R., Monteiro, G., Cabral,

J.M.S., Frade, R., Silva, M., Fernandes, P., “The

sunny side of Mycobacteria”, European Society of

Mycobacteriology, Porto, Portugal, July

Silva, A.I., Mateus, M., "Construction and perfor-

mance assessment of an in vitro testing device for

the development of vascular bio-artificial pancreas",

Membranes in Medicine, Workshop in the frame-

work of the of the European Network of Excellence

on Nanoscale-based Membrane Technologies, Lis-

bon, Portugal, February

Sousa, I.T., Taipa, M.A., “Triazine-scaffolded syn-

thetic affinity ligands as a novel tool to improve pro-

tein stability”, 8th International Conference on Pro-

tein Stabilisation | ProStab2009, Graz, Austria, April

National Conferences


da Silva, C.L., Cabral, J.M.S., “Ex-vivo expansion of

hematopoietic stem/progenitor cells in co-culture

with mesenchymal stem cells: optimizing the cyto-

kine cocktail", 4th International Meeting of the Portu-

guese Society of Stem Cells and Cell Therapies |

SPCE-TC, Lisbon, April

de Carvalho, C.C.C.R., “Rhodococcus: aplicações

em Biotecnologia”, V Dia de Biologia Marinha e

Biotecnologia, Escola Superior de Turismo e Tec-

nologia do Mar, Peniche, May

Eibes, G., dos Santos, F., Andrade, P.Z., da Silva,

C.L., Cabral, J.M.S., “A microcarrier-based stirred

culture system for the expansion of human mesen-

chymal stem cells", 4th International Meeting of the

Portuguese Society of Stem Cells and Cell Thera-

pies | SPCE-TC, Lisbon, April

Fernandes, P., Marques, M.P.C., Cattorini, S., Car-

valho, F., Cabral , J.M.S., “A dual purpose immobi-

lized biocatalyst for inulin and sucrose hydrolysis”,

Carbohydrates as Organic Raw Materials V (CORM

V), Lisbon, January

Fernandes-Platzgummer, A.M., Diogo, M.M., Bap-

tista, R.P., da Silva, C.L., Cabral, J.M.S. “Scale-up

of mouse embryonic stem cell expansion: from spin-

ner-flask to a fully controlled bioreactor”, 4th Interna-

tional Meeting of the Portuguese Society of Stem

Cells and Cell Therapies | SPCE-TC, Lisbon, April

Fonseca, L.P., “Biocatálise enzimática, dos reacto-

res industriais aos micro-reactores”, Jornadas Bio-

tecnologia ESB-UCP, Porto, October

Fonseca, L.P., “Biocatálise no desenvolvimento de

processos industriais mais sustentáveis e amigos

do ambiente”, Jornadas Tecnológicas da UNL-FCT

| JORTEC 2009, Lisboa, May

Fonseca, L.P., “New trends in biocatalysisin indus-

trial biotechnology”, Joint Meeting of the Portuguese

Society of Microbiology and of the Portuguese Society

of Biotechnology | MicroBiotec09, Vilamoura,

November

Lourenço, N.M.T., Vidinha, P., Cordas, C.M., Afon-

so, C.A.M., Barreiros, S., Cabral, J.M.S., Fonseca,

L.P., “Real-time pathogens bio-sensing based on

Ion Jelly® technology”, MIT-Portugal Program, Can-

tanhede, March


teiro, G.A., “Structural instability in plasmid vectors

for DNA vaccination”, Joint Meeting of the Portuguese

Society of Microbiology and of the Portuguese Society

of Biotechnology | MicroBiotec09, Vilamoura,

November

Rodrigues, C.A.V., Diogo, M.M., da Silva, C.L.,

Cabral, J.M.S., “Low oxygen tension enhances pro-

liferation of mouse neural stem cells", 4th Interna-



Teixeira, M.C., Mira, N.P., Raposo, L.R., Sintra,

E.R., de Carvalho, C.C.C.R., Lourenço, A.B., Sá-

Correia, I., “Global analysis of yeast determinants of

resistance to ethanol: the important role of FPS1”,

Joint Meeting of the Portuguese Society of Microbiol-

ogy and of the Portuguese Society of Biotechnology |

Microbiotec09, Vilamoura, November

Poster Communications

International Conferences


da Silva, C.L., Cabral, J.M.S., " Ex-vivo expansion

of hematopoietic stem/progenitor cells in co-culture


kine cocktail", 7th Annual Meeting of International

Society of Stem Cell Research | ISSCR, Barcelona,

Spain, July


da Silva, C.L., Cabral, J.M.S., “Synergistic cytokine

effects modulate ex-vivo expansion of hemato-

poietic stem/progenitor cells in co-culture with

mesenchymal stem cells”, 38th Annual Scientific

Meeting of the ISEH-Society for Hematology and

Stem Cells, Athens, Greece, September

Azevedo, A.M., Rosa, P.A.J., Aires-Barros, M.R.,

“Purification of antibodies by phenyl boronate affin-

ity chromatography”, 15th International Conference

on Biopartitioning and Purification | BPP2009, Ux-

bridge, UK, June

Azevedo, A.M., Rosa, P.A.J., Sommerfield, S.,

Baecker, W., Aires-Barros, M.R., “Affinity extraction

of human therapeutic antibodies in aqueous two-

phase systems”, 18th Biennial Meeting of the Inter-

national Society for Molecular Recognition | Affinity

2009, Reykjavik, Iceland, July

Azevedo, A.M., Rosa, P.A.J., Borlido, L., Aires-

Barros, M.R., “Purification of antibodies by phenyl

boronate affinity chromatography”, 18th Biennial

Meeting of the International Society for Molecular

Recognition | Affinity 2009, Reykjavik, Iceland, July

Azzoni, A., Monteiro, G.A., Prazeres, D.M.F.,

Souza, A.P., “Development of recombinant proteins

for non viral gene delivery. Taking advantage of

molecular motor proteins”, XXXVIII Annual Meeting

of the Brazilian Society for Biochemistry and Mo-

lecular Biology, Águas de Lindóia, São Paulo, Bra-

zil, May

Badenes, S.M., Lemos, F., Cabral, J.M.S., “Bio-

catalytic process using microencapsulated recombi-

nant cutinase in AOT-isooctane reversed micelles

for the synthesis of biodiesel”, 14th European Con-

gress on Biotechnology | ECB 14, Barcelona,

Spain, September


Badenes, S.M., Lemos, F., Cabral, J.M.S., “Bio-

diesel production by an enzymatic transesterifica-

tion using microencapsulated recombinant cutinase

in AOT-isooctane reversed micelles”, Enzyme Engi-

neering XX, Groningen, Netherlands, September


“Fed-batch operation mode as a tool for successful

biosynthesis of aroma esters catalyzed by cuti-

nase”, 9th International Symposium on Biocatalysis

and Biotransformations | BIOTRANS 2009, Berne,

Switzerland, July


Weiss, C.K., Landfester, K., “Great potential of mini-

emulsion system as a green chemistry reaction me-

dia for biosynthesis of aroma esters”, 9th Interna-

tional Symposium on Biocatalysis and Biotransfor-

mations | BIOTRANS 2009, Berne, Switzerland,

July


Weiss, C.K., Landfester, K., “Biosynthesis of fatty

acids alkyl esters in miniemulsion as a reaction me-

dia”, 14th European Congress on Biotechnology,

Barcelona | ECB 14, Spain, September


Weiss, C.K., Landfester, K., “Miniemulsion as a

green chemistry reaction media in biosynthesis of

fatty acids alkyl esters”, Enzyme Engineering XX,

Groningen, Netherlands, September


L.P., “Penicillin G acylase stabilization by immobili-

zation in sol-gel micro-particles: increase in hy-

drolase and synthetase activity using tris buffer”,

14th European Congress on Biotechnology, Barce-

lona | ECB 14, Spain, September


L.P., “Optimization of Penicillin G Acylase Immobili-

zation by Entrapment in Silica Xerogel with Mag-

netic Properties”, 9th International Symposium on

Biocatalysis and Biotransformations | BIOTRANS

2009, Bern, Switzerland, July


L.P., “Penicillin G Acylase: Tris or phosphate

buffer? What is the best option?”, 8th International

Conference on Protein Stabilisation | ProStab2009,

Graz, Austria, April

Borlido, L., Azevedo, A.M., Aires-Barros, M.R.,

“Studies on IgG stability in aqueous two-phase ex-

traction process”, 15th International Conference on

Biopartitioning and Purification | BPP2009, Ux-

bridge, UK, June

Chheub, V., Cattorini, S., Fonseca, L.P., Fernan-

des, P., “Immobilization in Lentikat® based parti-

cles: towards the production of fructose syrups”,

Enzyme Engineering XX, Groningen, Netherlands,

September

de Carvalho, C.C.C.R., Caramujo, M.J., “Tumor

metastasis as an adaptation of tumor cells to fulfill

their phosphorus requirements”, BIT's 2nd World

Cancer Congress, Beijing, P.R. of China, June

Cordas, C.M., Lourenço, N.M.T., Vidinha, P.,

Afonso, C.A.M., Barreiros, S., Fonseca, L.P.,

Cabral, J.M.S., “New conducting biomaterial based

on Ion Jelly technology for development of a new

generation of biosensors”, 14th European Congress

on Biotechnology | ECB 14, Barcelona, Spain, Sep-

tember


C.L., Cabral, J.M.S., "A microcarrier-based stirred


chymal stem cells", 7th Annual Meeting of Interna-

tional Society of Stem Cell Research | ISSCR, Bar-

celona, Spain, July

Fernandes, T.G., Fernandes-Platzgummer, A.M.,

Diogo, M.M., da Silva, C.L., Dordick, J.S., Cabral,

J.M.S., "Effects of low oxygen tension on mouse

embryonic stem cell expansion", 7th Annual Meeting

of International Society of Stem Cell Research |

ISSCR, Barcelona, Spain, July




embryonic stem cell expansion", 3rd International

Stem Cell Meeting: Biology and Clinical Applica-

tions, Tel-Aviv, Israel, June

Fernandes-Platzgummer, A.M., Diogo, M.M., Bap-



ner-flask to a fully controlled bioreactor”, 7th Annual

Meeting of International Society of Stem Cell Re-

search | ISSCR, Barcelona, Spain, July

Fonseca, L.P., Bernardino, S.M.S.A., Fernandes,

P., “Biocatalysts prepared by the entrapment of

inulinase and penicillin G acylase in sol-gel”, XVII

International Conference on Bioencapsulation,

Groningen, Netherlands, September


Prazeres, D.M.F., “RNA clearance from plasmid-

containing lysates by phenyl boronate adsorption”,

18th Biennial Meeting of the International Society for

Molecular Recognition | Affinity 2009, Reykjavik,

Iceland, July

Lourenço, N.M.T., Monteiro, C.M., Afonso, C.A.M.,

“Enzymatic kinetic resolution of sec-alcohols based

on ionic-acylating- agents”, 9th International Sympo-

sium on Biocatalysis and Biotransformations | BIO-

TRANS 2009, Bern, Switzerland, July


teiro, G.A., da Silva, L., Cabral, J.M.S.,

“Comparison of polyadenylation signals for use in


mal stem cells”, 38th Annual Scientific Meeting of

the ISEH-Society for Hematology and Stem Cells,

Athens, Greece, September

Madeira, C., Ribeiro, S., Boura, J., da Silva, C.L.,

Cabral, J.M.S., “Microporation of human mesenchy-

mal stem cells promotes high cellular recoveries

and efficient plasmid gene delivery”, XVII Annual

Congress of the European Society of Gene and Cell

Therapy | ESGCT, Hannover, Germany, Novem-

ber


teiro, G.A., da Silva, L., Cabral, J.M.S., “Non-viral

gene delivery to human mesenchymal stem cells

using cationic lipossomes”, 38th Annual Scientific

Meeting of the ISEH-Society for Hematology and

Stem Cells, Athens, Greece, September

Martins, S.A., Prazeres, D.M.F., Fonseca, L.P.,

Monteiro, G., “DNA biosensors: towards a micropar-

ticle based-platform for genomic DNA detection”,

14th European Congress on Biotechnology | ECB

14, Barcelona, Spain, September

Martins, V., Cardoso, F., Freitas, P., Germano, J.,

Cardoso, S., Souse, L., Piedade, M., Fonseca, L.,

“Magneto resistive biochip-based portable platform

for biomolecular recognition detection”, 14th Euro-

pean Congress on Biotechnology | ECB 14, Barce-

lona, Spain, September

Nascimento, K.S., Yelo, S., Azevedo, A.M.,

Cavada, B.S., Aires-Barros, M.R., “Liquid-liquid

equilibrium data for aqueous two-phase system-

scomposed by ethylene oxide propylene oxide co-

polymers”, 15th International Conference on Biopar-

titioning and Purification | BPP2009, Uxbridge, UK,

June

Nascimento, K.S., Azevedo, A.M., Cavada, B.S.,

Aires-Barros, M.R., “Optimization of aqueous two-

phase extraction of Canavalia brasilienses lectin by

response surface methodology”, 15th International

Conference on Biopartitioning and Purification |

BPP2009, Uxbridge, UK, June

Oliveira, P.H., Prazeres, D.M.F., Monteiro, G.A.,

“Hotspots for recombination are widespread among

plasmid vectors”, FEMS 2009, 3rd Congress of

European Microbiologists, Gothenburg, Sweden,

June-July

Raiado-Pereira, L., Prazeres, D.M.F., Mateus, M.,

"Exploring liposome affinity membrane for chroma-

tographic separation of plasmid DNA vaccines",

EUROMEMBRANE 2009 Conference, Montpellier,

France, September

Ribeiro, S., Mendes, R., Madeira, C., dos Santos,

F., da Silva, C.L., Cabral, J.M.S., “Establishment of

a protocol for the evaluation of mesenchymal stem

cell transfection using real-time PCR”, 7th Annual





liferation of mouse neural stem cells", 7th Annual





liferation of mouse neural stem cells", 3rd Interna-

tional Stem Cell Meeting: Biology and Clinical Appli-

cations, Tel-Aviv, Israel, June

dos Santos, F., Andrade, P.Z., da Silva, C.L., Abe-

casis, M., Cabral, J.M.S., "Ex-vivo expansion of

human mesenchymal stem cell (MSC) under hy-

poxia: effects on proliferation kinetics and metabo-

lism", 7th Annual Meeting of International Society of

Stem Cell Research | ISSCR, Barcelona, Spain,

July





poxia: effects on proliferation kinetics and metabo-

lism", Regenerative Medicine and Adult Stem Cell

Therapy | MSC2009, Cleveland, USA, August

da Silva, C.L., Eibes, G., dos Santos, F., Andrade,

P.Z., da Silva, C.L., Cabral, J.M.S., "A microcarrier-

based stirred culture system for the expansion of

human mesenchymal stem cells", Regenerative

Medicine and Adult Stem Cell Therapy | MSC2009,

Cleveland, USA, August

Silva, C.S.O., Lansalot, M., Afonso, C.A.M., Marti-

nho, J.M.G., Taipa, M.A., �”Bio-inspired affinity-

polymer systems for antibody recognition”, 18th Bi-

ennial meeting of the International Society for Mo-

lecular Recognition | Affinity 2009, Reykjavik, Ice-

land, July


Souza, A.P., Azzoni, A.R., “Development of recom-

binant proteins for non viral gene delivery taking

advantage of molecular motor proteins”, XVII Con-

gress of the European Society of Gene and Cell

Therapy | ESGCT, Hannover, Germany, November


Souza, A.P., Azzoni, A.R., “The use of recombinant

dynein light chains to mediate enhanced plasmidial

DNA transfection proteins for non viral gene deli-

very taking advantage of molecular motor proteins”,

XVII Congress of the European Society of Gene

and Cell Therapy | ESGCT, Hannover, Germany,

November

National Conferences


da Silva, C.L., Cabral, J.M.S., “Ex-vivo expansion of

hematopoietic stem/progenitor cells in co-culture


kine cocktail", 4th International Meeting of the Portu-

guese Society of Stem Cells and Cell Therapies |



Weiss, C.K., Landfester, K., “Synthesis of flavor

esters by cutinase in miniemulsion and organic sol-

vent media”, Joint Meeting of the Portuguese Society

of Microbiology and of the Portuguese Society of Bio-

technology | Microbiotec09, Vilamoura, November

Boura, J.S., dos Santos, F., da Silva, C.L., Abe-

casis, M.A., Vale, P., Cabral, J.M.S., “Culture me-

dium optimization for the ex-vivo expansion of adi-

pose tissue and bone marrow-derived mesenchy-

mal stem cells", 4th International Meeting of the Por-

tuguese Society of Stem Cells and Cell Therapies |



C.L., Cabral, J.M.S., “A microcarrier-based stirred


chymal stem cells", 4th International Meeting of the






embryonic stem cell expansion", 4th International

Meeting of the Portuguese Society of Stem Cells

and Cell Therapies | SPCE-TC, Lisbon, April

Fernandes-Platzgummer, A.M., Diogo M.M., Bap-



ner-flask to a fully controlled bioreactor”, 4th Interna-




Prazeres, D.M.F., “RNA clearance from plasmid-

containing lysates by phenyl boronate adsorption”,

Joint Meeting of the Portuguese Society of Microbiol-

ogy and of the Portuguese Society of Biotechnology |

Microbiotec09, Vilamoura, November

Mendes, R., Madeira, C., Ribeiro, S., dos Santos,

F., da Silva, C.L., Cabral, J.M.S. “Non-viral gene

delivery to human mesenchymal stem/progenitor

cells using cationic liposomes”, 4th International

Meeting of the Portuguese Society of Stem Cells

and Cell Therapies | SPCE-TC, Lisbon, April

Oliveira, P.H., Prather, K.J., Prazeres, D.M.F. and

Monteiro, G.A. “Rational engineering of E. coli

strains and vectors for improved manufacturing of

plasmid biopharmaceuticals”, 1st MIT-Portugal An-

nual Conference: Engineering for Better Jobs. Lis-

bon, July

Ribeiro, S., Madeira, C., dos Santos, F., Monteiro,

G., da Silva, C.L.L., M.S. Cabral, J.M.S.

“Comparison of polyadenylation signals for use in


mal stem cells”, 4th International Meeting of the




Cabral, J.M.S., “Low oxygen tension enhances

proliferation of mouse neural stem cells", 4th Inter-

national Meeting of the Portuguese Society of

Stem Cells and Cell Therapies | SPCE-TC, Lisbon,

April




poxia: Effects on proliferation kinetics and metabo-

lism", 4th International Meeting of the Portuguese

Society of Stem Cells and Cell Therapies | SPCE-

TC, Lisbon, April

Prizes

UTL/Deloitte Young Researcher Award

Cláudia Lobato da Silva received the UTL/Deloitte

Jovens Investigadores 2009 award in the scientific

domains of Biological Engineering, Biochemistry

and Biotechnology sponsored by the Technical

University of Lisbon and Deloitte (23rd June 2009).

Paula Rosa was distinguished with a Honourable

Mentions in UTL/Deloitte Jovens Investigadores

2009 award in the scientific domains of Biological

Engineering, Biochemistry and Biotechnology

sponsored by the Technical University of Lisbon

and Deloitte (23rd June 2009).

Filipa Ferreira was distinguished with a Honourable

Mentions in UTL/Deloitte Jovens Investigadores

2009 award in the scientific domains of Biological

Engineering, Biochemistry and Biotechnology

sponsored by the Technical University of Lisbon

and Deloitte (23rd June 2009).

UTL/Santander Totta Scientific Award

Miguel Prazeres was distinguished with a Honour-

able Mention in the Technical University of Lisbon

(UTL) scientific award competition in the Biological

Engineering, Biochemistry and Biotechnology area,

for the recognition of his scientific work and career,

sponsored by Santander Totta bank (16th Novem-

ber 2009).

Best Poster Award

Nuno Lourenço was named for best poster pre-

sented by a Young Researcher, at the 9th Interna-

tional Symposium on Biocatalysis and Biotransfor-

mation (Biotrans 2009) held in Bern, Switzerland

(9th July 2009).

Roche Younger Investigator Award

Cláudia Silva was distinguished with a Honourable

Mention of the Roche Younger Investigator Award

2009 at the Affinity 2009, the 18th biennial meeting

of the International Society for Molecular Recogni-

tion, in Reykjavik, Iceland (16th July 2009).

World Economic Forum's Annual Meet-

ing of the New Champions "Summer

Davos"

Cláudia Lobato da Silva was nominated as an out-

standing young scientist by the InterAcademy

Panel/World Economic Forum to participate in the

Annual Meeting of the New Champions "Summer

Davos" (10-12th September 2009).


Staff

Faculty

Joaquim M. S. Cabral

M. Raquel Aires-Barros

Luís P. Fonseca

D. Miguel F. Prazeres

Marília Mateus

Gabriel A. Monteiro

José A. L. Santos

Cláudia Lobato da Silva

M. Ângela Taipa

Frederico Ferreira

Research Scientists

Ana M. Azevedo

Carla C.C.R. de Carvalho

M. Margarida Diogo

Pedro Fernandes

Catarina Madeira

Post-doctoral Fellows

Ricardo Baptista

Cristina Cordas

Raquel Frade

Gemma Eibes

Nuno Lourenço

Sofia Ribeiro

PhD Students

Pedro Andrade

Susana Bernardino

Sara Badenes

Dragana Barros

Luís Borlido

Joana Carvalho

Nídia Estrela

Ana Fernandes

Tiago Fernandes

I. Filipa Ferreira

José Forte

Gabriela Gomes

Geisa Gonçalves

João Guerreiro

David Malta

Marco Marques

Sofia Martins

Verónica Martins

Patricia Soares

Kelany Nascimento

Pedro Oliveira

Carlos Rodrigues

Paula Rosa

Francisco Santos

Cláudia Silva

Ana Isabel Silva

Isabel Sousa

Jonathan de la Vega

Master Students

David Barata

Margarida Caras Altas

Filipe Carvalho

Rui Mendes

Johannes Österreicher

Sara Pereira

Irina Pinheiro

Ana Paula Sousa

Meritxell Turk

Research Assistants

Joana Biscaia

Joana Boura

Sofia Duarte

Jaqueline Garcia

João Martins

Luís Raiado-Pereira

Sofia Rebelo

Technicians

Ricardo Pereira

BERG

BioEngineering Research Group

Institute for Biotechnology and Bioengineering

Instituto Superior Técnico

Av. Rovisco Pais

1049-001 Lisbon

Portugal

www.ibb.pt/berg

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