2008 - fenix.tecnico.ulisboa.pt · the design of surfactant-stable cutinase mutants by direct...

2008

Annual Report

BERG | BioEngineering Research Group

3

20 08 Annual Report

Contents

4 Executive Summary

5 The Bioengineering Research Group

6 Bioprocess Engineering and Biocatalysis Laboratory

8 Nucleic Acid Bioengineering Laboratory

10 Stem Cell Bioengineering Laboratory

12 Research Highlights

20 Publications

26 Oral Presentations

30 Poster Presentations

34 Prizes

35 Staff

This annual report is the first of the “new” BioEngi-

neering Research Group (BERG) within the re-

cently created Institute for Biotechnology and Bio-

engineering (IBB), an Associated Laboratory ap-

proved by the Ministry of Science, Technology and

Higher Education in Portugal.

During 2008 major achievements were obtained on

Bioprocess Engineering and Biocatalysis, namely:

the design of surfactant-stable cutinase mutants by

direct evolution; an enzyme technology platform for

esters biosyntheses by cutinase in non-

conventional media; the use of microtiter plates as

platforms for multi-step bioconversions process

development; and the purification of human anti-

bodies from CHO cell cultures with high yields and

purity using aqueous two-phase systems. The Nu-

cleic Acid Bioengineering achievements include

the development of a plasmid DNA purification

chromatographic process based on DNA-amino

acid interactions and a bead-based hybridisation

assay for detection of traces of E. coli genomic

DNA present in purified plasmid DNA samples;

and the construction of DNA vaccine prototypes by

cloning parasitic antigenic proteins associated with

sleeping sickness and tested in mice models. Also

with impact on the Healthcare sector, the major

achievements on Stem Cell Bioengineering include

a high-throughput cell based screening device for

fast identification of small molecules to selectively

control mouse Embryonic Stem Cell (mESC) fate;

the development of serum-free culture systems for

the ex-vivo expansion of human Hematopoietic

Stem Cells (hHSC) in co-culture with human Me-

sebchymal Stem Cells (hMSC) and an integrated

culture system for mESC expansion and neural

differentiation; and the isolation and ex-vivo expan-

sion of human Mesenchymal Stem Cells (hMSC)

under GMP conditions for the treatment of graft

versus host disease, as well as adjuvant in hHSC

transplantation. The clinical trials, performed in

collaboration with Instituto Português de Oncologia

Francisco Gentil de Lisboa and Centro de Histo-

compatibilidade do Sul, are part of the European

Blood and Marrow Transplantation group activities

and represent a pioneer initiative in Portugal.

With the recent creation of IBB, a scientific strategy

was implemented to integrate complementary ex-

pertise in the group by hiring new Faculty mem-

bers and Postdoctoral researchers in emergent

scientific areas, through contracts with Instituto

Superior Técnico, the MIT-Portugal Program and

Programa Ciência 2007. These researchers will

contribute to reach our ambitious goals and

strengthen our research at international level on

bioengineering science. I would like also to ex-

press my confidence in BERG to promote excellent

quality research and advanced education pro-

grammes, ensuring national and international com-

petitiveness in the areas of Industrial and Health

Biotechnology.

Joaquim M.S. Cabral

BERG Head and

Director of IBB

Executive Summary Bioengineering Research Group | BERG

20 Annual Report 08 5

SCBL NABL BEBL 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, Gene/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)

Executive Summary Bioengineering Research Group | BERG

Bioprocess Engineering and Biocatalysis

Objectives

Bioprocess Engineering and Biocatalysis

aims to design and develop value-added bio-

products with potential application in key ar-

eas, such as food and feed, aroma, pharma-

ceutical industry and biofuels. Research is

focused on the production, purification and

stabilisation of proteins/enzymes and on the

design of improved bioconversion processes.

Research Topics

Research in BEBL is currently focused on the

development of technological platforms for

biocatalysis and biomolecules purification.

The projects under study are centred in three

major areas: i) Protein Stabilisation; ii) Bio-

catalysis; and iii) Production and purification

of proteins and biopharmaceuticals.

1. Protein Stabilisation - Approaches to en-

hance the stability of proteins/enzymes, in-

cluding synthetic mimetic affinity ligands and

encapsulation in biocompatible hydrogels, are

investigated in order to improve performance

and develop specific industrial and diagnostic

applications.

2. Biocatalysis - Ester biosyntheses (flavours,

biodiesel and macrocyclic esters) by cutinase

and engineered mutants are addressed in an

enzymatic platform. Nano/micro-biocatalysts

(biocomposites) are being developed based

on hydrogels, protein/cell assemblies and

nano-magnetic particles. The biocomposites

are used as nano/micro-bioreactors and their

performance is evaluated by on-line coupling

with analytical techniques (FIA/SIA) and mi-

crofluidic systems. New protein-ionic-

conducting-based biocompatible materials

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20 08 Annual Report

(Ion Jelly) with tailor-made properties have

been designed to build new planar am-

perometric biosensors.

Mini-scale devices are used to speed optimi-

sation of biotransformation/fermentation sys-

tems.

3. Production and Purification of Biopharma-

ceuticals - Alternative processes for the purifi-

cation of proteins and biopharmaceuticals

(antibodies) integrating aqueous two-phase

extraction with chromatographic steps are

being developed. Extraction separation units

using affinity materials and nanomagnetic

particles are being evaluated, characterised

and validated by modelling.

Stimuli-responsive beads based on PNIPAM

are also being developed as a strategy to

produce novel bio-inspired affinity polymer

systems for antibody recognition.

Major Achievements

The activity and stability of cutinase and

mutants obtained by direct evolution were

evaluated and compared. The S54D mu-

tant was much more resistant to AOT

denaturation than the native enzyme.

A new approach for protein stabilisation

was developed, through the design and

synthesis of combinatorial libraries of

ligands interacting with specific regions on

cutinase surface.

An enzyme technology platform was used

in esters biosyntheses by cutinase in non-

conventional media. A novel strategy us-

ing a green chemistry approach based on

mini-emulsions has also been developed

for aroma and flavours bioproduction.

Validation of microtiter plates as suitable

platforms for the characterisation of multi-

step bioconversions in conventional and

non-conventional media, using sitosterol

side-chain cleavage as model system.

Implementation of an effective system for

the production of inverted sugar syrup

using inulinase immobilised in PVA cap-

sules.

Human antibodies from CHO cell cultures

were purified using aqueous two-phase

system (ATPS) comprising a temperature

responsive polymer composed of ethylene

oxide and propylene oxide (EOPO), with

high yield and purity. The use of this

smart polymer has considerably simplified

the re-extraction step of antibodies.

Selected Publications

Baptista, R.P., Pedersen, S., Cabrita, G.J.,

Otzen, D.E., Cabral, J.M.S., Melo E.P., Bio-

polymers, 89, 538-547 (2008)

Brissos, V., Eggert, T., Cabral, J.M.S., Jae-

ger, K.E., Prot. Eng. Design Select., 21, 387-

393 (2008)

Claudino, M.J.C., Soares, D., Marques,

M.P.C., van Keulen, F., Cabral, J.M.S., Fer-

nandes, P., Bioresource Technol., 99, 2304-

2311 (2008)

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

Aires-Barros, M.R., J. Chromatogr. A, 1195,

94-100 (2008)

Teles, F.R.R., Fonseca, L.P., Talanta, 77,

606-623 (2008)

Objectives

Nucleic Acid Bioengineering is focused on: i)

plasmid vectors and their application in gene

therapy or DNA vaccination; and ii) micro-

chips for DNA detection. The specific objec-

tives are to address the scientific and techno-

logical challenges associated with plasmid

biopharmaceuticals by combining biomolecu-

lar engineering studies with bioprocess engi-

neering, and to co-develop (with INESC-MN)

thin-film microchip platforms for the manipula-

tion/detection of DNA.

Research Topics

In the case of plasmids, the following re-

search topics are pursued:

1. Structural stability of plasmids - Studies on

the nuclease barriers to gene expression

during plasmid trafficking through the cytosol

of mammalian cells are performed with the

goal of constructing plasmid vectors with an

increased resistance to nucleases and thus

with a higher transfection activity.

2. Manufacturing of plasmid vectors - Proc-

esses for the production of plasmids are con-

ceptually designed, developed, optimised and

compared. The impact of specific plasmid

structural elements in the performance of

upstream and downstream processes

(membranes, chromatography, aqueous two-

phases) and in the quality of the final product

is evaluated. Analytical procedures to monitor

manufacturing and control product quality are

also developed.

3. DNA vaccine prototyping - DNA vaccine

candidates are constructed by cloning para-

Nucleic Acid Bioengineering

9

20 08 Annual Report

sitic antigenic proteins associated with sleep-

ing sickness disease and tested in mice mod-

els for their ability to generate cellular and

humoral responses, and to provide immuni-

sation.

In the case of microchips for DNA detection

the following topics are addressed:

1. Immobilisation and handling of DNA - Thin

film technologies, chemical modification, mi-

crofluidics and electronic addressing are

used to develop microchips for the molecular

recognition of specific analytes via hybridisa-

tion. The core of the chips is a flat surface

with immobilised probe molecules. Other

features include the presence of micro-

electrodes to generate electric fields that ac-

celerate the kinetics of binding/recognition.

2. Photodetectors - Amorphous silicon

photodetectors are developed for the opto-

electronic detection of coloured, chemilumi-

nescent and fluorescent molecules in thin film

chips. The presence of these molecules ulti-

mately reports specific biorecognition events

such as DNA hybridisation.

Main Achievements

The feasibility of exploring DNA-amino

acid (histidine, arginine) interactions in

the context of plasmid DNA purification

by fixed-bed chromatography was dem-

onstrated (in collaboration with UBI).

The recombination of plasmid DNA vec-

tors harbouring direct repeats during rep-

lication in E. coli grown under increasing

antibiotic pressure was studied experi-

mentally. A simple non-linear mathemati-

cal function was developed to accurately

predict the corresponding recombination

frequencies.

A bead-based hybridisation assay was

developed for detection of traces of E.

coli genomic DNA present in purified

plasmid DNA samples

Miniaturised amorphous silicon thin-film

photodetectors were developed to quanti-

tate the light (colorimetry, fluorescence,

chemiluminescence) generated during

assays for the detection of molecular

recognition events such as DNA hybridi-

sation and antibody-antigen binding (in

collaboration with INESC-MN).

The possibility of using amorphous sili-

con-based ion-sensitive field-effect tran-

sistors (a-Si:H ISFETs) for the label-free

detection of DNA molecules was studied

in detail.


Gonçalves, D., Prazeres, D.M.F., Chu, V.,

Conde, J.P., Biosensors Bioelectronics, 24,

545-551 (2008)

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

Anal. Bioanal. Chem., 391, 2179-2187 (2008)

Oliveira, P.H., Lemos, F., Prazeres, D.M.F.,

Monteiro, G.A., Plasmid, 60, 159-165 (2008)

Pimentel, A.C, Prazeres, D.M.F., Chu, V.,

Conde, J.P., J. Applied Physics., 104,

054913 (2008)

Ribeiro, S.C., Oliveira, P.H., Prazeres,

D.M.F., Monteiro, G.A., Mol. Biotechnol., 40,

252-260 (2008)

Stem Cell Bioengineering

Objectives

The Stem Cell Bioengineering Laboratory

aims at the development of highly controlled

culture systems (e.g. bioreactors) for the ex-

vivo expansion of stem cells and their con-

trolled differentiation into specific cell types.

As stem cells are rare, their isolation and

expansion/differentiation in vitro significantly

increases the cell population available for

cellular and gene therapy settings, high-

throughput drug screening, tissue engineer-

ing and stem cell research. Human hemato-

poietic stem cells (HSC), human mesenchy-

mal stem cells (MSC), human and mouse

embryonic stem cells (ESC), and mouse neu-

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

tems.

Research Topics

1. Expansion of HSC in co-culture with MSC

under serum-free conditions - Current re-

search is focused on understanding the

mechanisms underlying the hematopoietic

supportive capacity of MSC combining prolif-

erative, functional and proteomic analysis.

The elucidation of those mechanisms will

have implications in terms of bioreactor de-

sign towards the maximization of human HSC

expansion in vitro.

2. Clinical-scale production of MSC -

By combining a cross-disciplinary approach

of Stem Cell Bioengineering and Experimen-

tal Hematology, culture protocols are opti-

mized for the expansion of human MSC,

while maintaining their multilineage and im-

munosuppressive capacities, for supplemen-

11

20 08 Annual Report

tation during HSC transplantation. MSC are

isolated from adult bone marrow (BM), adi-

pose tissue (AT) and umbilical cord blood

(UCB).

3. Bioreactor expansion of ESC and NSC -

The expansion of ESC and ESC-derived

NSC is addressed towards the definition of

highly controlled, efficient, reproducible and

cost-effective bioprocesses to obtain starting

material to generate mature cells (i.e. neu-

rons) for potential use in Regenerative Medi-

cine (e.g. treatment of neurological disor-

ders), as well as for high-throughput drug

screening. In a complementary approach,

high-throughput microarray systems are de-

veloped for studying the effect of the micro-

environment on self-renewal and neural dif-

ferentiation of ESC.

4. Gene delivery to stem cells - Aiming at the

maximization of the SC yield to reach mean-

ingful cell numbers for Cell or Gene Therapy

settings or high-throughput screening assays,

efficient plasmid DNA transfection protocols

are developed using non-viral vectors for the

transient over-expression of specific genes

involved in self-renewal or lineage commit-

ment, in collaboration with Nucleic Acid Bio-

engineering Laboratory.

5. Recombinant protein production for stem

cell research - A platform for recombinant

protein production and purification has been

developed, especially aiming to produce cyto-

kines/growth factors such as Leukemia Inhibi-

tory Factor (LIF) and Bone Morphogenetic

Protein 4 (BMP-4) for stem cell culture.

Main Achievements

● A consortium was established between IBB

-IST and Instituto Português de Oncologia

Francisco Gentil and Centro de Histocompati-

bilidade do Sul, focusing the isolation and ex-

vivo expansion of MSC under GMP condi-

tions for the treatment of graft versus host

disease, as well as adjuvant in HSC trans-

plantation. The clinical trials already per-

formed are part of the European Blood and

Marrow Transplantation group activities and

represent a pioneer initiative in Portugal,

since ex-vivo expanded allogenic MSC have

been infused into patients for the first time.

● A 3-D high-throughput cell based screening

device was developed for the fast identifica-

tion of small molecules that can be used to

selectively control mouse ESC fate, in col-

laboration with Jon Dordick, RPI, USA.

● Two serum-free culture systems were de-

veloped for: i) ex-vivo expansion of human

HSC in co-culture with human MSC and ii)

the integrated expansion and neural commit-

ment of mouse ESC.


Fernandes, T.G., Kwon, S.J., Lee, M.Y.,

Clark, D.S., Cabral, J.M.S., Dordick, J.S.,

Anal. Chem., 80, 6633-6639 (2008)

Diogo, M.M., Henrique D., Cabral, J.M.S.,

Biotechnol. Appl. Biochem., 49, 105-112

(2008)

Frias, A.M., Porada, C.D., Crapnell, K.B.,

Cabral, J.M., Zanjani, E.D., Almeida-Porada,

G., Exp. Hematol., 36, 61-68 (2008)

Aqueous Two-Phase Systems for Antibody

Purification

Raquel Aires-Barros and Ana M. Azevedo

The production of monoclonal antibodies (MABs)

has been chosen as a demanding and challenging

example process, since a large number of MABs

candidates in pre-clinical and clinical trials will

reach process development stage in a few years,

and production capacities will dramatically fall

short. The aim of this research is to evaluate the

use of aqueous two-phase systems (ATPS) as a

generic technology for the selective recovery and

purification of MABs.

Non-functionalised systems

A model system containing albumin, myoglobin

and IgG was used to investigate the feasibility of

using aqueous two-phase systems (ATPS) of

polyethylene glycol (PEG) and phosphate salts, to

recover human IgG. In order to improve the parti-

tion of IgG to the top phase different concentra-

tions of NaCl were added to the ATPS (Fig. 1).

For concentrations of NaCl higher than 10%, IgG

partitioned preferentially to the top phase while

the contaminant proteins remained in the bottom

phase. With 15% NaCl, about 90% of the IgG was

recovered in the top PEG rich-phase. A back-

extraction step was also performed and IgG was

recovered with a total yield of 76% and a purity of

100%.

The application of ATPS was expanded to the

initial recovery of human antibodies from both

Chinese Hamster Ovary (CHO) and hybridoma

cell culture supernatants composed of PEG 6000,

phosphate and NaCl (Fig. 2). ATPS was success-

fully used to partially purify monoclonal antibodies

from a hybridoma cell culture supernatant with a

total yield of 90% and a purification factor of 4.1.

In collaboration with Werner Bäcker from Bayer

Technology Services GmbHa, a counter-current

multi-stage extraction was performed in a mixer-

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

0 5 10 15

log

KP

NaCl %

Figure 1: Effect of NaCl concentration on the partition

coefficient of IgG (), HSA () and Myo (), with a sys-tem composition of 7.04% PEG 6000, 14.37% phosphate

pH 7.0, 0.1% protein.

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

0 5 10 15

log

KP

NaCl (%)

Figure 2: Effect of NaCl concentration on the partition

coefficient of IgG () and contaminant proteins (), in a system composition of 12% PEG 6000, 10% phosphate

pH 6, 40% hybridoma feed stock.

BEB

L

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20 08 Annual Report

Figure 3: Multi-stage mixer-settler battery used for aqueous two phase extraction .

settler battery using the optimised ATPS based in

PEG/ phosphate/ NaCl (Fig. 3). It was possible to

run successfully this type of system in a multi-

stage equipment with significant improvements in

the recovery yield, partition coefficient and purity

of IgG.

Functionalised systems

To improve the selectivity of aqueous two phase

extraction, the performance of several functional-

ised PEG and triethylene glycol (TEG) molecules

was evaluated for the capture of human antibod-

ies from a CHO cell supernatant. A screening of

ligands containing charged, hydrophobic and af-

finity groups was performed either in the form of

free ligands or as phase forming components.

Among the functionalised PEG3350 molecules

(Fig. 4), promising results were obtained with glu-

taric acid, amino and benzyl groups, with extrac-

tion yields higher than 75% and protein purities

around 90%. Among the free ligands, TEG diglu-

taric acid (TEG-COOH) displayed the highest af-

finity towards antibodies and facilitated an extrac-

tion yield of 96% and a protein purity of 95%.

Many of the diseases treated by antibodies re-

quire high doses or chronic administration, thus

economical process-scale production and purifica-

tion of these molecules is critical. This work can

contribute to important improvements in the down-

stream processing of antibodies, essential in order

to cut down the manufacturing costs, and to use

ATPS as a generic and efficient recovery and pu-

rification technology on a large scale in the phar-

maceutical industry.

-1.0

-0.5

0.0

0.5

1.0

1.5

Control GA NH2 Benzyl Pym MEP

log

KP

PEG-Ligand

Figure 4: Partition coefficient of IgG in the presence of

functionalised-PEG in ATPS composed of 8% PEG-ligand and () 5% dextran or () 8% dextran. GA: glu-

taric acid; Pym: pyrimidine; MEP: mercaptoethylpyridine.

R

esearch H

ighlights

BEB

L Enzymes In BioAnalytical Methods and

Monitoring Tools

Luís P. Fonseca

Rapid development of biotechnology and biotech-

nological applications in recent years resulted in

an increased need for methods for reliable biopro-

cess monitoring and control. Various techniques

have been investigated and proposed, but few are

widely accepted and implemented due to the in-

trinsic difficulties related to complexity of samples

from bioprocesses, asepticity requirements, and

the bioprocesses themselves. Enzymes In Bio-

Analytical Methods and Monitoring Tools (EIBAM-

MT) project has stemmed on the development of

directly interfaced enzyme based amperometric

detection devices for bioprocess monitoring. This

specific project focuses on-line monitoring of ana-

lyte(s), namely carbon source uptake and metabo-

lites formation in medium cultures and enzyme

activity (e.g. cutinase) by assessment with Flow

Injection Analysis (FIA) systems. These analytical

tools allow to improve process optimisation and

control according to the influence of stringent re-

sponse especially to define the substrate feeding

strategy and induction time of biosynthesis in fer-

mentation culture and enzyme yields on down-

stream processing, namely of Expanded Bed Ad-

sorption columns (Fig. 1). This led to a significant

reduction of variability in operation and more pre-

dictable yield and purification degree of enzyme.

The monitoring of nutrients and metabolites in

culture medium, (e.g. glucose, galactose, ethanol,

lactate, amino acids), as result of microbial cell

activity, such as S. cerevisiae and E. coli, and

animal cells, was carried out by multi-enzyme sys-

tems based on immobilised oxidase and horse-

Mixing

chamber

FIA system

Valve

Sample

injectorReagent A

Pump B

Pump A

Reagent B

Spectrophotometer

Sewage

Reaction coil

Reagent A

On/Off Valve

Sample poolSewage

Diluted sample

Off Off

Fermentation

broth

Sewage

vessel

Dilution

Mixing-module

Clarification

module

EBA column

Monitoring tool for EBA

adsorption at pH 4.5Yeast cells

Yeast cells

Mixing

chamber

FIA system

Valve

Sample

injectorReagent A

Pump B

Pump A

Reagent B

Spectrophotometer

Sewage

Reaction coil

Reagent A

On/Off Valve

Sample poolSewage

Diluted sample

Off Off

Fermentation

broth

Sewage

vessel

Dilution

Mixing-module

Clarification

module

EBA column

Mixing

chamber

FIA system

Valve

Sample

injectorReagent A

Pump B

Pump A

Reagent B

Spectrophotometer

Sewage

Reaction coil

Reagent A

On/Off Valve

Sample poolSewage

Diluted sample

Off Off

Fermentation

broth

Sewage

vessel

Dilution

Mixing-module

Clarification

module

EBA column

Monitoring tool for EBA

adsorption at pH 4.5Yeast cells

Yeast cells

Figure 1: Analytical tool for almost in real time monitoring cutinase activity come out from the

Expanded Bed Adsorption column.

15

20 08 Annual Report

Figure 2: Mini-analytical columns of CPG

based on immobilized of one oxidase and Figure 3: Integration of mini-analytical columns into Carrier

of the Flow Injection Analysis (FIA) system

Figure 4: Analytical step-up for monitoring of nutrients and

metabolites (e.g. glucose, galactose, ethanol, lacate, amino

acids) and cutinase activity in culture medium.

radish peroxidase (HRP) in mini-analytical reac-

tors (Fig. 2) integrated into a FIA system (Fig. 3).

The success of these mini-analytical reactors was

due to their high sensibility and stability, inclu-

sively in complex culture media. The development

of an in-situ stabilisation strategy of oxidases

against hydrogen peroxide, which is instantane-

ously and in-situ eliminated, represents a major

breakthrough. This stabilisation strategy is carried

out when the oxidation reaction is performed in

the presence of peroxidase, 4-aminoantipyrine

and phenol-4-sulfonic acid, which are reduced by

HRP to a quino-imine, a colorimetric compound

easily monitored in the spectrophotometric detec-

tor of the FIA system. The monitoring of cutinase

biosynthesis is also essential for process control

with great positive impact on process productivity

and efficiency, thus reducing production costs

(Fig. 4).

Recently, this project also focuses on monitoring

of environmental biological samples and water

quality by determination of total and speciation of

toxic metal elements based on enzymatic diges-

tion of biological samples assisted by probe soni-

cation (EPS), an emerging methodology that mini-

mises interferences on metal determinations in

contrast with traditional analytical methodologies.

It is also expected to design new protein-ionic-

conducting-based materials with tailor-made prop-

erties and biocompatibility according to the com-

position and conditions of preparation. Accuracy

of electrochemical signals, storage and opera-

tional stability of planar amperometric biosensors

based on peroxidases, dehydrogenases and

some O2 dependent oxidases (e.g. glucose, lac-

tate, alcohol, among others) and antibodies conju-

gated with peroxidase are being evaluated. These

new conducting materials, with tailor-made prop-

erties and efficient direct electron transfer be-

tween the biological element and the transducer,

can open a window of new opportunities for appli-

cations in chemistry and biology.

The Enzymes In Bioanalytical Methods and Moni-

toring Tools (EIBAM-MT) project shows how it is

important the use of enzymes in bioanalytical

methods and opens new perspectives on bioproc-

essing of enzymes and other biomolecules for

industrial and diagnostic applications.

R

esearch H

ighlights

NAB

L

Production and Design of DNA Vaccines

Duarte Miguel F. Prazeres and 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 and large scale production

of pDNA must be addressed. The research pro-

gram on “Nucleic Acid Bioengineering” addresses

some of these challenges.

DNA vaccine manufacturing

A patented process based on hydrophobic inter-

action chromatography (HIC) has been developed

and used for the manufacturing of pDNA. E. coli

cells harbouring the pDNA vaccine are cultured in

a bioreactor with a suitable medium. Here we

found out that: i) by extending cell culture up to 26

h it is possible to reduce RNA without compromis-

ing the pDNA yield and ii) pDNA is remarkably

stable when stored in cell pellets (>3 weeks at

4ºC, >12 weeks at –20ºC) prior to processing3.

Cells are then disrupted by alkaline lysis. If con-

venient, lysates can be stored at –20ºC within the

first 8 weeks without the onset of pDNA degrada-

tion. Next, pDNA is concentrated by isopropanol

precipitation and protein, endotoxin and RNA con-

tent is reduced by an ammonium sulphate precipi-

tation step that also acts as a conditioning step for

the subsequent HIC step. HIC is carried out with a

suitable support derivatised with hydrophobic

ligands. A typical chromatogram shows a first

sharp peak of DNA vaccine followed by a broader

peak of weakly retained contaminants: RNA, ge-

nomic DNA, proteins (Fig. 1). The process is ro-

bust, reproducible and amenable to scale-up and

delivers a product within the standard specifica-

tions and with adequate biological activity (Fig. 2).

It has been extensively used in our laboratory to

produce milligram quantities of pDNA vectors for

gene therapy and DNA vaccination, including pro-

totypes for immunization against rabies and sheep

Maedi-Visna virus.

Improvement of DNA vaccine stability

Extra- and intra-cellular nuclease degradation of

DNA vaccines after delivery and during trafficking

to the nucleus constitutes a barrier to gene ex-

pression and consequently to the elicitation of

immune responses. In vivo clearance of pDNA

occurs within a few hours. That barrier may be

circumvented by shielding the DNA vaccines from

the nuclease-rich environments with adjuvants like

cationic lipids and other biopolymers, or by using Figure 1: Purification of DNA vaccines by hydro-

phobic interaction chromatography

0

60

120

0 20 40 60

Ab

s. 2

80

nm

(%

)

time (min)

DNA

vaccine

impurities

a)

Ab

s 2

80

nm

(%

)

Time (min)

17

20 08 Annual Report

Figure 3: a) Resistance of plasmid vectors modified in the origin of replication, as measured by incubation with the single-

stranded specific S1 nuclease for different times; b) transfection of CHO cells with the modified vectors using Lipofectamine. Cells were analysed 24 hours post-transfection by flow cytometry. Error bars indicate standard deviation between four repli-

cates.

nuclease inhibitors. Another alternative which is

explored in our group relies on the construction of

pDNA variants that are more resistant to nuclease

action. Although DNA secondary structures play a

biologically relevant role by facilitating the binding

of specific proteins, our studies indicate that

pDNA vectors can be optimized without loss of

functionality, leading to vectors with higher trans-

fection efficiency. The choice of plasmid vector

sequences is important, not only for mRNA matu-

ration/stability, but also for pDNA resistance, and

should thus be taken into consideration in the de-

sign and evaluation of pDNA vectors. For in-

stances, in vitro and cell culture studies indicate

that pDNA nuclease resistance can be improved 2

-fold by changing the polyadenylation sequence.

This modification, however, led to a decrease in

transcription. The replacement of a few (seven)

specific nucleotides in the plasmid pMB1 origin of

replication could also increase (up to 2.5-fold) the

nuclease resistance (Fig. 3a), while simultane-

ously augmenting (1.5 fold) the levels of the ex-

pressed protein in cell culture (Fig. 3b). Moreover,

no significant functional loss of the modified origin

of replication was detected in E. coli.

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 veteri-

nary DNA vaccines. Furthermore, new develop-

ments are likely to surface within the coming

years because of increased investments from

academia and industry in the area. Our work is a

contribution to these efforts.

Figure 2: CHO cells transfected with GFP expressing plasmid DNA molecules purified by the HIC process.

0

10

20

30

40

50

pVAX1GFP pVAX1GFP-O1 pVAX1GFP-O2

Tra

nsfe

cti

on

Eff

icie

nc

y (

%)

b)

M 0 10 40 0 10 40 0 10 40 (min)

sc

pVAX1GFP pVAX1GFP-O1 pVAX1GFP-O2

a)

Research H

ighlights

SCB

L

Stem Cell Bioengineering Science aims to con-

tribute for a better knowledge of the ex‑vivo ex-

pansion of stem cells and their controlled differ-

entiation into specific cell types in bioreactor sys-

tems. As stem cells are rare, their isolation and

efficient expansion in vitro significantly increase

the cell population available for multiple cell

therapies. The development of ex-vivo culture

conditions capable of mimicking stem cell

“niches” in vivo, by facilitating the maintenance

and expansion of long-term transplantable stem

cells, as well as their commitment into a specific

cell lineage, is a major challenge in stem cell

research and its applications. Human hematopoi-

etic stem cells and mesenchymal stem cells, as

well as mouse embryonic stem cells (mESC)

have been used as model systems at the Stem

Cell Bioengineering Laboratory.

Expansion of ESC

In particular, ESC have the ability to differentiate

in vitro into a wide variety of cell types with po-

tential applications in regenerative medicine.

However, a large number of cells is required,

thus strengthening the need to develop large-

scale systems using chemically defined media

for cell expansion and/or controlled differentia-

tion. A stirred culture system was successfully

used to scale-up mESC expansion in serum-

containing or serum‑free media, using macropor-

ous microcarriers (Fig. 1).

After 8 days, maximal cell densities achieved

were 2.6 and 3.5×106 cells/mL for serum-

containing and serum-free media, respectively,

with fold increases (relatively to day 0) of 50 and

70. Importantly, mESC expanded using serum-

free medium retained their pluripotency and the

ability to commit to the neural lineage (Fig. 2).

High-throughput 3-D cell microarray

Although an effective system for the successful

expansion and/or differentiation of stem cells was

established, the potential therapeutic cell use is

contingent upon precise control of stem cell fate

in culture. We have recently developed in col-

Stem Cell Bioengineering Science:

Scaling-up or Scaling-down?

Joaquim MS Cabral, Cláudia Lobato da Silva and Margarida Diogo

Figure 1: mESC cell expansion on macroporous microcarriers under stirred culture conditions. Growth curve in terms of viable

cell densities per milliliter (A) and cell expansion in terms of fold increase in total cell number (B), respectively, are represented

for serum-containing () and serum-free () media.

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

0 1 2 3 4 5 6 7 8

Via

ble

cell

s/m

L

Time (days)

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8

Fo

ld In

cre

ase

Time (days)

19

20 08 Annual Report

laboration with J. Dordick, Department of Chemi-

cal and Biological Engineering at Rensselear

Polytechnic Institute, a miniaturized 3-D cell-

culture based chip for high-throughput screening

which consists of mESC encapsulated in 20 nL

alginate gels arrayed on a functionalized glass

slide (Fig. 3).

Our results show that this platform is suitable for

studying the expansion of mESC, while retaining

their pluripotent and undifferentiated state. In

addition, growth rate values obtained for different

culture systems are similar.

Overall, we expect this work will pave an impor-

tant role for the successful control of stem cell

fate in vitro, as well as for the design of efficient

culture systems with potential applications in

terms of regenerative medicine, as well as for

drug discovery in the pharmaceutical industry.

Figure 2: Evaluation of pluripotency and neural commitment potential of mESC cultured on macroporous micro-

carriers under stirred culture conditions. Pluripotency was evaluated by alkaline phosphatase staining (A). The percentage of neural progenitors was determined by flow cytometry (B, negative control; C, cells cultured in

neural differentiation medium).

Figure 3: 3-D cell culture microarray platform

R

esearch H

ighlights

Articles in International Peer-Reviewed

Journals

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

Barros, M.R., “Integrated process for the purification

of antibodies combining aqueous two-phase extrac-

tion, hydrophobic interaction chromatography and

size-exclusion chromatography”, J. Chromatogr. A,

1213, 154-161

Baptista, R.P., Pedersen, S., Cabrita, G.J., Otzen,

D.E., Cabral, J.M.S., Melo E.P., “Thermodynamics

and mechanism of cutinase stabilization by treha-

lose”, Biopolymers, 89, 538-547

Brissos, V., Eggert, T., Cabral, J.M.S., Jaeger,

K.E., “Improving activity and stability of cutinase to-

wards the anionic detergent AOT by complete satura-

tion mutagenesis”, Prot. Eng. Design Select., 21, 387-

393

Brissos, V., Melo, E.P., Martinho, J.M.G. , Cabral,

J.M.S., “Biochemical and structural characterisation

of cutinase mutants in the presence of the anionic

surfactant AOT”, Biochem. Biophys. Acta - Prot. Pro-

teom., 1784, 1326-1334

Cardoso, F.A., Germano, J., Ferreira, R., Cardoso,

S., Martins, V.C., Freitas, P.P., Piedade, M.S.,

Sousa, L., “Detection of 130 nm magnetic particles

by a portable electronic platform using spin valve and

magnetic tunnel junction sensors”, J. Appl. Phys.,

103, 07A310

Cardoso, M.A.T, Monteiro, G.A., Cardoso, J.P.,

Prazeres, T.J.V., Figueiredo, J.M.F., Martinho,

J.M.G., Cabral, J.M.S., Palavra, A.M.F.,

“Supercritical antisolvent micronization of minocycline

hydrochloride”, J. Supercritical Fluids, 44, 238-244

Cardoso, M.A.T., Geraldes, V., Cabral, J.M.S., Pa-

lavra, A.M.F., “Characterization of minocycline pow-

der micronized by a supercritical antisolvent (SAS)

process”, J. Supercritical Fluids, 46, 71-76

Cardoso, M.A.T, Cabral, J.M.S., Palavra, A.M.F.,

Geraldes, V., “CFD analysis of supercritical antisol-

vent (SAS) micronization of minocycline hydrochlo-

ride”, J. Supercritical Fluids, 47, 247-258

Carvalho, R.H., Lemos, M.A.N.D.A., Lemos, F.,

Cabral, J.M.S., Ribeiro, F.R., “Electro-oxidation of

phenol on zeolite/graphite composite electrodes -

Part 3. Influence of the electrolyte and of nonelectro-

active cations”, Catal. Today, 133, 855-862

Catarino, I., Minhalma, M., Beal, L.L., Mateus, M.,

de Pinho, M.N., "Assessment of saccharide frac-

tionation by ultrafiltration and nanofiltration", J.

Membr. Sci., 312, 34-40

Claudino, M.J.C., Soares, D., Marques, M.P.C., van

Keulen, F., Cabral, J.M.S., Fernandes, P.,

“Immobilization of mycobacterial cells onto silicone -

assessing the feasibility of the immobilized biocatalyst

in the production of androstenedione from sitosterol”,

Bioresource Technol., 99, 2304-2311

Conde, J.P., Pimentel, A.C., Pereira, A.T., Gouvêa,

A., Prazeres, D.M.F., Chu, V., “Detection of molecu-

lar tags with an integrated amorphous silicon

photodetector for biological applications,” J. Non-

Cryst. Solids, 354, 2594-2597

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

Cabral, J.M.S., “Comparing the effect of immobiliza-

tion methods on the activity of lipase biocatalysts in

ester hydrolysis”, Bioproc. Biosys. Eng., 31, 323-327

Publications

21

20 08 Annual Report


Cabral, J.M.S., “Following multi-component reactions

in liquid medium using spectral band-fitting tech-

niques”, Appl. Spectroscopy, 62, 932-935

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

J.M.S., “Zeolite screening for the racemization of 1-

phenylethanol”, Catal. Today, 133, 625-631

Diogo, M.M., Henrique D., Cabral, J.M.S., “Optimi-

zation and integration of expansion and neural com-

mitment of mouse embryonic stem cells”, Biotechnol.

Appl. Biochem., 49, 105-112

Fernandes, T.G., Kwon, S.J., Lee, M.Y., Clark, D.S.,

Cabral, J.M.S., Dordick, J.S., “An on-chip, cell-

based microarray immunofluorescence assay for high

-throughput analysis of target proteins”, Anal. Chem.,

80, 6633-6639

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

Barros, M.R., “Purification of human immunoglobulin

G by thermoseparating aqueous two-phase systems”,

J. Chromatogr. A, 1195, 94-100

Frias, A.M., Porada, C.D., Crapnell, K.B., Cabral,

J.M.S., Zanjani, E.D., Almeida-Porada, G., “Gene-

ration of functional natural killer and dendritic cells in

a human stromal-based serum-free culture system

designed for cord blood expansion”, Exp. Hematol.,

36, 61-68

Gonçalves, D., Prazeres, D.M.F., Chu, V., Conde,

J.P., “Detection of DNA and proteins using amor-

phous silicon ion-sensitive thin-film field effect transis-

tors”, Biosens. Bioelectron., 24, 545-551

Gonçalves, D., Prazeres, D.M.F., Chu, V., Conde,

J.P., “Amorphous silicon thin-film transistors gated

through an electrolyte solution”, IEEE Electron Device

Lett., 29, 1030-1033

Gouvêa, A., Pereira, A.T., Pimentel, A.C., Prazeres,

D.M.F., Chu, V., Conde, J.P., “Colorimetric detection

of molecular recognition reactions with an enzyme

biolabel using a thin-film amorphous silicon photodi-

ode on a glass substrate”, Sensors Actuators B:

Chemical, 135, 102-107

Lienqueo, M.E., Salazar, O., Calado, C.R.C.,

Fonseca, L.P., Cabral, J.M.S., “Influence of trypto-

phan tags on the purification of cutinase, secreted by

a recombinant Saccharomyces cerevisiae, using cati-

onic expanded bed adsorption and hydrophobic inter-

action chromatography”, Biotechnol. Lett., 30, 1353-

1358

Madeira, C., Loura, L.M.S., Prieto, M., Fedorov, A.,

Aires-Barros, M.R., "Effect of ionic strength and

presence of serum on lipoplexes structure monitor-

ized by FRET", BMC Biotechnol., 8: 20

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

“Online oxygen monitoring system - non-conventional

steroid fermentations in microtiter plates”, Bioforum

Europe, 9, 42-43

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

“Chemiluminescent bead-based hybridization assay

for the detection of genomic DNA from E. coli in puri-

fied plasmid samples”, Anal. Bioanal. Chem., 391,

2179-2187

Oliveira, P.H., Lemos, F., Monteiro, G.A., Prazeres,

D.M.F., “Recombination frequency in plasmid DNA

containing direct repeats - predictive correlation with

repeat and intervening sequence length”, Plasmid,

60, 159-165

Pimentel, A.C, Prazeres, D.M.F., Chu, V., Conde,

J.P., “Fluorescence detection of DNA using an amor-

phous silicon p-i-n photodiode”, J. Applied Physics.,

104, 054913

Prazeres, D.M.F., “Prediction of diffusion coefficients

of plasmids”, Biotechnol. Bioeng., 99, 1040-1044

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

Monteiro, G.A., “High frequency plasmid recombina-

tion mediated by 28 bp direct repeats”, Mol. Biotech-

nol., 40, 252-260

Sampaio, P.N., Fortes, A.M., Cabral, J.M.S., Pais,

M.S., Fonseca, L.P., “Production and characteriza-

tion of recombinant cyprosin B in Saccharomyces

cerevisiae (W303-1A) strain”, J. Biosci. Bioeng., 105,

305-312

Santos, A.M., Fedorov, A., Martinho, J.M.G., Bap-

tista, R.P., Taipa, M.A., Cabral, J.M.S., “Orientation

of cutinase adsorbed onto PMMA nanoparticles

probed by tryptophan fluorescence”, J. Phys. Chem.

B, 112, 3581-3585

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

chromatography approaches for overcoming the chal-

lenges of purifying plasmid DNA”, Trends Biotechnol.,

26, 518-525

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

recognition of supercoiled plasmid DNA by arginine

affinity chromatography”, Anal. Biochem., 374, 432-

434

Taipa, M.A., “Immunoassays: Biological tools for high

throughput screening and characterisation of combi-

natorial libraries”, CCHTS, 11, 325-335

23

20 08 Annual Report

Teles, F.R.R., Fonseca, L.P., “Trends in DNA bio-

sensors”, Talanta, 77, 606–623

Teles, F.R.R., Fonseca, L.P., “Applications of poly-

mers for biomolecule immobilization in electrochemi-

cal biosensors”, Mat. Sci. Eng. C, 28, 1530–1543

Vale, G., Mota, A., Fonseca, L., Capelo, J.L.,

“Ultrasonic assisted enzymatic digestion -USAED- for

total elemental determination and elemental speci-

ation: a tutorial”, Talanta, 75, 872-884

Vale, G., Pereira, S., Mota, A., Fonseca, L., Capelo,

J.L., “Enzymatic probe sonication as a tool for solid-

liquid extraction for total selenium determination by

electrothermal-atomic absorption spectrometry”, Ta-

lanta, 74,198-205

Vidinha, P., Augusto, V., Nunes, J., Lima, J.C.,

Cabral, J.M.S., Barreiros S., “Probing the microenvi-

ronment of sol-gel entrapped cutinase: The role of

added zeolite NaY”, J. Biotechnol., 135, 181-189

Vidinha, P., Barreiros, S., Cabral, J.M.S., Nunes,

T.G., Fidalgo, A., Ilharco, L.M., “Enhanced biocata-

lytic activity of ORMOSIL-encapsulated cutinase: The

matrix structural perspective”, J. Phys. Chem. C, 112,

2008-2015

Vidinha, P., Lourenço, N.M.T., Pinheiro, C., Brás,

A.R., Carvalho, T., Silva, T.S., Mukhopadhyay, A.,

Romão, M.J., Parola, J., Dionísio, M., Cabral

J.M.S., Afonso, C.A.M., Barreiros, S., “Ion Jelly: a

tailor-made conducting material for smart electro-

chemical devices”, Chem. Commun, 44, 5842–5844

Vojinović, V., Cabral, J.M.S., Fonseca, L.P., “Ex-

situ bioprocess monitoring techniques”, Chemical

Industry & Chemical Engineering Quarterly, 13, 1-15

Articles in Conference Proceedings

Barros, D.P.C., Bernardino, S.M.S.A., Fernandes,

P., Cabral, J.M.S., Fonseca, L.P., “Studies of fed-

batch operation mode on synthesis of short chain

ethyl esters catalyzed by cutinase”, Proceedings of

the 10th International Chemical and Biological Engi-

neering Conference - CHEMPOR 2008, E.C. Ferreira

and M. Mota (eds.), 4-6 September 2008, Braga, Por-

tugal, pp 471-476

Bernardino, S.M.S.A., Gallegos, J.F.M., Maduro,

F., Fernandes, P., Cabral, J.M.S., Fonseca, L.P.,

“Nano and micro-biocatalysts manufacture and their

impact on the synthesis of β-lactamic antibiotics”,

Proceedings of the 10th International Chemical and

Biological Engineering Conference - CHEMPOR

2008, E.C. Ferreira and M. Mota (eds.), 4-6 Septem-

ber 2008, Braga, Portugal, pp 489-494


Cabral, J.M.S., “Monitoring multi-component liquid

reaction systems containing highly dispersible hetero-

geneous catalysts using in situ diode array spectro-

photometry and band-fitting techniques”, Proceedings

of the 10th International Chemical and Biological Engi-

neering Conference - CHEMPOR 2008, E.C. Ferreira

and M. Mota (eds.), 4-6 September 2008, Braga, Por-

tugal, pp 213-218

Fernandes, P., Cattorini, S., Carvalho, F.,

Marques, M.P.C., Bernardino, S., Maduro, F.,

Badenes, S., Barros, D., de Carvalho, C.C.C.R.,

Fonseca, L.P., Cabral, J.M.S., “A multipurpose hy-

drogel system for biocatalyst immobilization”, Pro-

ceedings of the 10th International Chemical and Bio-

logical Engineering Conference - CHEMPOR 2008,

E.C. Ferreira and M. Mota (eds.), 4-6 September

2008, Braga, Portugal, pp 1935-1940

Fernandes, P., Cattorini, S., Cabral, J.M.S.,

“Enzymatic inulin hydrolysis using PVA-based matri-

ces”, Proceedings of the 10th International Chemical

and Biological Engineering Conference - CHEMPOR

2008, E.C. Ferreira and M. Mota (eds.), 4-6 Septem-

ber 2008, Braga, Portugal, pp 1873-1878

Pereira, A.T., Pimentel, A., Loureiro, J., Freitas,

P.P., Chu, V., Prazeres, D.M.F., Conde, J.P.,

"Colorimetric detection of antigen-antibody recogni-

tion in a microfluidic channel with an integrated photo-

diode", Proceedings of the XXII EUROSENSORS, 7-

10 September, Dresden, Germany, Verein Deutscher

Ingenieure, pp 1212-1215

Patents

Ribeiro, I., Afonso, C.A.M., Cabral, J.M.S.,

Lourenço, N.M.T., Vidinha, P., Barreiros, S.,

“Synthesis and application of a family of new materi-

als resulting from the chemical cross-linking between

gelatine and organic salts”, European (EP2006321),

United States (US2008/0319164), South Korea (10-

2008-0058173) and Japan (2008-160947) Patent

Applications.

Book chapters

Fernandes, P., Cabral, J.M.S., "Biocatalysis in Bi-

phasic Systems: General", in: Organic synthesis with

enzymes in non-aqueous media, G. Carrea, S. Riva

(eds.), Wiley-VCH, Weinheim, pp. 191-209

Fernandes, P., Cabral, J.M.S., "Immobilization –

Microencapsulation", in: Advances in Fermentation

Technology, A. Pandey, C. Larroche, C.R. Soccol, C.-

G. Dussap (eds.), Asiatech Publishers, Inc., New

Delhi, pp. 45-84

Ph.D. Thesis

Dina Isabel Viegas Gonçalves, “Label-free detection

of biomolecules using amorphous silicon ion-sensitive

field-effect transistors”, PhD Thesis, Technical Uni-

versity of Lisbon, IST, Lisbon (advisors: João P.E.R.

Conde and D. Miguel F.T. Prazeres; IST, Lisbon)

25

20 08 Annual Report

Luisella Ruiu, “De novo design, synthesis and

screening of combinatorial libraries of affinity ligands

directed towards the surface of cutinase from Fusa-

rium solani pisi”, PhD Thesis, Technical University of

Lisbon, IST, Lisbon (advisor: M. Ângela C.G. Taipa;

IST, Lisbon)

Fani Pereira Sousa, “Affinity chromatography proc-

esses in nucleic acids”, PhD Thesis, Universidade da

Beira Interior, Covilhã (advisors: João A.S.R. Quei-

roz, UBI, Covilhã and D. Miguel F.T. Prazeres; IST,

Lisbon)

Sofia de Medina Aires Martins, “Development of

quantitative micro-plate-based assays for DNA hy-

bridization”, PhD Thesis, Technical University of Lis-

bon, IST, Lisbon (advisors: Gabriel A.A. Monteiro and

Luís J.P. Fonseca; IST, Lisbon)

M.Sc. Thesis

Ana Mafalda Nunes Rodrigues, “Application of elec-

tric field assisted hydridization to peptide nucleic ac-

ids”, MSc Thesis, Technical University of Lisbon, IST,

Lisbon (advisors: João P.E.R. Conde and D. Miguel

F.T. Prazeres; IST, Lisbon)

Joana Brissos Magalhães Lima, “Efeito do grau de

superenrolamento de plasmídeos na sua estabilidade

estrutural e função biológica”, MSc Thesis, Faculdade

de Ciências, Universidade de Lisboa, Lisbon

(advisors: D. Miguel F.T. Prazeres, IST, Lisbon and

Maria do Céu Correia, FCUL, Lisbon)

Joana Filipa Sobrinho Boura, “Ex-vivo expansion of

human myoblasts and fibroblasts for potential use in

urinary incontinence treatment”, MSc Thesis,

Faculdade de Ciências, Universidade de Lisboa, Lis-

bon (advisors: Cláudia A.M. Lobato da Silva, IST,

Lisbon and Maria Gabriela G.M. Rodrigues, FCUL,

Lisbon)

Mauro José Castanho Claudino, “Use of miniature

reactors for the characterization of the side-chain

cleavage of β-sitosterol using immobilized cells”, MSc

Thesis, Technical University of Lisbon, IST, Lisbon

(advisors: Pedro Fernandes, IST and Joaquim M.S.

Cabral; IST, Lisbon)

Milene da Silva Santos, “Cell micropatterning for cell

chips applications”, MSc Thesis, Technical University

of Lisbon, IST, Lisbon (advisors: João P.E.R. Conde

and D. Miguel F.T. Prazeres, IST, Lisbon)

Salomé Alexandra de Sá Magalhães, “Construction,

optimization and testing of African Tripanosomiasis

DNA vaccine prototypes with improved nuclease re-

sistance”, MSc Thesis, Faculdade de Ciências, Uni-

versidade de Lisboa, Lisbon (advisors: D. Miguel F.

T. Prazeres, IST, Lisbon and Maria do Céu Correia,

FCUL, Lisbon)

International Conferences

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

“Efficient ionic acylating agents for enzymatic resolu-

tion of alcohols in ionic liquids”, 236th National Meet-

ing and Exposition of the American Chemical Society,

Philadelphia, USA, August

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

J., Korporaal, R., Verhoef, H.J., Visser, T.J., Aires-

Barros, M.R., “Affinity-enhanced partitioning of hu-

man antibodies in aqueous two-phase systems”, 7th

European Symposium on Biochemical Engineering

Science – ESBES7, Faro, Portugal, September

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

Barros, M.R., “An alternative process for the purifica-

tion of therapeutic antibodies comprising aqueous two

-phase extraction”, 28th International Symposium on

the Separation of Proteins, Peptides and Polynucleo-

tides – ISPPP2008, Baden-Baden, Germany, Sep-

tember


P., Cabral, J.M.S., Fonseca, L.P., “Studies on fed-

batch operation mode on biosynthesis of short chain

ethyl esters catalyzed by cutinase”, 10th International

Chemical and Biological Engineering Conference -

CHEMPOR 2008, Braga, Portugal, September

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

Weiss, C., Landfester, K., “Enzymatic synthesis of

fatty acid alkyl esters in miniemulsion”, 7th European

Symposium on Biochemical Engineering – ESBES 7,

Faro, Portugal, September

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

L.P., “Cephalexin synthesis by Penicillin G Acylase

immobilized in sol-gel”, 7th European Symposium on

Biochemical Engineering – ESBES 7, Faro, Portugal,

September

Bernardino, S.M.S.A., Gallegos, J.F.M., Maduro,

F., Fernandes, P., Cabral, J.M.S., Fonseca, L.P.,

“Nano and micro-biocatalysts manufacture and their

impact on the synthesis of β-lactamic antibiotics”, 10th

International Chemical and Biological Engineering

Conference – CHEMPOR 2008, Braga, Portugal,

September

Fernandes, A.M., Diogo M.M., Lobato da Silva, C.,

Henrique, D., Cabral, J.M.S., “Mouse embryonic

stem cell expansion in a microcarrier-based stirred

culture system”, Tissue Engineering and Regenera-

tive Medicine International Society - 2008 Annual

TERMIS, Porto, Portugal, June

Fernandes, A.M., Diogo M.M., Lobato da Silva, C.,

Henrique D., Cabral, J.M.S., “Mouse embryonic


culture system”, 10th International Chemical and Bio-

logical Engineering Conference – CHEMPOR 2008,

Braga, Portugal, September

Oral Communications

20 Annual Report 08 27

Fernandes, T.G., Kwon, S.J., Lee, M., Diogo, M.M.,

Lobato da Silva, C., Clark, D. S., Cabral, J.M.S.,

Dordick, J.S., " High-throughput 3D cell microarray to

study stem cell fate", SBE's First International

Conference on Stem Cell Engineering, San Diego,

USA, January

Fernandes, T.G., Kwon, S.J., Lee, M.Y., Diogo,

M.M., Lobato da Silva, C., Clark, D.S., Cabral,

J.M.S., Dordick, J.S., "A high-throughput 3D cell

microarray to study stem cell fate", 7th European

Symposium on Biochemical Engineering – ESBES 7,




J.M.S., Dordick, J.S., "Exploring stem cell fate using

three-dimensional cellular microarrays", 7th European

Symposium on Biochemical Engineering Science –

ESBES 7, Malcolm Lilly Award Lecture, Faro,

Portugal, September

Fernandes, P., “Whole cell biocatalysis”, III Congreso

Interuniversitario de Biotecnología, Léon, Spain, July

Fonseca, L.P., Fernandes, P., Lourenço, N.,

Cordas, C., Bernadino, S., Barros, D., Marques,

M., “Recent trends in enzyme and cell immobilization

by entrapment and encapsulation”, XVI International

Conference on Bioencapsulation, Dublin, Irland,

September

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

Prazeres, D.M.F., “Plasmid DNA purification by

phenyl boronate affinity chromatography”, 7th Euro-

pean Symposium on Biochemical Engineering Sci-

ence - ESBES7, Faro, Portugal, September

Lobato da Silva, C., dos Santos, F., Andrade, P.Z.,

Gonçalves, R., Almeida-Porada, G., Cabral, J.M.S.,

“Maximization of the ex-vivo expansion of human

hematopoietic stem/progenitor cells by direct contact

culture with mesenchymal stem cells", SBE's First

International Conference on Stem Cell Engineering,

San Diego, USA, January

Madeira, C., Ferreira, J.A.B., Andrade, S., Cabrita,

G.J.M., Costa, S.M.B., Melo, E.P., “Fluorescence

studies of the pair CFP-YFP: FLIM-FRET in vitro

studies and expression in neuronal cells to probe the

prion protein at the cell surface”, Focus on Micros-

copy, Osaka-Awaji, Japan, April

Martins, V.C., Cardoso, F.A., Fonseca, L.P.,

Freitas, P.P., “Femptomolar sensitivity for magneti-

cally assisted DNA hybridisation”, 1st International

Conference from Nanoparticles and Nanomaterials to

Nanodevices and Nanosystems - IC4N 2008,

Halkidiki, Greece, June

Martins, V.C., Cardoso, F.A., Loureiro, J., Ger-

mano, J., Cardoso, S., Ferreira, R., Fonseca, L.P.,

Sousa, L., Piedade, M.S., Freitas, P.P., “Integrated

spintronic platforms for biomolecular detection, sepa-

ration and counting”, Joint European Magnetic Sym-

posia - JEMS08, Dublin, Ireland, September

Monteiro, G.A., “Bringing DNA vaccines closer to the

bedside”, Vaccines Europe, Brussels, Belgium, De-

cember

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

Gouvêa, A., Chu, V., Conde, J.P., “Integrated opto-

electronic detection of molecular recognition reac-

tions”, 7th European Symposium on Biochemical Engi-

neering Science - ESBES7, Faro, Portugal, Septem-

ber

Rosa, P.A.J., Azevedo, A.M., de Vries, J., Korpo-

raal, R., Verhoef, H.J., Visser, T.J., Aires-Barros,

M.R., “Optimisation of affinity-enhanced purification of

antibodies using aqueous two-phase extraction”, 12th

International Symposium on Preparative and Indus-

trial Chromatography and Allied Techniques - SPICA

2008, Zurich, Switzerland, September - October

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

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

stage-enhanced recovery of human antibodies by

aqueous two-phase extraction”, 7th European Sympo-

sium on Biochemical Engineering Science - ESBES

7, Faro, Portugal, September

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

Barros, M.R., “Purification of human antibodies using

affinity aqueous-two phase systems”, 10th Interna-

tional Chemical and Biological Engineering Confer-

ence - CHEMPOR 2008, Braga, Portugal, September

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

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

stage aqueous-two phase extraction of human anti-

bodies”, Jahrestreffen des ProcessNet – Fachauss-

chusses Extraktion und des Arbeitskreises Phytoex-

trakte, Clausthal-Zellerfeld, Germany, April

Samatou, J. A., Wentink, E.A., Hoffmann, A., Rosa,

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

W., Górak, A., “Modellierung und simulation der

mehrstufigen extraktion von monoklonalen

antikörpern in wässrigen zweiphasensystemen”,

Jahrestreffen der Fachgemeinschaft Prozess-,

Apparate- und Anlagentechnik, Bad Honnef,

Germany, November

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

“Temperature-induced conformational changes of

pDNA and their influence on histidine-agarose reten-

tion: A circular dichroism study”, 7th European Sympo-

sium on Biochemical Engineering Science - ESBES7,


Vidinha, P., Lourenço, N.M.T., Carvalho, T., Brás,

A.R., Silva, T.S., Mukhopadhyay, A., Cordas, C.M.,

Romão, M.J., Dionisio, M., Cabral, J.M.S., Fonse-

ca, L.P., Afonso, C.A.M., Barreiros, S., "Ion jelly®:

A tailor-made conducting material for smart electro-

chemical devices", 7th International Symposium on

Polyelectrolytes - Polyelectrolytes 2008, Coimbra,

Portugal, June

29

20 08 Annual Report

National Conferences

Andrade, P.Z., Temtem, M., dos Santos, F.,

Lobato da Silva, C., Aguiar-Ricardo, A., Cabral,

J.M.S., “"Green" chitosan membranes for the ex-vivo

expansion of human bone marrow mesenchymal

stem cells”, 3rd Annual International Meeting of the

Portuguese Society for Stem Cells and Cellular

Therapies, Faro, Portugal, April

Diogo, M.M., Fernandes, A.M., Fernandes, T.G.,

Lobato da Silva, C., Henrique, D., Dordick, J.S.,

Cabral, J.M.S., "Large-scale and nano-scale

approaches for the expansion of mouse embryonic

stem (mES) cells", 3rd Annual International Meeting

of the Portuguese Society for Stem Cells and Cellu-

lar Therapies, Faro, Portugal, April

dos Santos, F., Lobato da Silva, C., Andrade,

P.Z., Miranda, N., Teixeira, G., Guimarães, A., Fer-

reira, I., Rodriguez, E., Oiveira, J., Trindade, H.,

Abecassis, M., Cabral, J.M.S., “Treatment of steroid

and extracorporeal resistant acute graft-versus-host

disease with donor mesenchymal stem cells”, 3rd

Annual International Meeting of the Portuguese Soci-

ety for Stem Cells and Cellular Therapies, Faro, Por-

tugal, April

Eibes, G., Fernandes, A.M., Diogo, M.M., Lobato

da Silva, C., Cabral, J.M.S., “Modeling of mouse

embryonic stem cell expansion in a stirred culture

system”, 3rd Annual International Meeting of the Por-

tuguese Society for Stem Cells and Cellular Thera-

pies, Faro, Portugal, April

Estrela, N.L., Chen, L.Y., Gunna, S.M.C., Cabrita,

G.J.M., Otzen, D.E., Melo, E.P., “Folding and amy-

loidosis of proteins: The prevention of amyloidosis by

osmolytes”, XVIth National Congress of Biochemis-

try, São Miguel, Portugal, October

Fernandes, P., “Biocatálise industrial”, Workshop IV

Dia de Biologia Marinha e Biotecnologia, Peniche,

Portugal, May

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

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

zed biocatalyst for inulin and sucrose hydrolysis”,

Carbohydrates as Organic Raw Materials V - CORM

V, Lisbon, Portugal, February

Lourenço, N.M.T., Vidinha, P., Brás, A.R.,

Carvalho, T., Silva, T.S., Mukhopadhyay, A.,

Cordas, C., Dionisio, M., Romão, M.J., Cabral,

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

S., "Ion Jelly®- A tailor-made Material for

Electrochemical Applications", 1st Portuguese Young

Chemists Meeting, Lisbon, Portugal, October

Marques, M.P.C., Caramujo, M.J., de Carvalho,

C.C.C.R., "Bioremediação de amostras da Base

Naval de Lisboa", Jornadas do Mar 2008, Escola

Naval, Lisbon, Portugal, November

International Conferences

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

Barros, M.R., “Aqueous two-phase extraction of hu-

man antibodies”, Bioprocess Technology Europe:

Development and Production of Antibodies, Vaccines

and Gene Vectors, Amsterdam, The Netherlands,

June-July

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

lysed transesterification of triolein using microencap-

sulated cutinase in AOT-reversed micelles for bio-

diesel production”, 4th International Congress on Bio-

catalysis - BIOCAT 2008, Hamburg, Germany, Au-

gust-September

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

mization of biodiesel production by triolein transesteri-

fication using microencapsulated cutinase in AOT-

reversed micelles”, 7th European Symposium on Bio-

chemical Engineering Science – ESBES 7, Faro, Por-

tugal, September


P., Fonseca, L.P., Cabral, J.M.S., “Comparison of

cutinase bioencapsulation in sol-gel and PVA versus

lyophilized form on biosynthesis of ethyl caproate in

organic solvent”, XVI International Conference on

Bioencapsulation, Dublin, Ireland, September

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

Weiss, C., Landfester, K., “Mini-emulsion and or-

ganic solvent media in biosynthesis of flavours esters

- studies on stepwise addition of substrates”, 4th Inter-

national Congress on Biocatalysis – BIOCAT 2008,

Hamburg, Germany, August-September

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

L.P., “Preparation of micro-porous silica xerogel with

magnetic properties and its application to penicillin G

acylase immobilization”, XVI International Conference

on Bioencapsulation, Dublin, Ireland, September

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

Pinho, M.N., “Membrane fractionation of galacto-

oligosaccharides produced enzymatically in lactose

solutions”, Engineering with Membranes - Membrane

Processes: Development, Monitoring and Modelling;

From the nano to the macroscale - EWM2008, Vale

de Lobo, Portugal, May

Cabeça, R., Prazeres, D.M.F., Chu, V., Conde, J.P.,

“Electrical and Chemical Control of Surfaces for DNA

Immobilization and Hybridization”, 2008 Materials

Research Society Spring Meeting, São Francisco,

USA, March

Carapuça, E., Azzoni, A., Prazeres, D.M.F., Mon-

teiro, G.A., Mergulhão, F.J.M., “In vivo plasmid sta-

bility: production host versus target cell. Implications

on DNA vaccine development and protein produc-

tion”, 5th Recombinant Protein Production Meeting: An

integrate view of host physiology, Sardinia, Italy, Sep-

tember

Carvalho, J.A., Monteiro, G.A., Atouguia, J., Pra-

zeres, D.M.F., Rodgers, J., “Developing a vaccine

for African trypanosomiasis: only wishful thinking or a

definite possibility?”, Infectious diseases of the nerv-

ous system: pathogenesis and worldwide impact,

Paris, France, September

Carvalho, J.A., Rodgers, J., Atouguia, J., Pra-

zeres, D.M.F., Monteiro, G.A, “Screening of DNA

vaccines prototypes encoding antigen targeting se-

quences against African Trypanosomiasis”, Vaccine

Technology, Albufeira, Portugal, June

Poster Presentations

31

20 08 Annual Report

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

Afonso, C.A.M., Barreiros, S., Cabral, J.M.S.,

Fonseca, L.P., “Immobilization of proteins in new-

ionic-conducting-based materials - Ion Jelly®”, XVI

International Conference on Bioencapsulation, Dub-

lin, Ireland, September

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

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

Cabral, J.M.S., "Electrochemical detection of immobi-

lized proteins in ion jelly films", 7th International Sym-

posium on Polyelectrolytes - Polyelectrolytes 2008,

Coimbra, Portugal, June

de Carvalho, C.C.C.R., Marques, M.P.C., "Bioreme-

diation of samples from a naval base", 7th European

Symposium on Biochemical Engineering Science -

ESBES 7, Faro, Portugal, September

dos Santos, F., Lobato da Silva, C., Andrade, P.Z.,

Abecasis, M., Cabral, J.M.S, “Effect of hypoxia on

human mesenchymal stem cell (MSC) expansion and

metabolism”, 7th European Symposium on Biochemi-

cal Engineering Science – ESBES 7, Faro, Portugal,

September

dos Santos, F., Lobato da Silva, C., Andrade, P.Z.,

Abecasis, M., Cabral, J.M.S, “Effect of hypoxia on

human mesenchymal stem cell (MSC) expansion and

metabolism”, 36th International Annual Scientific

Meeting, ISEH Society for Hematology and Stem

Cells, Boston, USA, July

Fernandes, P., Cattorini, S., Carvalho, F.,

Marques, M.P.C., Bernardino, S., Maduro, F.,

Badenes, S., Barros, D., Carvalho, C.C.C.R.,

Fonseca, L.P., Cabral, J.M.S., “A multipurpose

hydrogel system for biocatalyst immobilization”, 10th

International Chemical and Biological Engineering

Conference - CHEMPOR 2008, Braga, Portugal, Sep-

tember

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

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

mobilization using PVA based hydrogel particles”, 7th


Science - ESBES 7, Faro, Portugal, September

Fernandes, P., Cattorini, S., Cabral, J.M.S.,

“Enzymatic inulin hydrolysis using PVA-based

matrices”, 10th International Chemical and Biological

Engineering Conference - CHEMPOR 2008, Braga,

Portugal, September

Fernandes, A.M., Diogo, M.M., Lobato da Silva, C.,

Henrique D., Cabral, J.M.S., “Mouse embryonic


culture system”, SBE's First International Conference

on Stem Cell Engineering, San Diego, USA, January

Fernandes, T.G., Kwon, S.J., Lee, M.Y., Clark,

D.S., Cabral, J.M.S., Dordick, J.S., “An on-chip, cell-

based microarray immunofluorescence assay for high

-throughput analysis of target proteins” 7th European





J.M.S., Dordick, J.S., "Expansion and neural com-

mitment of mouse embryonic stem cells on a microar-

ray platform", SBE's First International Conference on

Stem Cell Engineering, San Diego, USA, January



J.M.S., Dordick, J.S., "A high-throughput 3D cell

microarray to study stem cell fate", 6th ISSCR Annual

Meeting, Philadelphia, USA, June

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

Barros, M.R., “Temperature induced affinity aqueous

two-phase extraction for the purification of human

immunoglobulin G”, 7th European Symposium on Bio-

chemical Engineering Science – ESBES 7, Faro, Por-

tugal, September


Barros, M.R., “Optimisation of temperature induced

aqueous-two phase extraction of human antibodies”,

28th International Symposium on the Separation of

Proteins, Peptides and Polynucleotides – ISPPP

2008, Baden-Baden, Germany, September

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



12th International Symposium on Preparative and

Industrial Chromatography and Allied Techniques -

SPICA 2008, Zurich, Switzerland, September-

October

Fonseca, L.P., Barros, D.P.C., “Biosynthesis of ethyl

caproate and other short alkyl esters catalyzed by

cutinase”, COST 865 - Spring 2008 Workshop, Bioen-

capsulation Sciences to Applications, Ljubljana, Slo-

venia, April

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

Santos, J.A.L., “Recovery of Plasmid DNA from Pre-

treated Lysates Using Tangential Flow Filtration”, 7th


Science – ESBES 7, Faro, Portugal, September

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

Santos, J.A.L., “Purification of plasmid DNA vectors

by hydrophobic interaction chromatography using

sodium citrate in the mobile phase”, 28th International

Symposium on the Separation of Proteins, Peptides

and Polynucleotides – ISPPP 2008, Baden-Baden,

Germany, September

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

D.M.F., Santos, J.A.L., “Intermediate Recovery of

Plasmid DNA Using Tangential Flow Filtration”, Engi-

neering with Membranes 2008, Vale do Lobo, Portu-

gal, May

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

Prazeres, D.M.F., “Plasmid DNA purification by

phenyl boronate affinity chromatography”, 28th Inter-

national Symposium on the Separation of Proteins,

Peptides and Polynucleotides – ISPPP2008, Baden-

Baden, Germany, September


Miranda, N., Teixeira, G., Guimarães, I.,

Rodriguez, E., Oliveira, J., Trindade, H., Cabral,

J.M.S, Abecasis, M., “Treatment of steroid and

extracorporeal resistant acute graft-versus-host

disease with donor mesenchymal stem cells”, 36th

International Annual Scientific Meeting, ISEH Society

for Hematology and Stem Cells, Boston, USA, July


Miranda, N., Teixeira, G., Guimarães, I., Rodri-

guez, E., Oliveira, J., Trindade, H., Cabral, J.M.S,

Abecasis, M., “Treatment of steroid and

extracorporeal resistant acute graft-versus-host

disease with donor mesenchymal stem cells”, 7th


Science – ESBES 7, Faro, Portugal, September

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

Afonso, C.A.M., Barreiros, S., Cabral, J.M.S.,

Fonseca, L.P., “Ion Jelly® - A suitable material for

biosensing detection", 7th International Symposium on

Polyelectrolytes – Polyelectrolytes 2008, Coimbra,

Portugal, June

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

novel online-monitoring system for oxygen in non-

conventional fermentations”, 7th European Sympo-

sium on Biochemical Engineering Science – ESBES


33

20 08 Annual Report

Marques, M.P.C., Carvalho, F., Claudino, M.J.C.,

Fernandes, P., Cabral, J.M.S., “Multi-step biotrans-

formations throughout scales”, 7th European Sympo-

sium on Biochemical Engineering Science - ESBES


Martins, V.C., Cardoso, F.A., Cardoso, S.,

Fonseca, L.P., Freitas, P.P., “Biological detection

limit of a SV-based biochip for pathogenic analysis”,

Nanoiberian Conference – Nanospain 2008, Braga,

Portugal, April

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

“Enzymatic resolution and separation of sec-alcohols

based on sustainable acylating agents”, 10th Interna-

tional Chemical and Biological Engineering Confer-

ence, CHEMPOR 2008, Braga, Portugal, September

Monteiro, G.A., Carvalho, J., Rodgers, J., Praz-

eres, D.M.F., “Screening of DNA Vaccine Prototypes

Encoding Antigen Targeting Sequences Against

Sleeping Sickness”, Vaccine Technology II, Albufeira,

Portugal, June

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

Aires-Barros, M.R., “Partitioning of Canavalia brasil-

iensis lectin in polyethylene glycol – sodium citrate

aqueous two-phase systems”, 7th European Sympo-

sium on Biochemical Engineering Science – ESBES


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

“Plasmid DNA instability mediated by direct repeats

and type-2 insertion sequences”, EMBO conference

Recombination Mechanisms, Il Ciocco, Italy, May

Pereira, A.T., Pimentel, A., Loureiro, J., Freitas,

P.P., Chu, V., Prazeres, D.M.F., Conde, J.P.,

“Colorimetric detection of antigen-antibody recogni-

tion in a microfluidic channel with an integrated photo-

diode”, Eurosensors XXII, Dresden, Germany, Sep-

tember

Prazeres, D.M.F., Azzoni, A.R, Freitas, S.S., San-

tos, J.A.L., Monteiro, G.A., “On the design and pro-

duction of more stable and efficient plasmid DNA

vectors”, Vaccine Technology II, Albufeira, Portugal,

June

Prazeres, D.M.F., “Prediction of Diffusion Coefficients

of Plasmids”, 28th International Symposium on the

Separation of Proteins, Peptides and Polynucleotides

– ISPPP 2008, Baden-Baden, Germany, September

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

process for the manufacturing of plasmid biopharma-

ceuticals: alkaline lysis, microfiltration, salt precipita-

tion and hydrophobic interaction chromatography”,

Bioprocess Technology Europe: Development and

Production of Antibodies, Vaccines and Gene Vec-

tors, Amsterdam, The Netherlands, June-July

Raiado-Pereira, L., Santos, J.A.L., Prazeres,

D.M.F., Mateus, M., “Prospective hydrophobic inter-

action and pseudo-affinity membranes for chroma-

tographic purification of plasmid DNA”, 7th European



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

tinho, J.M.G., Taipa, M.A., “Biomimetic affinity poly-

mer-particles for antibody recognition”, 7th European



National Conferences

Marques, M.P.C., Carvalho, F., Monteiro, G.A.,

Cabral, J.M.S., Fernandes, P., “Maintenance of

catalytic activity in complex steroid bioconversion”,

XVI National Congress of Biochemistry, Ponta

Delgada, S. Miguel, Portugal, October

Malcolm Lilly Award

Tiago G. Fernandes was awarded with the 2008 Mal-

colm Lilly award on the occasion of the 7th European

Symposium on Biochemical Engineering Science

(ESBES-7, September 7-10, 2008), in Faro, Portugal,

for the contribution “Exploring stem cell fate using

three-dimensional cellular microarrays”, supervised

by J.M.S. Cabral (IBB-IST) and J.S. Dordick

(Renssealaer Polytechnic Institute, Troy, NY, USA).

Best Oral Presentation

Martins, V.C., Cardoso, F.A., Loureiro, J., Germano,

J., Cardoso, S., Ferreira, R., Fonseca, L.P., Sousa,

L., Piedade, M.S., Freitas, P.P., “Integrated spintronic

platforms for biomolecular detection, separation and

counting”, Joint European Magnetic Symposia -

JEMS08, Dublin, Ireland.

Best Poster Awards




28th International Symposium on the Separation of

Proteins, Peptides and Polynucleotides - ISPPP2008,

Baden-Baden, Germany.

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



12th International Symposium on Preparative and

Industrial Chromatography and Allied Techniques -

SPICA 2008, Zurich, Switzerland.

Raiado-Pereira, L., Santos, J.A.L., Prazeres, D.M.F.,

Mateus, M., “Prospective hydrophobic interaction and

pseudo-affinity membranes for chromatographic puri-

fication of plasmid DNA”, 7th European Symposium on

Biochemical Engineering Science - ESBES 7, Faro,

Portugal,

UTL/Santander Totta Scientific Award

Professor Joaquim M. Sampaio Cabral won the sci-

entific award from the Technical University of Lisbon

(UTL) in Biological Engineering, Biochemistry and

Biotecnology area. This award is the recognition of

the scientific work and career and is sponsored by

Santander Totta bank. The award ceremony took

place on the 4th December 2008 at IST.

FLAD Educational Innovation Awards

Cláudia Lobato da Silva (Professor at IST) was distin-

guished with the First Annual FLAD Educational Inno-

vation Award. This award from the Luso-American

Foundation based in Lisbon recognizes the extraordi-

nary efforts of Portuguese faculty members in the MIT

-Portugal Program to create educational programs at

the highest standards of excellence.

Idea to Product Competition

Nuno Lourenço won the 2nd Place on 6th Annual Idea

to Product Competition -Cockrell School of Engineer-

ing Int'l Challenge with the project “Ion Jelly®- Thin-

Film Batteries”, held at University of Texas, Austin.

BES Innovation Award

Nuno Lourenço was distinguished with the BES Inno-

vation award, area of Energy, with the Project “Ion

Jelly®- Development of Thin-Film Flexible Batteries”.

Young researcher Award

M. Margarida Diogo and Cláudia A. M. Lobato da

Silva received Honourable Mentions in “Prémio Jov-

ens Investigadores UTL/Deloitte” in the scientific area

of Biological Engineering, Biochemistry and Biotech-

nology.

Prizes

35

20 08 Annual Report

Staff

Faculty

Joaquim M. S. Cabral

M. Raquel Aires-Barros

Luís P. Fonseca

D. Miguel F. Prazeres

Marília Mateus

Gabriel A. Monteiro

José L. A. Santos

Cláudia Lobato da Silva

M. Ângela Taipa

Research Scientists

Ana M. Azevedo

M. Margarida Diogo

Pedro Fernandes

Post-doctoral Fellows

Ricardo Baptista

Cristina Cordas

Gemma Eibes

Nuno Lourenço

Catarina Madeira

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

João Guerreiro

David Malta

Marco Marques

Sofia Martins

Verónica Martins

Kelany Nascimento

Pedro Oliveira

Carlos Rodrigues

Paula Rosa

Francisco Santos

Cláudia Silva

Ana Isabel Silva

Isabel Sousa

Master Students

Joana Boura

Joana Lima

Salomé Magalhães

Research Assistants

Luís Raiado-Pereira

Evandro Tavares

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

2008 - fenix.tecnico.ulisboa.pt · the design of surfactant-stable cutinase mutants by direct...

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