2008 - fenix.tecnico.ulisboa.pt · the design of surfactant-stable cutinase mutants by direct...
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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
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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.
Selected Publications
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.
Selected Publications
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
Costa, L., Brissos, V., Lemos, F., Ribeiro, F.R.,
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
Costa, L., Brissos, V., Lemos, F., Ribeiro, F.R.,
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
Barros, D.P.C., Bernardino, S.M.S.A., Fernandes,
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
stem cell expansion in a microcarrier-based stirred
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,
Faro, Portugal, September
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., "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,
Faro, Portugal, September
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
Barros, D.P.C., Bernardino, S.M.S.A., Fernandes,
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
European Symposium on Biochemical Engineering
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
stem cell expansion in a microcarrier-based stirred
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
Symposium on Biochemical Engineering Science –
ESBES 7, Faro, Portugal, September
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., "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
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", 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
Ferreira, I.F., Azevedo, A.M., Rosa, P.A.J, Aires-
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-
Barros, M.R., “Optimisation of temperature induced
aqueous-two phase extraction of human antibodies”,
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
European Symposium on Biochemical Engineering
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
Lobato da Silva, C., dos Santos, F., Andrade, P.Z.,
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
Lobato da Silva, C., dos Santos, F., Andrade, P.Z.,
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
European Symposium on Biochemical Engineering
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
7, Faro, Portugal, September
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
7, Faro, Portugal, September
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
7, Faro, Portugal, September
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
Symposium on Biochemical Engineering Science –
ESBES 7, Faro, Portugal, September
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
Symposium on Biochemical Engineering Science –
ESBES 7, Faro, Portugal, September
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
Ferreira, I.F., Azevedo, A.M., Rosa, P.A.J, Aires-
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 - ISPPP2008,
Baden-Baden, Germany.
Ferreira, I.F., Rosa, P.A.J., Azevedo, A.M., Aires-
Barros, M.R., “Optimisation of temperature induced
aqueous-two phase extraction of human antibodies”,
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