STRATEGIC PROGRAM

Thematic Line 1

 

Stem Cell Engineering: Tools and Applications

 

The main goal of this thematic line is to achieve new developments in Stem Cell-based therapies, as well as treatment options and diagnostics by Stem Cell bioprocessing. Ultimately this will promote the development of novel Advanced Therapy Medicinal Products (ATMPs) and in vitro platforms with high technological impact in the healthcare sector.

 

1. Clinical Manufacturing of Stem Cell-based Therapies

Our focus is the manufacturing of clinical-grade Stem cells/progenitors to be used as ATMPs for first-in-human studies in collaboration with our clinical partners, specifically human Mesenchymal Stem Cells (MSC) for Acute Myocardial Infarction (AMI) treatment and Umbilical Cord Blood Hematopoietic Stem/Progenitor Cells expanded in co-culture with MSC to be infused in hemato-oncological patients. The main goal is to optimize our previously microcarrier-based platform for MSC cultivation and its GMP implementation. A systematic analysis of Stem/Progenitor cells will be pursued through culture parameters control and their impact on cell product potency. Specifically we aim to set-up a quality control platform by identifying reliable potency biomarkers to assure safe and clinically effective expanded cells. We propose to generate pre-clinical data for the development of both ATMPs and dosages to be tested will be determined based on potency levels, according to EMA/INFARMED.

 

2. Multi-Scale Strategies for Human Pluripotent Stem Cell Bioprocessing

The main objective is to develop Integrated Bioprocesses to overcome the main technological barriers that presently limits the application of human Pluripotent Stem Cells (PSC), embryonic (ESC) and induced pluripotent (iPSC), and their derivatives. Large-scale production of PSC-derived neural precursors and cardiomyocytes is focused to leverage the emergence of alternative therapies and screening tools for neurodegenerative and cardiovascular diseases. Bioreactors are used to integrate PSC expansion and differentiation based on tightly-controlled microenvironments using tailor-made niche-inspired microcarriers. Modeling is used to uncover complex interactions between different niche components and systems biology approaches are applied for better understanding of pluripotency at the molecular level. Affinity-based separation methods are integrated with bioreactors for purification of PSC derivatives, including the depletion of tumorigenic PSC.

 

3. Ex-vivo Gene Therapy for Regenerative Medicine Applications

Novel gene carriers (minicircles) have been tested into Stem/Progenitors cells to prolong gene expression and augment cell survival while guaranteeing extremely low probability of genome integration. The main focus is the development of Gene Activated Matrices, composed by hydrogels embedding minicircles, encoding genes involved in specific differentiation fates. With this approach, these genes are released in a sustained mode into Stem/Progenitors cells and expressed at low levels assuring the formation of native-like tissue and/or able to promote endogenous repair in vivo. By using human MSC our main goal is to promote cartilage regeneration and angiogenesis in ischemic heart for cardiac regeneration.

 

4. Tailoring Biomaterials to Support Stem Cell Cultivation

The aim is to mimic the natural features of extracellular matrix to promote cell-cell and cell-material interactions and cellular adhesion, as well as to add artificial functions facilitating cell processing. The envisaged Key Enabling Technologies include i) Electrospun aligned nanofiber scaffolds and scalable plate&frame bioreactors, to direct cell organization and function targeting neural or cardiac regeneration ii) Encapsulation/hydrogel-based systems that will be tailored for chondrogenesis, iii) Microcarriers for Stem/Progenitors cells cultivation, and iv) stimulus-responsive materials facilitating cell harvesting or controlled delivery of small molecules and bio-macromolecules (genes and proteins).

 

Thematic Line 2

 

Advanced Bioprocess Engineering

 

The main objective of the Advanced Bioprocess Engineering thematic line is to design and develop the tools, technologies and materials required to support the biologically based manufacturing of the future. The focus is placed on solving current industrial bottlenecks and on developing new bioprocess engineering methodologies to translate recent scientific developments into new products and processes. IBB contributes to solve challenges in three major areas: 1. Harnessing microbial factories and enzymes, 2. Unlocking downstream processing bottlenecks and 3. Advancing bioprocess monitoring. These challenges are addressed by using and developing miniaturized platform technologies and methodologies compatible with a fast screening of microbial expression and enzymatic activities, and with the rapid definition of upstream and downstream operating conditions.

 

1. Harnessing microbial factories and enzymes: to use cell-free expression, engineer microbial strains and develop microscale experimentation to speed up the design and optimization of i) OOL-catalyzed cascade multi-reactions and ii) microbial production of minicircles, polyhydroxyalkanoates, OOLs and bioenergy based on BioFuel Cells. Emphasis is placed on the use of micro- and minibioreactors to screen for the best producer strains and reaction conditions. The different bioreactor configurations have been used aiming to provide the most adequate framework according to the stage of process development.

 

2. Unlocking downstream processing bottlenecks: to speed up the development and optimization of DSP of OOLs, mAbs and DNA minicircles by resorting to microscale experimentation and screening. Emphasis is placed on the use of microtiter plate formats (e.g. classic, membrane and deep-well plates) and microfluidics to screen for alternative unit operations like ATPE, nanomagnetic separations and membrane/monolithic chromatography. Multimodal ligands, tailor-made synthetic ligands and affinity tags (e.g. carbohydrate binding modules) have been used to improve biomolecule purification and function.

 

3. Advancing bioprocess monitoring: to develop miniaturized, enzyme-based analytical tools for the on-line monitoring of glucose, lactate, amino acids and alcohols during cell growth and of enzymatic activities during downstream processing. Efforts are also directed towards the implementation of spectroscopic techniques (e.g., vibrational, dielectric, 2D fluorescence) and system engineering approaches (whole-process analysis techniques) within the PAT framework as a means to enhance understanding and control of manufacturing process (QbD).

 

Thematic Line 3

 

Post-Genomic Technologies to boost Biological Sciences and Bioengineering Research

 

Post-genomic technologies, including functional and comparative genomics, transcriptomics, expression proteomics, metabolomics, lipidomics, and metagenomics combined with bioinformatic tools are essential in biotechnology and biomedical research and applications. These tools are crucial for the exploitation of Systems and Synthetic Biology strategies to gain full understanding of biological systems and to enable their redesign and construction in order to display functions more suited to specific biotechnological applications.

 

The main objective of this thematic line is to further the implementation of post-genomic approaches, providing the necessary support and leverage to iBB research activities in biological sciences and in bioengineering, in the framework of the other thematic lines. Our successful research programmes in the fields of Yeast Toxicogenomics and Microbial Pathogenomics will be pursued and extended to other relevant biological systems and problems. One of the challenges is the implementation/development of the necessary competitive post-genomics platform technologies to cement the Omics analyses and the systems biology level research already in place and to boost the other research lines. Another challenge is the implementation of sustainable research in the fields of: i) Synthetic Biology, given its unprecedented power to enable the construction and redesign of biological systems that display functions unknown in nature, and ii) Metagenomics, given its influence on how we view and study the microbial world and all the important contributions to many areas at the heart of iBB research programs.

 

To boost the research programs in the 3 other research lines, massive genome sequencing will contribute to the fulfilment of a wide-range of objectives. The implementation of a Next Generation Sequencing (NGS) platform will allow the fulfilment of the strategic plan. It suits the needs for: 1) massive sequencing of genomes of specific organisms that are on the focus of ongoing research; 2) massive sequencing associated to metagenomic projects; and 3) RNA sequencing for transcriptomic analysis. This genomics platform will be able to support several research activities, namely at the level of microbial metagenomics, related with microbial genome variation in pathogenesis, biotechnological applications, adaptive evolution research as well as in the stem cell field.

 

Another post-genomic tool of paramount importance to increase the competitiveness and visibility of our research and to support the work is the YEASTRACT database (www.yeastract.com). This web resource was originally developed to support the analysis of transcription regulatory associations in S. cerevisiae and resulted from a joint effort between the BSRG and computer scientists. The update and upgrade of this public database on gene and genomic regulation in yeast will be pursued.

 

As detailed in the YEASTRACT+: proposal for an e-infrastructure for research on gene and genomic regulation in yeasts, recently included in the Portuguese infrastructures roadmap, the YEASTRACT database is being extended to the S. cerevisiae pan-genome, considering all the variations in gene occurrence and expression that are found from strain to strain – and to other selected yeast species of biomedical and biotechnological interest (e.g. Candida glabata, C albicans, Zygosaccharomyces bailii), in a comparative genomics approach.

 

Metagenomics, as a powerful complementation of culture-dependent methods, is planned to be used in iBB projects in the fields of environmental microbiology and biotechnology to allow the identification of the microbial communities present, the interactions between its members, and catabolic genes and their regulation under the conditions existing in contaminated sites, in particular envisaging the bioremediation of soils contaminated with herbicides or of oil contaminated sites. Given the strategic research agenda towards an integrated Sea research, the launch of a marine metagenomics program will be conducted to complement the screen for novel marine microbial metabolites for biotechnological and biomedical applications.

 

The emerging multidisciplinary field of Synthetic Biology bridges biological sciences and bioengineering research, and is being explored to enable the construction and redesign of biological systems with applications in the scope of the activities of IBB, fromregulon-specific transcription factor engineering to improve yeast robustness to microbial cells´ engineering by redirecting metabolic fluxes for efficient production of novel metabolites for biotechnological and biomedical applications.

 

Thematic Line 4

 

Response and Resistance to Environmental Changes

 

This wide-spanning thematic line aims at understanding the complexity of cellular responses to environmental alterations and insults, which is one of the major challenges in Biology and is also crucial for successful biotechnological applications. In fact, the survival and performance of living cells depends on their ability to sense alterations in the environment and to appropriately respond to the new stressing situations by remodelling genomic expression. Also, cellular resistance to multiple drugs/xenobiotics is implicated in the failure of many therapeutic, food-preservation and crop protection actions but may help to improve the productivity of biotechnological processes.

 

This thematic line includes research programs aiming at uncovering the complexity of cellular responses to new situations, in particular to stress, which might be instrumental to improve biotechnological processes performance, food preservation and crop protection actions, and the control of drug resistance and microbial pathogenicity.

 

Mechanistic insights and a genome-wide view on the responses to selected environmental challenges have been gathered to identify critical genes, proteins and metabolites and to characterize new signalling pathways that are affected by, and respond to, a specific stress, and to identify molecular biomarkers of drug/toxicant exposure. A molecular systems-level approach, based on the combination of Omics analyses (transcriptomics, expression- and phospho- proteomics, chemogenomics, metabolomics, and lipidomics) with bioinformatics are used to get an integrative view on how microbial cells with important roles in biotechnology, human health, agriculture and the environment, respond and resist to selected drugs, toxic metabolites and other relevant environmental stresses. Suitable biological systems already on the focus of our current research, such as the eukaryote model S. cerevisiae, the opportunistic pathogenic yeasts C. albicans and C. glabrata, the acidic food spoilage yeast Zygosaccharomyces bailii, and Sinorhizobium meliloti, Burkholderia cepacia complex (Bcc) and Rhodococcus bacteria, which are human and plant pathogens, plant symbionts or environmentally important bacteria, will be examined.

 

The identification of candidate genes and signalling pathways involved in stress response and resistance is essential to: i) find targets for genetic manipulation to increase microbial strain robustness for biotechnological processes (emphasis on second generation bio-ethanol and wine fermentation-related challenges), ii) guide food preservation strategies (emphasis on weak acid preservation), iii) predict toxicological outcomes of exposure to environmental pollutants/pesticides (emphasis on microbial toxicogenomics and bioremediation of environmental pollutants), iv) increase crop robustness and productivity (emphasis on multixenobiotic resistance transporters and regulators, and on symbiosis between nitrogen fixing S. meliloti cells and leguminous plants), v) identify pesticide off-targets and their relationship with human diseases (emphasis on the Parkinson-linked agriculture fungicide mancozeb and different herbicides), vi) identify microbial virulence determinants (emphasis on bacterial isolates leading to persistent life-threatening respiratory infections in cystic fibrosis and on C. albicans and C. glabrata), vii) identify direct and off-target effects of drugs and predictive biomarkers of drug toxicity (emphasis on bacterial proteins as anticancer agents and relevant antifungal and antibacterial drugs), viii) assist stem cell engineering for regenerative medicine (emphasis on the response to cell culture conditions).

 




 

 

Research Units