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Molecular and Cellular Mechanisms of Disease

To investigate normal and deregulated systems and pathways that characterize the...

Molecular and Cellular Mechanisms of Disease

To investigate normal and deregulated systems and pathways that characterize the pathology of serious diseases and genetic disorders.

Despite the major advances in medicine, many diseases remain a challenge for both the individual and society. To improve prevention, diagnosis, prognosis and therapy, we need to understand both how the system functions normally and how this system is subverted in disease. The Molecular and Cellular Mechanisms of Disease (MCMD) thematic strand investigates a range of systems, processes and mechanisms that underlie several clinically and economically important diseases and disorders. As such, several collaborative research projects have been developed as a joint effort between a number of research groups within the CBMR. This effort ensures that these complementary research groups are included within various national and international research efforts.

The derivation of human embryonic stem cells and induced pluripotent stem cells has created great interest due to their potential ability to differentiate into any cell type of the adult body. Pluripotent stem cells can be cultured in vitro and expanded indefinitely. By manipulating the substrate and culture conditions, the cells can be induced to abandon the pluripotent state and commit to a given germ layer. Further manipulation allows differentiation to multipotent progenitors and eventually to mature differentiated cell types. The production of large numbers of specific differentiated cell types for transplantation has now become a possibility within reach, opening the door to the development of therapies for diseases that currently have little or no treatment. At the CBMR within the RMP, we will focus on cardiovascular disease, neurodegenerative disorders, diabetes and lysosomal storage disorders. In particular, our goal is to produce cardiomyocytes for cardiac cell therapy, neuronal cell types for neurodegenerative disease, insulin producing cells for diabetes and the creation of cell models of lysososmal storage disorders that will facilitate the development of therapeutic compounds for the treatment of these serious diseases.

On the path from bench to bedside, a long list of technical challenges must be overcome in order to make regenerative medicine a clinical reality. It will be crucial to obtain a thorough understanding of the molecular basis of pluripotency and the regulatory mechanisms involved in maintaining the pluripotent state. To this end, the RMP is developing methods to culture large amounts of these cells in bioreactors while maintaining their genomic integrity and pluripotency.

In addition to the production of specific cell types, specific disease models are also being developed within the RMP to be used to investigate the underlying mechanism of disease, allowing the development of novel therapeutic compounds. This approach involves taking fibroblasts from patients with genetic disorders and reprogramming them to the pluripotent state. In a later step within the RMP, these cells are differentiated to disease relevant cell type(s) for basic mechanistic studies and as a platform to develop novel therapies. Within the RMP, this approach will be applied to both cardiovascular diseases and lisosomal storage diseases.

MCMD Main Research Lines

Cancer

Cancer is a disease with great individual and societal impact, and a major topic of MCMD research. The regulation of proliferation and dedifferentiation are central to cancer, and several research projects are focused on these processes. The role of overexpressed proteins (e.g., Hes1), the incorrect localization of transcription factors (e.g., FOXO, p53), aberrant cell signalling, deregulation of onco-, and tumor-suppressor proteins, tissue invasion, metastasis, protein-microenvironment interactions, antioxidant protein interactions and signalling, inflammation and chemotherapy resistance. To complement this fundamental research, network-based approaches are also incorporated to identify the cellular pathways that are involved in the interactions between the tumour and surrounding stromal cells. The MCMD also examines the mechanisms underlying cancer susceptibility and to identify new risk factors and gene loci for cancer. Additional CBMR projects examine the role of cis-regulatory transcription variation affecting tumour biology and the response to chemotherapeutic drugs. All of these important areas are aggressively investigated within the MCMD.

Developmental and genetic diseases

The development of the heart is a finely regulated, critical process. When this process is perturbed children are born with congenital heart defects. The MCMD is focused on understanding the regulatory mechanisms that drive cardiac progenitor cell differentiation and how alterations in this process yields cardiac defects. The MCMD has also developed cellular models of lysosomal storage disorders (e.g., Gaucher’s disease) to investigate the enzymes responsible for these diseases and to explore molecular methods to restore normal enzymatic activity. The MCMD is also examining the influence of cholesterol content on cell membrane homeostasis, fluidity and on enzymatic (ATPase) activity, critical to understand the development of cell membrane-based pathologies such as cancer, lysosomal storage disorders and atherosclerosis. The MCMD is also examining diabetic retinopathy and the factors that promote retinal neovascularization, a critical complication associated with diabetes.

Developmental and neurodegenerative brain disorders

Developmental dyslexia is one of the most common neurodevelopmental disorders with significant consequences for both the individual and society in terms of educational under-achievement, loss of professional opportunity, social-emotional and behavioural problems in adulthood resulting from chronic school failure. Dyslexia shows comorbidity with several other neurodevelopmental disorders (e.g., attention-deficit/hyperactivity disorder, specific language impairment). Current work within the MCMD is focused on the characterization of the cognitive profiles of poor readers in Portugal. The research on implicit learning is in certain respect world leading and the research efforts has recently characterized an association between the FOXP2-regulated gene CNTNAP2, implicit learning and the activity levels in Broca’s region (a classical neocortical language region) measured with fMRI. The CBMR has developed a unique AGL paradigm based on the structural mere-exposure effect in combination with preference/grammaticality classification which we are investigating with eye-tracking, FMRI, and EEG methodology. The role of implicit learning in dyslexia and language development, more generally, is currently a strong research focus and is being conducted with all modern tools available in cognitive neuroscience and functional neuroimaging. Neurodegenerative disorders are a growing problem in the aging populations of modern society. MCMD research is dedicated to the understanding of the molecular mechanisms governing the structure and function of proteins implicated in such disorders. Research is underway within the MCMD to examine the mutated prion protein that, in infected cells, aggregates and triggers neuronal death. Pathway deregulation in neurodegenerative disease is also being investigated through network-based computational analyses. Further MCMD research is dedicated to the clarification of the mechanisms that regulate neural stem cell proliferation and differentiation (neurogenesis) involving brain injury and neuro-inflammation, including epilepsy and stroke.

Host-pathogen interactions in disease

Pathogenic and non-pathogenic microorganisms play a vital role in human health and disease. Microbes not only cause infectious disease but also have a significant influence on body homeostasis and the organism’s response to other diseases. Within the CBMR, the MCMD research focuses on virulence factors that assist bacterial pathogens to circumnavigate the host defensive barriers. Other studies focus on the characterization and function of the intestinal microbiota in diabetes and autoimmune disorders. The MCMD is also investigating the role of host gene polymorphisms that encode drug metabolizing enzymes and transporters that effect the efficiency and safety of therapeutics directed against pathogens (e.g., Plasmodium [malaria]).

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Molecular Biomedicine and Technology

To investigate normal and deregulated systems and pathways that characterize the...

Molecular Biomedicine and Technology

To investigate normal and deregulated systems and pathways that characterize the pathology of serious diseases and genetic disorders.

The Molecular Biomedicine and Technology (MBT) area focus on the study of the properties and interactions of biomedically relevant molecules, and on the design and development of novel biomolecular devices. In general terms, the MBT integrates molecular engineering and biophysical characterization to develop better systems for detection, diagnostics, and delivery of pharmaceuticals.

One of the characteristics of this thematic area is that the MBT produces the needed biomolecules for the systems under study. The MBT has extensive expertise as well as the required technology for production and purification of such biomolecules, in particular large-scale protein expression, purification and analysis followed by the biophysical characterization of proteins and lipids.

MBT main research lines

Development and characterization of novel polymeric drug delivery systems

Polymers of either natural or synthetic origin are used to produce vehicles for the delivery of molecules of interest, for example proteins, antigens, genetic material (DNA, siRNA) and low molecular weight drugs that face delivery limitations. Particular focus within the MBT is directed towards systems that address the treatment of pulmonary diseases, such as tuberculosis and cystic fibrosis, and the ocular delivery of gene expressing systems targeting the retinal area.
Dynamics and structure of membranes

The MBT is also examining biomembrane structure, organization and dynamics comprising the influence of key components such as cholesterol, known to be involved in many clinically important pathologies. Understanding how protein and solutes partition into lipid bilayers is also of interest within the MBT due to its relevance to membrane properties and function.
Protein nanoparticles for molecular therapy

Protein nanoparticles are developed within the MBT for the targeted delivery of proteins to treat disease. Based on the full virus genome the MBT focuses on the selection and optimization of gene constructs responsible for nanoparticle assembly. These nanoparticles are further engineered within the MBT to incorporate scFvs and single domain recombinant antibodies as therapeutic motifs and to present specific target motifs at their surface. We develop such systems in order to generate nanodelivery systems suitable for further manipulation and for targeted molecular delivery in most relevant disease settings, in particular HIV1.

Development of biosensor technology driven to biomedical applications

New scientific instruments and techniques including acoustic, optical and microelectronic transducers are developed within the MBT for the study of biomedical relevant interactions. This includes HIV antigens and single chain antibodies that are integrated with engineering principles and concepts enabling the MBT to design novel biosensing devices. Biosensors are also being developed to assess the mechanism of cell adhesion and differentiation. Based on the propagation of acoustic waves, the MBT has developed models that describes the biosensor signal as a response to the morphological alteration of the cell during adhesion. Such sensors are being developed for detection of the effect of drugs and also to optimize and evaluate the effect of differentiation factors The MBT is also screening of transcription factors using biosensors, addressing protein-DNA interactions and conformational changes. A prototype technique integrating an acoustic sensor and a flow calorimeter (QCMHCC) is being developed within the MBT for the rapid and complete thermodynamic characterization of biomolecular interactions, aimed at various applications in biomedicine, in particular drug screening/design.

Protein folding in human disease

Protein folding is crucial to the study of human disease that arises from protein misfolding, the storage and use of therapeutic proteins to treat disease, the development of novel protein delivery systems and the application of proteins as bio-sensors. The study of oxidative protein folding in the endoplasmic reticulum is particularly important within neurological disorders. Studies of protein folding, stability and misfolding are conducted for several proteins, including the prion protein involved in prion diseases, haemoglobin, oxidoredutases, and small model proteins such as bovine serum albumin.

Molecular Modelling and Simulation

The MBT also incorporates computational and bioinformatics studies that significantly support, complement and allow the in-depth analysis of our on-going projects. Particular focus within the MBT has been the examination of in-silico protein-protein interactions, ligand-protein docking studies and relevance of protein electrostatics to protein structure and function.

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Regenerative Medicine

The use of cells and biomaterials to reconstruct cell compartments, tissues and ...

Regenerative Medicine

The use of cells and biomaterials to reconstruct cell compartments, tissues and organs affected by trauma, genetic mutations, disease or age.

The Regenerative Medicine Research Program (RMP) centres on the use of cells and biomaterials to reconstruct cell compartments, tissues and organs affected by trauma, genetic mutations, disease or age. In essence, its aim is to replace, renew, regenerate or reconstruct tissues, organs or body parts when required, an endeavour that will require contributions from multiple fields, including stem cell biology, cell biology, biomaterials, bioengineering, immunology and medicine.

The derivation of human embryonic stem cells and induced pluripotent stem cells has created great interest due to their potential ability to differentiate into any cell type of the adult body. Pluripotent stem cells can be cultured in vitro and expanded indefinitely. By manipulating the substrate and culture conditions, the cells can be induced to abandon the pluripotent state and commit to a given germ layer. Further manipulation allows differentiation to multipotent progenitors and eventually to mature differentiated cell types. The production of large numbers of specific differentiated cell types for transplantation has now become a possibility within reach, opening the door to the development of therapies for diseases that currently have little or no treatment. At the CBMR within the RMP, we will focus on cardiovascular disease, neurodegenerative disorders, diabetes and lysosomal storage disorders. In particular, our goal is to produce cardiomyocytes for cardiac cell therapy, neuronal cell types for neurodegenerative disease, insulin producing cells for diabetes and the creation of cell models of lysososmal storage disorders that will facilitate the development of therapeutic compounds for the treatment of these serious diseases.

On the path from bench to bedside, a long list of technical challenges must be overcome in order to make regenerative medicine a clinical reality. It will be crucial to obtain a thorough understanding of the molecular basis of pluripotency and the regulatory mechanisms involved in maintaining the pluripotent state. To this end, the RMP is developing methods to culture large amounts of these cells in bioreactors while maintaining their genomic integrity and pluripotency.

In addition to the production of specific cell types, specific disease models are also being developed within the RMP to be used to investigate the underlying mechanism of disease, allowing the development of novel therapeutic compounds. This approach involves taking fibroblasts from patients with genetic disorders and reprogramming them to the pluripotent state. In a later step within the RMP, these cells are differentiated to disease relevant cell type(s) for basic mechanistic studies and as a platform to develop novel therapies. Within the RMP, this approach will be applied to both cardiovascular diseases and lisosomal storage diseases.

RMP Main Research Lines

Control of the pluripotent regulatory state and study of neural, cardiac and pancreatic progenitors

Robust differentiation protocols will require detailed understanding of the pluripotent state, its maintenance and how to exit towards particular lineages. Importantly, this stage is the main point of expansion of the cell populations for eventual scale up of production. Of particular interest is the work developed in genes involved in temporal and spatial integration of molecular clocks with cell cycle associated exit points from pluripotency and their relation to commitment to a particular germ layer. The RMP is actively investigating the effect of different biomaterials and nanoparticles for neuronal and cardiomyocyte differentiation. Within the RMP, biosensors and microfludic chips are used to study the effect of different biomaterials and control the addition of differentiation factors in order to optimize methodologies to control the pluripotent state of ESC, improving differentiation and reprograming efficiency.

Development of Induced Pluripotent Stem Cell (iPSc) models of Gaucher’s and Fabry Diseases

The RMP is actively developing iPSc disease models for Gaucher’s (neuronopathic form) and Fabry Disease. The RMP has already developed a proof of principle iPSc based model of the neuronopathic form of Gaucher’s Disease, differentiated these cells dopaminergic neurons and tested candidate chaperone compounds using this model.

Development and characterization of pharmacological compounds for Gaucher’s Disease

The RMP is investigating chaperone compounds to treat Gaucher’s Disease. The compound-protein interactions of candidate molecules are being investigated and biophysically characterized. This includes the optimization of the pharmacokinetic profile for these compounds within the RMP.

 

 

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Translational Medicine

The development and promotion of fundamental research discoveries for practical ...

Translational Medicine

The development and promotion of fundamental research discoveries for practical applications and commercial development to treat disease.

The translational research program (TRP) is a cornerstone of CBMR´s effort to use basic research knowledge for practical applications to improve patient health. The TRP is composed of a wide variety of scientific and technological expertise in biomedical and bioengineering sciences that bridge the gap between basic science and clinical applications, the development of new technologies and products for commercialization and industrial development. A primary focus of the TRP is the discovery and repurposing of therapeutic drug candidates to treat disease. This focus extends to include the development of biomarkers, biosensors and drug delivery systems to improve patient treatment.

The TRP expertise and collaborative drug discovery partnership allows us to continue compound screening efforts for a broad range of diseases examining crucial molecular targets. These targets include the nuclear export receptor CRM1, tumor suppressors (FOXO and p53), oncogenic TRIB2, the plasminogen receptor annexin A2 heterotetramer (involved in cancer cell invasion/metastasis), beta-glucocerebrosidase (mutated in Gaucher’s disease) and infectious prion protein (PrSC) using complex chemical compound libraries, natural products and approved drug molecules (for drug repurposing).

The TRP is investigating effective ways to prevent and diagnose diseases with particular emphasis directed towards cancer, including breast, ovarian, colon, pancreatic and skin cancer. Furthermore the TRP is driving the development of personalized medicine and in particular, the identification of biomarkers (the first step in this process).

The TRP is actively identifying, validating and developing disease and drug related biomarkers to enhance individual patient disease prediction as well as patient management for a range of cancers that currently present with poor patient prognosis. Included within this line of investigation, the TRP is using newly identified biomarkers to predict chemotherapeutic resistance to current, as well as a novel anti-cancer drugs.

The TRP is also actively exploring new methods to administer biomolecules and pharmaceutical compounds to achieve increase their therapeutic effect. This includes unique technologies based on virus like particles for the targeted delivery of therapeutics to specific cells or tissues. In addition the TRP is continuing to develop polymeric drug delivery systems, both at nano and micro scale to encapsulate therapeutic agents (e.g., antibiotics for the treatment of tuberculosis).

TRP Main Research Lines

Pre-clinical development of lead molecules /compounds with pharmaceutical companies

The TRP is conducting compound screening efforts to identify small molecules that potently inhibit the nuclear export machinery. We are investigating the efficacy of these novel nuclear export inhibitors in a broad range of cancer cell line and xenograft models to evaluate their anti-cancer and pharmacokinetic properties.

Discovery and repurposing of therapeutic drug candidates

Based on collaborative partnerships and previously established assay technology the TRP is conducting drug discovery campaigns identifying lead compounds capable of assisting intracellular glucocerebroside protein folding to treat Gaucher disease, excluding FOXO factors from the cell nucleus (ablating chemotherapy resistance in a range of cancers), extending life span of C. elegans and depleting the mutated prion protein from the cell membrane. Specifically the CBMR´s drug discovery effort is the continued development of lead compounds to treat several diseases including lipid storage diseases, neurodegenerative diseases and cancer.

Novel Biomedical Instrumentation

The new technique of Quartz Crystal Microbalance with Heat Conduction Calorimetry (QCMHCC) can overcome the limitations of classical calorimetric techniques to characterize molecular interactions by combining a microcalorimeter with a QCM biosensor. The TRP have developed and built a QCMHCC prototype that enables the rapid screening of chemical compounds compared to an immobilized pharmacological target to complement the CBMR´s drug-discovery platform.

Development of disease-related and drug-related biomarkers

The oncobiology group in collaboration with the TRP revealed that the kinase like protein TRIB2 confers resistance to several anti-cancer agents. Alongside several clinical research teams and pharmaceutical companies the TRP is validating and developing TRIB2 as a diagnostic marker for several human malignancies as well as a predictive biomarker for chemotherapy resistance.

Susceptibility genetic markers for breast cancer

The TRP is currently investigating genetic susceptibility to breast cancer, with particular attention directed towards regulatory genetic variation. The TRP is pioneering the first genome-wide association study in breast cancer using differential allelic expression, to precisely characterise the effect of cis-regulatory variation in breast cancer as a quantitative trait.

Drug delivery systems

The TRP is actively developing encapsulation strategies to provide alternate therapeutic administration approaches against local pulmonary diseases like cystic fibrosis and tuberculosis. The TRP is evaluating the effectiveness of several packaging formulations that have yielded promising results in our in vitro and ex vivo models.

Protein Nanoparticles for Molecular Delivery

The TRP has developed a unique panel of virus-like particles (VLPs) that are assembled within out in vitro systems. The TRP is developing these for the targeted intracellular delivery of biomolecules to specific cells or tissues, in particular these are being developed for treatment for HIV1 molecular therapy, cancer treatment, and iPS differentiation.

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