New roles of Rab GTPases on eye diseases: targeting VEGF secretion

RESUMO: A retina �� composta, entre outras estruturas, pelo epit��lio pigmentar da retina (EPR)e pela cor��ide. A regi��o central da retina denomina-se m��cula, e �� a zona mais afetada na degeneresc��ncia macular relacionada com a idade, a forma mais comum de degeneresc��ncia da retina. Nesta doen��a, a secre����o de fatores de crescimento pelo EPR �� afetada, nomeadamente a do fator de crescimento vascular endotelial (VEGF), e pouco se sabe ainda sobre os mecanismos moleculares conducentes a esta condi����o. A fam��lia de prote��nas Rab GTPases est�� envolvida nas vias intracelulares de sinaliza����o e tr��fego membranares, essenciais na transdu����o de sinais extracelulares em respostas biol��gicas. A sua crucial import��ncia nestes mecanismos levou-nos a considerar o seu potencial envolvimento nas vias de secre����o do VEGF, e a questionar-nos se teriam algum papel regulador sobre as mesmas. O principal objetivo deste trabalho �� identificar Rab GTPases importantes para as vias de secre����o e endocitose do VEGF no EPR. Essa identifica����o ajudar�� a esclarecer a patog��nese da degeneresc��ncia macular da retina, e poder�� servir para uma procura mais direcionada de novos agentes terap��uticos. A caracteriza����o de dois modelos in vitro do EPR, c��lulas prim��rias isoladas de murganho e a linha celular B6-RPE07,levou-nos a concluir que s��o ambos semelhantes. Contudo, a linha celular foi escolhida como prot��tipo do EPR por permitir o acesso a um n��mero ilimitado de c��lulas. No decurso deste trabalho, desenvolvemos e caracteriz��mos uma biblioteca de ferramentas moleculares que nos permitiram reduzir os n��veis proteicos das prote��nas Rab GTPases, com base na tecnologia de ��cido ribonucleico (ARN) de interfer��ncia. O papel das prote��nas Rab GTPases na secre����o do VEGF no EPR foi estudado com base no silenciamento de apenas uma prote��na, ou combinando v��rias, segundo a sua localiza����o e fun����es intracelulares descritas. Este trabalho permitiu-nos concluir que as prote��nas Rab GTPases s��o importantes intervenientes no processo de secre����o de VEGF pelo EPR, e confirmar dados anteriores que relatam o envolvimento de algumas Rab GTPases endoc��ticas no processo. Propomos ainda um novo modelo para a intera����o destas prote��nas no EPR, e sugerimos que a Rab10 e a Rab14 atuam negativamente sobre a Rab8, controlando o seu funcionamento. Os nossos resultados evidenciam a import��ncia das prote��nas Rab GTPases na secre����o do VEGF pelas c��lulas do EPR, e servem de base a futuros estudos que melhor procurem compreender este mecanismo e de que modo a sua altera����o se relaciona com a degeneresc��ncia da retina.--------ABSTRACT: Retinal pigment epithelium (RPE) and choroid are components of the mammalian retina, of which the central region is called macula. The most common form of retinaldegeneration, age-related macular degeneration (AMD), involves primarily deregulation of growth factors secretion by the RPE. Very little is known about the molecular mechanisms that lead to impairment of RPE���s homeostatic intracellular processes, namely the secretion of vascular endothelial growth factor (VEGF). Rab GTPases��� family regulates membrane targeting and traffic, being essential in the transduction of signal pathways. Given Rab proteins��� role in intracellular trafficking, we propose to identify key regulatory Rab proteins involved in either the secretory or the recycling pathways of VEGF in RPE. Understanding how Rab proteins��� function disruption could lead to retinal and choroidal pathology would ultimately contribute to find new therapeutic agents. Here, we characterized two mouse RPE in vitro cell models, primary cells and B6-RPE07 cell line, and concluded that both display important epithelial features as the RPE presents in vivo. Considering unlimited cell number and results reproducibility, we chose B6-RPE07 cells to further study Rab proteins��� function. To scrutinize the consequences of Rab proteins��� absence or diminished levels, we have developed novel molecular tools to achieve silencing of these key proteins using miRNA technology. We further addressed the effect of Rab proteins��� absence on VEGF secretion by performing an extensive screening where different Rab proteins were silenced, both individually and in multiple combinations considering their cellular/ compartment location. We conclude that Rab GTPases are important intervenients in VEGF secretion by RPE cells, confirming endocytic Rab proteins��� role in regulation of VEGF biology. We also propose a novel model for Rab proteins��� interaction in RPE. Our results suggest that Rab10 and Rab14 might influence Rab8 in a negative feedback mechanism, important for controlling VEGF secretion. Our achievements��� unravel Rab proteins��� role in VEGF secretion by RPE cells and are the basis for future studies to better understand RPE molecular secretory machinery.

Analysis of PAX2 gene alterations in renal cell tumors

Dissertation for applying to a Master���s Degree in Molecular Genetics and Biomedicine submitted to the Sciences and Technology Faculty of New University of Lisbon

T cell Maturation and Regulatory T Cell Differentiation:From the Thymus to the Periphery

Immunological tolerance, that is, the failure to mount an immune response to an otherwise immunogenic molecule, is one of the fundamental questions in immunology. The fact that lymphocytes express antigen receptors that are generated randomly and have the potential to recognize any conceivable antigen, adds another puzzle to the physiology of immunological tolerance. The other side of the coin, the general absence of immune responses to self antigens, is ensured by a tight regulation and several selection steps during T and B cell differentiation. One of these processes is the differentiation of regulatory T cells (Treg). While developing in the thymus, T cell clones bearing receptors with high affinity/avidity to antigens present at the time of differentiation may be eliminated by apoptosis or, alternatively, express Foxp3 and become Treg. Treg are key players in the regulation of immunological tolerance since humans and mice with complete loss of function variants of this gene develop fatal autoimmune conditions early in life.(...)

Establishing a cell biology platform: isolation and preservation of human blood products

Disserta����o para obten����o do Grau de Mestre em Gen��tica Molecular e Biomedicina

Bioprocess engineering of induced pluripotent stem cells for application in cell therapy and pre-clinical research

Dissertation to obtain Master Degree in Biotechnology

Collecting data for indoor mapping of the university of M��nster via a location based game

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Variation in protein expression levels in cell populations

Dissertation presented to obtain the Ph.D degree in Systems Biology

Inheritance of histone H3 variants across mitotic cell divisions

Dissertation presented to obtain the Ph.D degree in Molecular Biology.

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology

Cell-to cell Communication in Pectobacterium wasabiae

Dissertation presented to obtain the PhD degree in Biology

Estudo da actividade angiog��nica na nurse cell de trichinella spiralis no decurso da triquinelose em modelo roedor

A triquinelose �� uma zoonose parasit��ria que �� transmitida aos humanos e animais, atrav��s da ingest��o de carne crua ou insuficientemente cozinhada, que contenha larvas infectantes de Trichinella spp., sendo actualmente considerada uma doen��a emergente e/ou re-emergente. O sucesso do parasitismo do nem��tode Trichinella spiralis est�� intimamente ligado com o processo de angiog��nese, ou seja, a forma����o de novos vasos a partir de vasos pr��-existentes. Com o objectivo de estudar a actividade angiog��nica na nurse cell de T. spiralis, realizaram-se t��cnicas imunohistoqu��micas e imunofluorescentes para o factor de crescimento endotelial vascular (VEGF), mol��cula-1 de ades��o celular endotelial a plaquetas (PECAM-1) e actina m��sculo liso (AML), em tecido muscular de Rattus rattus infectado com T. spiralis. Atrav��s destas t��cnicas observou-se marca����o intensa no infiltrado inflamat��rio adjacente �� nurse cell e tamb��m na larva. J�� o citoplasma da nurse cell apresentou uma marca����o moderada. Este padr��o de marca����o manteve-se desde os 45 at�� aos 120 dias ap��s a infec����o. A avalia����o da densidade vascular (PECAM-1) e da densidade da express��o de c��lulas positivas para AML permitiu estabelecer uma correla����o positiva entre o aumento da densidade vascular e o n��mero de dias de infec����o. Adicionalmente estabeleceu-se uma correla����o negativa entre o aumento da densidade de c��lulas que expressam AML e o n��mero de dias de infec����o. Os resultados indicam uma produ����o constante de VEGF pela larva, pelo citoplasma da nurse cell e pelo hospedeiro (infiltrado inflamat��rio), durante todo per��odo de infec����o, levando �� forma����o de uma rede vascular crescente (com um aumento m��dio de 79% face ao controlo), acompanhada de c��lulas murais que promovem a sua estabiliza����o (com um aumento m��dio de 50% face ao controlo) com particular incid��ncia no primeiro per��odo estudado (45 a 55 dias).

Amyotrophic Lateral Sclerosis:Mammalian Cell Models,Copper-Zinc Superoxide Dismutaseand Biological Characteristics

"Amyotrophic Lateral Sclerosis (ALS) is the most severe and common adult onset disorder that affects motor neurons in the spinal cord, brainstem and cortex, resulting in progressive weakness and death from respiratory failure within two to five years of symptoms onset(...)

Novel Cryopreservation Strategies for Cell���Therapies and Pre���Clinical Research

The expanding need for complex biologics for therapeutic applications, in���vitro pharmacology and toxicology studies and fundamental research demands the production of banks of well���characterized and safety���tested stocks of a large number of cell/tissue samples. This implies the development of effective cryopreservation methodologies that can cope with process scalability and automation and must reflect the biological and physical properties of the cells as these can be significantly altered by the process.

Manipulation of host cell functions by Salmonella: the role of the bacterial effector protein SteA

Salmonella enterica serovars are Gram-negative facultative intracellular bacterial pathogens that infect a wide variety of animals. Salmonella infections are common in humans, causing usually typhoid fever and gastrointestinal diseases. Salmonella enterica serovar Typhimurium (S. Typhimurium), which is a leading cause of human gastroenteritis, has been extensively used to study the molecular pathogenesis of Salmonella, because of the availability of sophisticated genetic tools, and of suitable animal and tissue culture models mimicking different aspects of Salmonella infections.(...)

Can location-based mobile marketing enhance the customer experience and increase customer satisfaction and loyalty?

This study intends to explore the impact of customer experience on customer satisfaction and loyalty by trying to understand how location-based mobile marketing might enhance the customer experience. Primary data was collected from 201 smartphone users in 24 countries. Results have indicated that targeted location-based marketing positively influences customers��� experiences. Besides, the analysis has also shown a favorable impact on customers��� satisfaction and self-perceived loyalty. This suggests that location-based mobile marketing has the potential to positively add value to a customer���s experience and should therefore be considered an important tool in marketing communications.