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Browsing UAlg-Teses by Field of Science and Technology (FOS) "Ciências Médicas::Biotecnologia Médica"
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- Analysis of the transcriptional regulatory network: underlying heart developmentPublication . Machado, Rui Sotero Rodrigues; Futschik, Matthias E.; Bragança, JoséHeart development is a highly complex process with a series of precisely spatially and temporally ordered events on molecular level. To understand how these events are controlled and coordinated, it is necessary to study the underlying gene expression and its regulation. While many studies have been carried out in the examination of single genes and their expression patterns, comprehensive analyses of genome-wide expression profiles associated with cardiomyogenesis (i.e. the differentiation of stem cells into cardiomyocytes) are still rare. In fact, no study exists to date which compares and consolidates the publicly available genome-wide measurement for cardiomyogenesis. Such endeavour however is important, as it is well known that individual microarray studies can be seriously compromised by artefacts. In contrast, the combination of various expression studies, which was performed in my study, can lead to more reliable results and help elucidate the different aspects of heart development and repair. Furthermore, a brief study was performed regarding the potential risk of originating cancer or teratomas from stem cell therapy. Finally, I carried out a network-based analysis, to identify regulatory actions between genes, based on published interaction data. This type of analysis can also help to identify novel genes with a role in heart development and provide new valuable targets to future experimental laboratorial analysis. The combination of the multiple dataset is thus an important approach to gain better insights of the different heart development processes as well as regenerative medicine applied to the heart.
- Changes in components of the brain extracellular matrix after experimental ischemic strokePublication . Guerreiro, Carla Sofia de Jesus; Wieloch, Tadeusz; Araújo, Inês; Quattromani, Miriana JleniaStroke is the 3rd cause of death in the world. During stroke, there is a disruption in the blood supply to the brain leading to rapid loss of brain function. Ischemic strokes are caused by obstruction of the blood supply, while hemorrhagic strokes results from rupture of a blood vessel. Eight-five percent of the strokes are ischemic. The only treatment recommended for acute ischemic stroke is the recombinant tissue activator of plasminogen but only a few percentages of patients are eligible for rtPA administration. Approximately 30% of the ischemic stroke victims die and 30% become severely disabled, resulting in among others deficits in motor function in the contralateral musculature. Spontaneous recovery occurs during weeks to months following injury. There are many physiological and anatomical examples of cortical brain plasticity and one of the most potent modulators of cortical structure and function is behavioral experience. Functional recovery after stroke can be enhanced by physical training in stroke patients. In the animal settings, physical training can be accomplished by enriched environment (EE). EE refers to housing conditions, either home cages or exploratory chamber, that facilitate enhanced sensory, cognitive and motor stimulation relative to standard housing conditions. The extracellular matrix (ECM) is important in the regulation of brain plasticity but is also a potential hampering factor for recovery after stroke. It is known that EE affects chondroitin sulfate proteoglycans (CSPGs) present in ECM, leading to functional recovery. Matrix metalloproteinases (MMPs) are able to cleave ECM components. There are some evidences that beta-dystroglycan (β-DG) is a MMP-9 target. After the degradation of β-DG, there is a 30 kDa product. The aim of this work is to explore how EE affects β-DG and gelatinases over 1 week of recovery after experimental stroke, performed as photothrombosis (PT). We show that EE does not affect the infarct size and improves tactile/proprioceptive response to limb stimulation. We found that β-DG is mostly present in vessels across the brain cortex and animals housed in an EE had a higher degradation than STD animals when comparing to sham non-operated animals. β-DG can be related with changes in the ECM that leads to brain plasticity, promoting functional recovery after experimental stroke, possibly due to MMPs enzymatic activity.
- Efeito do stresse oxidativo na miosina - estudo das modificações moleculares, funcionais e estruturais induzidas pelo agente oxidante peroxinitritoPublication . Palma, Pedro Filipe dos Santos; Power, DeborahO presente trabalho consiste na elaboração de um Relatório de Atividade Profissional para obtenção do grau de Mestre em Biotecnologia, de acordo com os termos do despacho RT.033/2011 para obtenção do grau de Mestre pelos Licenciados Pré-Bolonha da Universidade do Algarve.
- Evaluation of pEPI-1 and pEPito expression systems for gene transfer to the retinaPublication . Calado, Sofia de Amaral Melo; Silva, GabrielaO objectivo de estudo do nosso laboratório é o desenvolvimento de vectores não virais para a terapia génica ocular. O principal objectivo da terapia génica é a introdução de material genético no interior das células. Em teoria, o veículo ideal para a terapia génica é aquele que consegue penetrar eficientemente a membrana celular e libertar o material genético, sem desencadear uma resposta imunológica agressiva.
- Identification of novel molecular determinants of tissue mineralization in fishPublication . Rosa, Joana Alexandra Teixeira; Cancela, Leonor; Laizé, VincentThe identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.
- Interferindo com o "checkpoint" mitótico para potenciar a ação de agentes anti-mitóticosPublication . Teixeira, Joana Filipa Tomás; Bousbaa, Hassan; Silva, Patrícia; Tavares, ÁlvaroAtualmente, várias terapias anti-cancro apresentamuma toxicidade associada e são confrontadas com resistências por parte de muitos cancros. Por exemplo,em quimioterapia vários cancros exibem resistência a agentes anti-mitóticos como o Taxol. Estes agentesinterferem com a formação ou a dinâmica dos microtúbulos, o que leva a uma paragem das células em mitose, devido à ativação do “checkpoint” mitótico, o mecanismo que permite a correta segregação dos cromossomas.No entanto as célulastumorais resistentes ao taxolconseguem contornar esta paragem e continuar a sua proliferação. Posto isto, o“checkpoint” mitóticotem sido sugerido como um potencial alvo no tratamento anti-cancro. Assim este trabalho teve comoobjetivo inicial a avaliação do potencial inibidor de pequenas moléculas contra uma proteína envolvida no checkpoint mitótico (BubR1). Os resultados obtidos mostraram que uma das pequenas moléculas analisadas, demonstrou ter uma atividade anti-proliferativa em células HeLa, demonstrando uma possível interferência com a função da proteína BubR1. O objetivo principal deste trabalho consistiu emsensibilizar células tumorais à ação do taxol, depletando proteínas envolvida no silenciamento do“checkpoint” mitótico (Spindly) e na saída da mitose (Cdc20). Os resultadosdemonstraram que a depleção das proteínas leva a uma paragem das células em mitose, mais eficiente após a depleção da proteína Spindly. Foi demonstrado também uma redução da viabilidade e da capacidade proliferativa das células,com uma reduçãomais acentuada apósa combinação da depleção com o Taxol. Assim este trabalho sugere que a depleção destas duas proteínas ajuda a sensibilizar as células à ação do taxol nas concentrações usadas em quimioterapia.
- Investigation of the presence and expression of the cyclophilin a pseudogene in NCI-H596 lung cell line and its expression in other cell linesPublication . Vaz, Ana Rita Diogo Martins; Bailey, Tracey; Belo, José AntónioLung câncer is one of the most studied cancers and it is the number one cause of câncer deaths in Europe and United States. Prostate câncer is the most common malignancy among the males in the Western world and both of them are associated with several risk conditions.
- Lymphotoxin-beta receptor and RANK signaling in TEL-JAK2-induced T-cell leukemiaPublication . Fernandes, Mónica Alexandra Teotónio; Santos, Nuno Rodrigues dosT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy that arises from the combination of genetic and epigenetic alterations in thymic T-cell precursors and extracellular signals provided by the microenvironment. It was previously found that RelB expression in non-hematopoietic stromal cells promoted T-cell leukemogenesis in the EμSRalpha-TEL-JAK2 transgenic (TJ2-Tg) mouse model. In thymic stromal cells, RelB is a transcription mediator of lymphotoxin-beta receptor (LTβR). Lymphotoxin-mediated activation of LTβR has been implicated in physiological crosstalk between T cells and lymphoid organ stromal cells, but also pathological processes, including carcinogenesis. Since its role in T-ALL has remained elusive, we aimed to determine whether LTβR signaling is activated and playing a role in TEL-JAK2-induced leukemogenesis. In TJ2-Tg thymic lymphomas, activation of LTα1β2-LTβR signaling axis was supported by LTβRencoding gene expression, while the genes encoding its cognate ligand, lymphotoxin (LT)-α and LTβ, were found to be expressed by leukemic T cells, in an NF-κB-dependent manner. LTα1β2 protein was detected at the surface of TJ2-Tg leukemic cells only upon ex vivo culture or mitogenic stimulation. Moreover, we found that cell-surface LTα1β2 is downmodulated in vivo, indicating ongoing signaling. Further supporting a role for lymphotoxin signaling, LTβR genetic deficiency delayed TEL-JAK2-induced leukemia onset, but the tumor load in lymphoid organs and leukemia cell surface phenotype were comparable in end-stage disease. In accordance, the detection of reduced proportions of malignant thymocytes in TJ2-Tg;Ltbr-/- mice with no signs of disease implicated LTβR in early stages of leukemia development. Together, these data indicate that T-ALL-derived lymphotoxin activates LTβR signaling in thymic stromal cells, promoting leukemogenesis. Importantly, lymphotoxin-encoding genes were expressed in T-ALL patient samples, indicating that these may be also involved in human disease. Thus, future studies should provide a better understanding on how cellular crosstalk mediated by the lymphotoxin-LTβR axis supports T-ALL and assess the utility of blocking LTβR signaling as a novel therapeutic approach.
- MEF2C: expression, regulation and interaction with target genes in health and diseasesPublication . Adrião, Andreia Lúcia Gonçalves; Cancela, Leonor; Grazina, Manuela; Conceição, NatérciaMEF2C is the first MEF2 family factor to be expressed during embryonic development. Zebrafish has been proven to be an excellent model for human genetic diseases and has two MEF2C orthologues, mef2ca and mef2cb. The involvement of MEF2C in different developmental processes was studied in zebrafish through expression analysis and usage of mutant lines as loss of function models. Alterations in this gene were related to cardiac defects and its haploinsufficiency was linked to a human disease (MRD20) whose patients show abnormal neuronal and craniofacial development. Recently this gene was reported to be involved in dementia conditions such as Alzheimer disease, Parkinson disease and amyotrophic lateral sclerosis. Our work aims to further validate zebrafish as an animal model to study MEF2C related pathologies focusing on craniofacial and neuronal issues. Our in silico analysis demonstrated a high conservation of mef2c pattern of chromosome localization, protein structure and sites of mRNA expression throughout evolution. We also identified two transcriptional start sites for mef2cb that were conserved through evolution. These are related to the occurrence of alternative promoters that appear to be differentially regulated by Sox9b, Sox10 and Runx2, three nuclear factors associated to craniofacial or neuronal development. Through the use of Mef2ca and Mef2cb loss of function mutant lines, we described the craniofacial phenotype resulting from the absence of both mef2c isoforms in zebrafish. We concluded that both orthologues are involved in cartilage, bone and brain development in zebrafish and we described the molecular profile of specific marker genes resulting from mef2c loss of function. Our results allow us to consider zebrafish as a valuable animal model for analysis concerning MEF2C related pathologies. To complement our work we performed a pilot study in which we describe for the first time an alteration in MEF2C gene in a patient with frontotemporal lobar degeneration, suggesting an association between MEF2C and this disease, a result that extends the involvement of this gene to a previously unsuspected human pathology.
- Molecular and functional analysis of DAND5 in human Congenital Heart Disease (CHD)Publication . Cristo, Fernando Jorge Pego; Belo, JoséThe majority of congenital heart disease (CHD) is sporadic, with a minority of cases associated with a known genetic abnormality. Combinations of geneticenvironmental factors are implicated in the etiology of the disease. Recently, several studies, using mostly animal models, unraveled that perturbations in the molecular processes that precede the beginning of heart development might also be at the origin of CHD. In fact, some of the most complex CHDs are found associated with laterality defects, a disorder resulting from abnormal Left-Right axis formation. In our laboratory, the identified mouse Cerberus-like2 (Cerl2 – human DAND5), a protein that inhibit Nodal signaling, prompt us to study cardiac and laterality diseases, since the generated Cerl2 KO mice display a wide range of laterality defects and CHD. Considering the high conservation of genetic pathways regulating cardiac development in mouse and human, the main objective of the present thesis was the study of human genes involved in the Nodal pathway, focusing mostly in DAND5, in a CHD and/or laterality defects patients cohort. The sequence analysis of DAND5 revealed two patients displaying the same p.R152H variant, resulting in a substantial decreased in the function of the protein. We propose that p.R152H acts as a risk allele for CHD and/or laterality defects. In addition, we found two alterations in NODAL, two alterations in PITX2C and one alteration in CFC1. We hypothesized that the NODAL p.H165R variant can act as a common modifier and the intronic variants in NODAL and PITX2C might cause alterations in the splicing pattern of the mRNA molecules. Moreover, we generated patient-specific iPSCs to understand the molecular mechanisms of disease behind the DAND5 nucleotide variant. Although we cannot make a clearly genotype-phenotype correlation, the variants here identified probably increase the disease susceptibility due to the resulting abnormal Nodal signaling. Because most of the patients presented more than one alteration, the cumulative effect of each variant within the pathway seems to enhance even more these risk. Therefore, the imbalance in dosage-sensitive Nodal signaling is a common denominator for laterality defects and associated CHDs.