Dissection of the early steps in the assembly of the 20S proteasome in Saccharomyces cerevisiae

Tese de dout., Bioquímica (Biologia Celular e Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Autophagy in hypothalamic cells: role of Neuropeptide Y

Dissertação de mest.Ciências Biomédicas. Departamento de Ciências Biomédicas e Medicina, Univ. do Algarve, 2011

Caractérisation des mécanismes d’action du microARN 169 chez Arabidopsis thaliana

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

The role of lymphotoxin-B receptor signaling in t-cell acute lymphoblastic leukemia development

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Produção de bioetanol a partir de resíduos industriais de alfarroba: cinética de inibição do etanol para diferentes concentrações do nutriente limitante. Fermentação com recirculação de células livres e células imobilizadas

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2012

Effects of nanoparticles exposure in the mussel Mytilus Galloprovincialis

Tese de dout., Ciências do Mar, Terra e Ambiente (Ecotoxicologia), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2012

Identification of genes regulated by mir-363*, a regulator of bone marrow endothelial cell properties

As células estaminais hematopoiéticas residem na medula óssea e possuem capacidade para se auto-renovar e dar origem a todos os tipos de células sanguíneas. O endotélio da medula óssea é constituído por células endoteliais de medula óssea (BMEC) e compreende dois nichos com funções distintas: o nicho osteoblástico e o nicho vascular. O nicho osteoblásctico proporciona condições para a quiescência de células estaminais hematopoiéticas, enquanto no nicho vascular ocorre proliferação e diferenciação das mesmas. Quando ocorre um desequilíbrio na expressão de genes que codificam para proteínas envolvidas na mobilização de células do nicho osteoblástico para o nicho vascular – factores angiócrinos – ocorre uma desestabilização do microambiente medular, que se pode traduzir num processo tumoral. Os microRNAs (miRNAs) são uma classe de RNAs não codificantes, de cadeia simples, que regula a expressão génica. Os miRNAs são sequências endógenas de RNA que possuem entre 19 e 25 nucleótidos de tamanho. Os miRNAs são reguladores da expressão genica, induzindo o silenciamento a nível da pós-transcrição, através da sua ligação com uma sequência específica para a qual possuem afinidade, na região 3’ não traduzida (3’ UTR) dos seus mRNA alvo, conduzindo à inibição da tradução ou à sua degradação. Os miRNAs estão envolvidos na regulação de genes de diversas vias afectando processos fundamentais como hematopoiese, apoptose, proliferação celular e tumorigénese. Os níveis de expressão dos miRNAs estão alterados no cancro, podendo actuar directamente como supressores de tumor ou como oncogenes, sendo neste caso denominados de oncomirs. Os perfis dos níveis de expressão de vários miRNAs foram estudados, tendo-se verificado que se alteram durante o processo de carcinogénese, podendo actuar directamente como supressores de tumor ou como oncogenes, sendo neste caso denominados de oncomirs. Apesar do miR-363* estar envolvido na regulação da expressão de genes que regulam propriedades das células endoteliais e medula óssea, os genes sobre os quais exerce a sua função ainda não foram identificados.O objectivo do presente estudo é a identificação dos genes directamente regulados pelo miR-363* (genes alvo) e a sua relevância para a disfunção medular e a sua caracterização nos síndromes mielodisplásicos. A estratégia usada baseou-se na redução ou aumento forçados dos níveis de miR-363* em células endoteliais e subsequente análise da expressão génica através de microarrays de cDNA do genoma humano. A redução do miR-363* vai implicar o aumento da expressão dos seus genes alvo, assim como o aumento dos níveis do miR-363* vai induzir a degradação e consequente redução dos seus genes alvos. A intersecção dos dados gerados através do estudo da expressão com bases de dados que possuem algoritmos para previsão de genes alvo directos dos miRNAs (miRBase e MicroCosm Targets) permitiu restringir os genes a analisar a sete genes, nomeadamente BST1, ESAM, FCER1G, IKBKG, SELE, THBS3 e TIMP1. A interacção directa destes candidatos a alvos directos do miR-363* foi posteriormente validada. Para tal, as 3’UTR dos genes foram clonadas num vector que contém o gene da luciferase. Uma vez as clonagens realizadas, efectuaram-se ensaios funcionais em células endoteliais, nomeadamente HUVEC, nas quais se co-transfectaram os vectores gerados, anti-miRs ou pre-miRs (para diminuir ou aumentar o nível de miRNA) e o plasmídeo controlo da Renilla para normalização dos ensaios de luciferase. A variação da luminescência obtida em presença do aumento ou redução do miR-363* deu uma forte indicação da regulação directa do miR-363* nesses alvos. No entanto, a confirmação desta interacção directa foi efectuada através de ensaios de mutagénese, nos quais de induziram mutações na 3’UTR nos locais de ligação do miRNA, seguidos dos ensaios funcionais como acima descritos. Esta estratégia sugere que o TIMP1, inibidor da metaloprotease-9 (MMP-9), é regulado directamente pelo miR-363*. Adicionalmente, os níveis de expressão dos alvos directos do miR-363* foram estudados em 17 amostras de aspirados de medula óssea de doentes com síndromes mielodisplásicos. Os síndromes mielodisplásicos são caracterizados como um grupo heterogéneo de condições, que apresentam citopenias (produção deficiente de eritrócitos, leucócitos e/ou megacariócitos) e medula óssea displástica e hipercelular. A escalonagem dos doentes foi feita de acordo com o sistema de prognóstico IPSS elaborado pela Organização Mundial de Saúde, e que consiste numa tabela de risco de progressão de síndromes mielodisplásicos para leucemia mielóide aguda (LMA) e que agrupa os doentes em baixo risco – que compreende os níveis baixo e intermédio 1 – e em alto risco – que compreende os níveis intermédio 2 e alto. Dos genes regulados pelo miR-363*, o destacam-se o TIMP1, estando aumentando em doentes com mau prognóstico, e o THBS3 que apresenta um aumento nos doentes com prognóstico intermédio. Em suma, os estudos realizados permitiram a identificação de genes regulados pelo miR-363* e contribuiram para o conhecimento de como o miR-363* contribui para a disfunção medular, particularmente em síndromes mielodisplásicos, pela desregulação das propriedades endoteliais.

Differential requirements of aPKC activity within different epithelial tissues

Epithelial tissues are essential during morphogenesis and organogenesis. During development, epithelial tissues undergo several different remodeling processes, from cell intercalation to cell change shape. An epithelial cell has a highly polarized structure, which is important to maintain tissue integrity. The mechanisms that regulate and maintain apicobasal polarity and epithelial integrity are mostly conserved among all species and in different tissues within the same organism. aPKC-PAR complex localizes in the apical domain of polarized cells, and its function is essential for apicobasal polarization and epithelial integrity. In this work we characterized two novel alleles of aPKC: a temperature sensitive allele (aPKCTS), which has a point mutation on a kinase domain, and another allele with a point mutation on a highly conserved amino acid within the PB1 domain of aPKC (aPKCPB1). Analysis of the aPKCTS mutant phenotypes, lead us to propose that during development different epithelial tissues have differential requirements of aPKC activity. More specifically, our work suggests de novo formation of adherens junctions (AJs) is particularly sensitive to sub-optimal levels of apkc activity. Analysis of the aPKCPB1 allele, suggests that aPKC is likely to have an apical structural function mostly independent of its kinase activity. Altogether our work suggests that although loss of aPKC function is associated to similar epithelial phenotypes (e.g., loss of apicobasal polarization and epithelial integrity), the requirements of aPKC activity within these tissues are nevertheless likely to vary.

Effect of polyunsaturated fatty acids (PUFAs) on the proliferation and extracellular matrix mineralization and gene expression of gilthead seabream skeletal cell lines

The aquaculture industry aims at replacing significant amounts of marine fish oil by vegetable oils in fish diet. Dietary lipids have been shown to alter the fatty acid composition of bone compartments, which would impact the local production of factors controlling bone formation. Knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is however scarce in fish. Two in vitro bone-derived cell systems developed from seabream (an important species for aquaculture in the Mediterranean region) vertebra, capable of in vitro mineralization and exhibiting prechondrocyte (VSa13) and pre-osteoblast (VSa16) phenotype, were used to assess the effect of certain polyunsaturated fatty acids (PUFAs; arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids) on cell proliferation, extracellular matrix (ECM) mineralization and gene expression. While all PUFAs promoted morphological changes in both cell lines, VSa16 cell proliferation appeared to be stimulated by PUFAs in a dose dependent manner until 100M, whereas proliferation of VSa13 cells was impaired at concentrations above 10M. AA, EPA and DHA inhibited VSa13 ECM mineralization, alone and in combination, while VSa16 ECM mineralization was only inhibited by AA and EPA. DHA had the opposite effect, increasing mineralization almost by 2 fold. When EFAs were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. Expression of marker genes for bone and lipid metabolisms has been investigated by qPCR and shown to be regulated in pre-osteoblasts exposed to individual PUFAs. Our results show that PUFAs are effectors of fish bone cell lines, altering cell morphology, proliferation and mineralization when added to culture medium. This work also demonstrates the suitability of our in vitro cell systems to get insights into mineralization-related effects of PUFAs in vivo and to evaluate the replacement of fish oils by vegetable oil sources in fish feeds.

A proteomic study of the effects of nutrient restriction in utero on postnatal metabolism

It is widely recognized that protein restriction in utero may cause metabolic and endocrine adaptations, which may be of benefit to the neonate on a short-term basis but may cause adverse long-term conditions such as obesity, Type 2 diabetes, metabolic syndrome, hypertension and cardiovascular diseases. Adequate foetal and early post natal nutrient and energy supply is therefore essential for adult animal health, performance and life span. In this project it was investigated the progressive adaptations of the hepatic proteome in male mink offspring exposed to either a low protein (FL) or an adequate protein (FA) diet in utero fed either on a low protein (LP) or on an adequate (AP) diet from weaning until sexual maturity. Specifically, the aim was to determine the metabolic adaptations at selected phases of the animal’s first annual cycle and establish the metabolic priorities occurring during those phases. The three different morphological stages studied during the first year of development included, end of bone growth at 4 months of age, maximal fat accretion at 6 months of age and sexual maturity at 12 months of age. A reference proteome of mink liver coming from these different animal groups were generated using 2D electrophoresis coupled to MALDI-TOF analysis and the way in which dietary treatment affect their proteome was established. Approximately 330 proteins were detected in the mink liver proteome. A total of 27 comparisons were carried out between all different animal groups which resulted in 20 differentially expressed proteins. An extensive survey was conducted towards the characterization of these proteins including their subcellular localization, the biological processes in which they are involved and their molecular functions. This characterization allowed the identification of proteins in various processes including the glycolysis and fatty acid metabolism. The detailed analysis of the different dietary treatment animal groups was indicative of differences in metabolism and also to changes associated with development in mink.

The effect of sucrose on protein stability and fibrillation of prion and S6 protein

Aggregation and fibrillation of proteins have a great importance in medicine and industry. Misfolding and aggregation are the basis of many neurodegenerative diseases like Alzheimer and Parkinson. Osmolytes are molecules that can accumulate within cells and act as protective agents and they can inclusively act as protein stabilizers when cells are exposed to stress conditions. Osmolytes can also act as protein stabilizers in vitro. In this work, two different proteins were studied, the ribosomal protein from Thermus thermophilus and the mouse prion protein. The existence of an unstructured N-terminal on the prion protein does not affect its stability. The effect of the osmolyte sucrose on the fibrillation and stabilization of these two proteins was studied through kinectic and equilibrium measurements. It was shown that sucrose is able to compact the native structure of S6 protein in fibrillization conditions. Sucrose affects also folding and unfolding kinetic of S6 protein, delaying unfolding and increasing folding rate constants. The mechanism of stabilization by sucrose is non-specific because it is distributed for all protein structure, as it was demonstrated by a protein engineering approach. Sucrose delays the process of formation and elongation of S6 and prion protein from mouse. This delay is the result of the compaction of the native structure refered above. However, cellular toxicity studies have shown that fibrils formed in the presence of sucrose are more toxic to neuronal cells.

Exploring an uncommon host-parasite interaction to unveil the uniqueness of Perkinsus sp as a model organism

We report the exploration of some unique metabolic pathways in Perkinsus olseni a marine protist parasite, responsible to significant mortalities in mollusks, especially in bivalves all around the world. In Algarve, south of Portugal carpet shell clam Ruditapes decussatus mortalities can reach up to 70%, causing social and economic losses. The objective of studying those unique pathways, is finding new therapeutic strategies capable of controlling/eliminating P. olseni proliferation in clams. In that sense metabolic pathways, were explored, and drugs affecting these cycles were tested for activity. The first step involved the identification of the genes behind those pathways, the reconstitution of the main steps, and molecular characterization of those genes and later on, the identification of possible targets within the genes studied. Metabolic cycles were screened due to the fact of not being present in host or differ in a critical way, such as the following pathways: shikimate, MEP-­‐ isoprenoids, Leloir cycle for chitin production, purine biosynthesis (unique among protists), the de novo synthesis of folates (absent in metazoa) and some unique genes like, the alternative oxidase (a branch of respiratory chain) and the hypoxia sensor HPH. All those pathways were covered and possible chemical inhibition using therapeutic drugs was tested with positive results. The relation between the common host Ruditapes decussatus and P. olseni was also explored in a dimension not possible some years ago. With the accessibility to second generation sequencers and microarray analysis platforms, genes involved in host defense or parasite virulence and resistance to the host were deciphered, allowing aiming to new targets (mechanisms and pathways), offering new possibilities for the control of Perkinsus in close environments. The thousands of genes, generated by this work, sequenced and analyzed from this commercial valuable clam and for Perkinsus olseni will be an important and value tool for the scientific community, allowing a better understanding of host-­‐parasite interactions, promoting the usage of P. olseni as an emerging model for alveolata parasites.

Comportamento in vitro de formulações lipossomais de um aminoglicosídeo com actividade anti-parasitária: citotoxicidade e interacção lipossoma-célula

Os sistemas de veiculação de fármacos, presentemente, apresentam-se como uma alternativa viável às terapias convencionais. De entre os diversos sistemas de transporte passíveis de associar substâncias farmacologicamente activas, destacam-se os de base lipídica, em particular, os lipossomas, os quais constituem um dos sistemas mais estudados e com maior sucesso, comprovado pelo número de produtos em fase clínica ou já aprovados para utilização em humanos. Os lipossomas são estruturas constituídas por membranas lipídicas organizadas em bicamadas, fechadas e concêntricas, que permitem a encapsulação de moléculas hidrofílicas no espaço interno aquoso e hidrofóbicas na bicamada lipídica. No presente trabalho, foram desenvolvidas metodologias com vista à encapsulação em lipossomas do aminoglicosídeo, a Paromomicina (PRM). Este fármaco está indicado para o tratamento de doenças infecciosas nomeadamente parasitárias e bacterianas. Algumas das principais desvantagens resultantes da sua utilização em clínica são, o reduzido tempo de circulação na corrente sanguínea, rápida excreção renal e consequentemente insuficiente concentração intracelular do fármaco. Como forma de ultrapassar algumas das desvantagens apresentadas, foram desenvolvidas formulações lipossomais de PRM com vista a melhorar o desempenho deste antibiótico. Para tal, foram preparadas e caracterizadas diversas formulações lipossomais de PRM com vista à selecção daquelas que apresentem maiores valores de eficácia de encapsulação (E.E.), e superior estabilidade. Com as formulações seleccionadas foram realizados estudos in vitro de interacção lipossoma-célula, utilizando uma linha celular humana monocítica leucémica, THP-1. De entre as formulações desenvolvidas e seleccionadas para os estudos in vitro de a formulação DPPC:DPPG, foi uma das que apresentou uma E.E. superior a 80% e valores de internalização celular superiores a 90%.

Insights into molecular and cellular events involved in normal and pathological vertebral development and fusion using zebrafish as model organism

The vertebral column and its units, the vertebrae, are fundamental features, characteristic of all vertebrates. Developmental segregation of the vertebral bodies as articulated units is an intrinsic requirement to guarantee the proper function of the spine. Whenever these units become fused either during development or postsegmentation, movement is affected in a more or less severe manner, depending on the number of vertebrae affected. Nevertheless, fusion may occur as part of regular development and as a physiological requirement, like in the tetrapod sacrum or in fish posterior vertebrae forming the urostyle. In order to meet the main objective of this PhD project, which aimed to better understand the molecular and cellular events underlying vertebral fusion under physiological and pathological conditions, a detailed characterization of the vertebral fusion occurring in zebrafish caudal fin region was conducted. This showed that fusion in the caudal fin region comprised 5 vertebral bodies, from which, only fusion between [PU1++U1] and ural2 [U2+] was still traceable during development. This involved bone deposition around the notochord sheath while fusion within the remaining vertebral bodies occur at the level of the notochord sheath, as during the early establishment of the vertebral bodies. A comparison approach between the caudal fin vertebrae and the remaining vertebral column showed conserved features such as the presence of mineralization related proteins as Osteocalcin were identified throughout the vertebral column, independently on the mineralization patterns. This unexpected presence of Osteocalcin in notochord sheath, here identified as Oc1, suggested that this gene, opposing to Oc2, generally associated with bone formation and mature osteoblast activity, is potentially associated with early mineralization events including chordacentrum formation. Nevertheless, major differences between caudal fin region and anterior vertebral bodies considering arch histology and mineralization patterns, led us to use RA as an inductive factor for vertebral fusion, allowing a direct comparison of equivalent structures under normal and fusion events. This fusion phenotype was associated with notochord sheath ectopic mineralization instead of ectopic perichordal bone formation related with increased osteoblast activity, as suggested in previous reports. Additionally, alterations in ECM content, cell adhesion and blood coagulation were discussed as potentially related with the fusion phenotype. Finally, Matrix gla protein, upregulated upon RA treatment and shown to be associated with chordacentrum mineralization sites in regular development, was further described considering its potential function in vertebral formation and pathological fusion. Therefore with this work we propose zebrafish caudal fin vertebral fusion as a potential model to study both congenital and postsegmentation fusion and we present candidate factors and genes that may be further explored in order to clarify whether we can prevent vertebral fusion.

The role of hypoxia in the regulation of membrane transporters

ATP binding cassette (ABC) and solute carrier (SLC) transporters are responsible for the majority of the transcellular movement of various substrates, including drugs, among epithelial cells. Despite the well characterized regulation of influx (SLC) and efflux (ABC) transporters by endogenous mediators, such as inflammatory cytokines, little is known about how changes in oxygen levels may affect expression of these transporters. In this study we showed that the expression of SLC22A4, SLC22A5, SLC22A1, SLC02B1, SLC10A2, ABCC2 and ABCC3 transporters is upregulated by hypoxia in HT29 colon carcinoma cells, but not in HepG2 hepatocarcinoma cells. Moreover, OCTN1 (SLC22A4), OCT1 (SLC22A1) and OATP-B (SLC02B1) transporter expression is also induced by inflammatory cytokines but in a smaller extent than in hypoxia. Furthermore our experiments indicate that there is no cross talk between HIF-1 and NF-κB pathways in HT-29 cells, but these two pathways act simultaneously activating common genes, such as, some SLC and ABC transporters. Our preliminary results from studies with an in vivo murine model of colitis, suggest that HIF-1is stabilized and OCTN1 is strongly induced during severe inflammation, which can be relevant for a recovery from the inflammatory process. We have also been interested in the distribution of HIF-1α variants among different ethnic groups as well as their contribution for cancer risk. Thus, we have demonstrated that there is an ethnicity-related variation in the frequency of the C1772T (P582S) single nucleotide polymorphism (SNP) in the HIF-1α gene. Furthermore, we performed a case-control study in a breast cancer population and our results suggest that there is no association between this SNP or the rare G1790A (A588T) SNP and the incidence of breast cancer. Taken together, the results obtained in this study contribute to a better knowledge of drug influx and efflux during hypoxia and inflammation as well as to the understanding of the pharmacogenetic variability of the HIF-1.