http://hdl.handle.net/10362/1054 DCV_PhD Fri, 24 May 2013 02:39:27 GMT 2013-05-24T02:39:27Z http://hdl.handle.net/10362/9682 Title: Signal transduction pathways involving the hypertension-related WNK1 and WNK4 protein kinases Authors: Mendes, Ana Isabel Vieira Lopes Abstract: The genes WNK1 and WNK4 belong to the subfamily of WNK protein kinases and their mutation causes pseudohypoaldosteronism type II, a rare familial form of hypertension with hyperkalemia and hypercalciuria. The molecular mechanisms underlying this condition involve the regulation of renal electrolyte homeostasis and the modulation of diverse ion channels and transporters via WNK kinases. Additionally, WNKs have also been reported to participate in signal transduction pathways related to cell survival and proliferation. The objective of the present thesis was to identify novel WNK1 and WNK4 interacting proteins and the underlying signal transduction pathways. First, it was found that WNK1 forms a protein complex with the Rab-GAP TBC1D4 and phosphorylates it in vitro. It was shown that the expression levels of WNK1 regulate surface expression of the constitutive glucose transporter GLUT1 in HEK293 cells. WNK1 is shown to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. Moreover, these effects were kinase activity-dependent. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. Second, WNK4 was found to promote the cell surface expression of the CFTR chloride channel in mammalian cells. The mechanism by which WNK4 acts on CFTR involves interaction with the tyrosine kinase Syk, which we found to phosphorylate tyrosine 512 (Tyr512) in the first nucleotide-binding domain of CFTR. The presence of WNK4 prevents this in vitro phosphorylation in a kinase-independent manner. In BHK21 cells stably expressing CFTR, Syk reduces, while WNK4 promotes, the cell surface expression of CFTR. Mutation of Tyr512 revealed that its phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk play an antagonistic role in this process. Finally, ten WNK4 variants were detected in a cohort of Portuguese patients and control individuals, which subsequently were tested for association to hypertension and/or osteoporosis. Despite none of the variants yield any significant association to hypertension, a rare missense alteration (rs56116165) in a highly conserved arginine residue showed a nominal association to osteoporosis. This finding advocates that this polymorphism is a rare allelic variant, in a candidate gene with a biological function in renal calcium homeostasis, that may contribute to a genetic predisposition to osteoporosis. Description: Dissertação apresentada para obtenção do Grau de Doutor em Biologia, na especialidade de Genética Molecular, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10362/9682 2011-01-01T00:00:00Z http://hdl.handle.net/10362/9424 Title: Molecular mechanisms of sexual development in basidiomycetes: exploring connections with lifestyles Authors: Coelho, Marco António Dias Abstract: This work concerns the investigation of the molecular mechanisms of sexual reproduction in fungi and their possible implication for fungal lifestyles (parasitic vs. saprobic) and for the emergence of asexual fungal lineages. The association between pathogenicity and sexuality is well-known in the basidiomycete plant parasite Ustilago maydis (subphylum Ustilaginomycotina), an economically important smut fungus. However, Ustilago species are phylogenetically interspersed with species of the genus Pseudozyma, which are considered saprobic and asexual. In this work, a study focused on genes involved in determining sexual identity (mating type or MAT genes), showed that Pseudozyma prolifica retains full sexual competence and pathogenicity, being therefore indistinguishable from U. maydis. For other Pseudozyma species, molecular analyses of PRF1, a gene that encodes a master regulator of sexual reproduction in U. maydis, showed no substantial evidence of loss of sexual reproduction. However, some clues were also found suggesting that some Pseudozyma species may be evolving towards a saprobic lifestyle. The earliest derived lineage of Basidiomycota (subphylum Pucciniomycotina) includes also important plant pathogens (rust and anther smut fungi) as well as lineages composed solely of saprobic organisms. Among the latter, the red yeasts of the order Sporidiobolales have the advantage of completing their life cycle in culture media, but have remained very little explored concerning the characterization of mating systems, the identification of MAT genes and the evolutionary relationships between sexual and asexual species. A comprehensive analysis of more than 200 strains belonging to 32 species of the Sporidiobolales indicated that asexuality seems to originate frequently from sexual lineages, but does not seem to persist long enough to form truly asexual species devoid of MAT genes. A more in-depth investigation of the red yeasts Rhodosporidium toruloides and Sporidiobolus salmonicolor allowed the identification for the first time in the Pucciniomycotina of the complete set of MAT genes. A detailed and multidisciplinary characterization of the mating system in the latter species yielded surprising results. A novel mating system that differs substantially from the two mating paradigms in basidiomycetes, the bipolar and tetrapolar systems, was brought to light. Given the basal phylogenetic position of the Pucciniomycotina within the Basidiomycota, this new system designated pseudo-bipolar, constitutes a significant contribution to the study of the evolution of MAT systems in fungi. Description: Dissertação para obtenção do Grau de Doutor em Biologia Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10362/9424 2011-01-01T00:00:00Z http://hdl.handle.net/10362/8798 Title: Analysis of the intranuclear life of nonsense transcripts Authors: Morgado, Ana Sofia João Abstract: Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs). Mammalian NMD depends on both splicing and translation, and requires recognition of the premature stop codon by the cytoplasmic ribosomes. Surprisingly, some published data have suggested that nonsense codons may also affect the nuclear metabolism of the nonsense-mutated transcripts. Therefore, we hypothesized that human β-globin transcripts sensitive to NMD could have a singular subcellular localization and processing state in mammalian cells nuclei. To determine if PTCs could influence nuclear events, we have established mouse erythroleukemia (MEL) cell lines stably transfected with wild-type or PTC-containing human β-globin genes. Subsequently, we analyzed the accumulation of NMD-competent β-globin transcripts versus wild-type counterparts using two different approaches: visualization of transcripts localization by fluorescence in situ hybridization (FISH); and quantification of pre-mRNA steady-state levels by ribonuclease protection assays (RPA) and reverse transcription-coupled quantitative polymerase chain reaction (RT-qPCR). FISH analysis shows that MEL cells stably expressing PTC-containing β-globin transcripts present a marked tendency to display an abnormal speckled-like pattern of localization in the nucleus. However, in addition to the presence of the PTC, other effectors may act on the β-globin transcripts localization, as some wild-type β-globin MEL cells presented this abnormal FISH phenotype as well. On the other hand, our analyses by RPA and RT-qPCR clearly show that β- -globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated β-globin pre-mRNA. Conversely, in non-erythroid HeLa cells, human β-globin pre-mRNAs carrying NMD-competent PTCs accumulate at normal levels. Half-life analysis of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control. Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected. In conclusion, our set of data highlights potential nuclear pathways that induce a selective downregulation of PTC-containing β-globin pre-mRNA in MEL cells, albeit not affecting their stability or splicing effectiveness. These specialized nuclear pathways, which may act in concert with the general NMD mechanism, might discriminate the NMD-sensitive transcripts as abnormal in a promoter- and/or cell line-specific manner, probably to obtain optimal NMD activity. Description: Dissertação para obtenção do Grau de Doutor em Biologia, Especialidade de Biologia Molecular Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10362/8798 2013-01-01T00:00:00Z http://hdl.handle.net/10362/8779 Title: Structural and functional studies of PpcA: a key protein in the electron transfer pathways of Geobacter sulfurreducens Authors: Morgado, Maria Leonor Carvalho Abstract: Geobacter species show an impressive respiratory versatility and can sustain their growth by using insoluble extracellular compounds as terminal electron acceptors. The genome of Geobacter sulfurreducens has been completely sequenced and it revealed an unprecedented number of putative c-type cytochromes, 73 of which with more than one heme binding site. Although numerous electron transfer proteins have been identified, the electron transfer pathways that allow G. sulfurreducens to obtain energy are still far from being understood. Five homologous triheme cytochromes (PpcA-E) were identified in G. sulfurreducens periplasm and gene knockout studies revealed their involvement in Fe(III) and U(VI) extracellular reduction. This thesis focuses on the characterization of these proteins, with special emphasis on PpcA, the most abundant in G. sulfurreducens’ periplasm. PpcA, PpcB, PpcD and PpcE were thermodynamically characterized in detail using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The results obtained showed that PpcA and PpcD were able to perform e-/H+ energy transduction in addition to their role in the electron transfer pathways. No evidence for coupling of e-/H+ transfer was observed for PpcB and PpcE. The functional implications of these results are discussed. PpcA solution structure in the fully reduced state was determined using NMR spectroscopy and the redox-Bohr center responsible for controlling the e-/H+ transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. In order to elucidate the physiologic function of PpcA individual key residues and understand its functional mechanism, a family of mutants covering the entire protein was prepared using site-directed mutagenesis. The results obtained revealed how proper tuning of the reduction potentials of the heme groups is fundamental to achieve concerted e-/H+ transfer. Description: Dissertação para obtenção do Grau de Doutor em Bioquímica, ramo de Biotecnologia Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10362/8779 2011-01-01T00:00:00Z