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O camundongo mdx, modelo murino para a Distrofia Muscular de Duchenne (DMD) possui uma mutação de ponto no gene da distrofina que resulta na ausência da proteína no músculo, porém seu fenótipo é brando o que o torna um bom modelo genético e molecular, mas não um bom modelo funcional. Esperando obter um modelo para DMD que tivesse um fenótipo mais fiel ao apresentado pelos pacientes humanos, optou-se por transferir a mutação mdx para o background 129/Sv. Através de cruzamentos sucessivos foram obtidas 3 gerações de animais mdx com background 129/Sv (mdx129) e cada geração foi avaliada funcionalmente por 6 meses. Desde a primeira geração é possível observar que os animais mdx129 são mais fortes do que os mdx originais em background C57BL (mdxC57BL), sendo o oposto do esperado no início dos experimentos. O estudo então foi redirecionado para tentar entender o motivo dessa melhora. Em relação ao padrão histológico, em geral há diferenças entre o mdxC57BL e mdx129. Observa-se também que os animais mdx129 entram no processo de degeneração mais tardiamente que os animais mdxC57BL e seu processo de regeneração se estende por mais tempo. Através de estudos de microarray foi possível observar que os animais 129/Sv apresentam poucos genes diferencialmente expressos (GDEs) em relação aos animais C57BL, portanto os dois backgrounds são muito semelhantes. O mdxC57BL apresenta muito mais GDEs em relação ao seu selvagem (C57BL) do que o mdx129 em relação ao 129/Sv, entretanto, ambos os modelos apresentam mais genes superexpressos do que subexpressos, indicando que as alterações distróficas e regenerativas estão mais associadas com a ativação do que a repressão de genes. Quando os GDEs de ambos os modelos de mdx são distribuídos em categorias funcionais, há o predomínio de genes ligados ao sistema imune e quando essa categoria é omitida para melhor visualização das restantes, observa se que ambos os modelos apresentam categorias funcionais semelhantes, porém com proporções diferentes. No modelo mdx129 se destaca a diminuição da participação da categoria de rota endo/exocítica (tráfego de vesículas) e homeostase e aumento da participação das categorias de matiz extracelular e atividade enzimática. Cada modelo apresenta genes exclusivos, destacando os genes SPP1 e IL1RN na comparação 129/Sv x mdx129F3. O gene SPP1 codifica a proteína osteopontina (OPN) e o polimorfismo rs28357094 neste gene é utilizado como biomarcador de prognóstico para DMD. O papel da OPN na progressão da distrofia não é bem conhecido. Alguns estudos afirmam que a ausência dessa proteína melhora a força muscular de camundongos mdx, enquanto outros apontam que sua participação é necessária para a regeneração muscular. Assim sendo, mais estudos serão necessários para verificar qual seria a via responsável pela melhora fenotípica do modelo mdx129. Já o gene IL1RN codifica a proteína IL-1Ra, a qual é um antagonista de interleucina 1 (citocina pró-inflamatória e pró fibrótica). Portanto o aumento da expressão do gene de seu antagonista sugere que os animais mdx129F3 podem estar mais protegidos do processo inflamatório causado por essas moléculas. Quando analisadas as listas filtradas para músculo esquelético das comparações C57BL x mdxC57BL e 129/Sv x mdx129F3 para a formação de vias metabólicas, foi gerada apenas uma via em ambas as comparações com número relevante de moléculas. A via gerada pela análise da lista C57BL x mdxC57BL possui mais moléculas do que a via gerada pela analise da lista 129/Sv x mdx129F3, porém, todas as moléculas presentes nesta via, estão presentes na via C57BL x mdxC57BL, indicando que mesmo com número diferente de moléculas envolvidas, os genes participam das mesmas vias. Tanto a comparação de cada geração de mdx129 com o 129/Sv como a comparação das gerações entre si mostram que os efeitos da mudança de background estão presentes desde a primeira geração e não se alteram significativamente com os cruzamentos sucessivos. / The mdx mouse, murine model for Duchenne Muscular Dystrophy (DMD) has a point mutation in the dystrophin gene that results in the absence of the protein in the muscle, however its phenotype is mild, which makes it a good genetic and molecular model, but not a good functional model. Hoping to obtain a model for DMD with a phenotype that is more similar the patients\', it was chosen to transfer the mdx mutation to the 129/Sv background. Through successive breedings, 3 generations of mdx animals with 129/Sv background were obtained and each generation was functionally evaluated for 6 months. Since the first generation it is possible to observe that the mdx129 animals are stronger than the original mdx with C57BL background. The results were the opposite of what was expected in the beginning of the experiments, therefore the study was redirectioned to try to understand the reason of the improved phenotype. About the general histological pattern, there are differences between mdxC57BL and mdx129. It can be observed that the mdx129 animals enter the degenerative process later than the mdxC57BL animals and the regenerative process lasts longer. Through microarray studies it was possible to observe that the 129/Sv animals present few differentially expressed genes (DEGs) in comparison to the C57BL animals; therefore both backgrounds are very similar. The mdxC57BL presents many more DEGs in comparison to C57BL than mdx129 in comparison to 129/Sv, however both models present more super expressed genes than sub expressed, indicating that the dystrophic and regenerative alterations are more associated to the activation rather than the repression of genes. When the DEGs of both mdx models are distributed in functional categories, there is the predominance of genes related to the immune system and when this category is omitted for the better visualization of the remaining, it can be observed that both models present similar functional categories, but with different proportions. In the mdx129 model we can highlight the decrease in participation of the endo/exocytic pathway (vesicle traffic) and homeostasis categories, and increase in participation of the extracellular matrix and enzymatic activity categories. Each model presents exclusive genes, highlighting SPP1 and IL1RN in the comparison 129/Sv x mdx129F3. SPP1 encodes the protein osteopontina (OPN) and the polymorphism rs28357094 in this gene is used as a DMD prognostic biomarker. The role of OPN in the dystrophy progression is not well known. Some studies claim that the absence of OPN increases the muscle strength of the mdx mouse, while others indicate that its participation is necessary to muscle regeneration. More studies are needed to ascertain what pathway is responsible for the phenotypic improvement of the mdx129 model. The IL1RN gene encodes the protein IL-1Ra, and interleukin 1 antagonist, which is a pro-inflammatory and pro-fibrotic cytokine. Therefore, the increase in the expression of its antagonist suggests that the mdx129F3 animals may be more protected from the inflammatory process caused by these molecules. When the filtered lists for skeletal muscle of the comparisons C57BL x mdxC57BL e 129/Sv x mdx129F3 were analyzed for the formation of metabolic pathways, only one pathway was generated in both comparisons. The pathway generated in the analysis C57BL x mdxC57BL has more molecules that the one generated by the 129/Sv x mdx129F3 list, but all molecules present in the latter are also present in the former, indicating that even with different numbers of molecules involved, the genes participate in the same pathways. The comparisons of each generation of mdx129 with the 129/Sv and the comparison of the generations among each other show that the effects of the background change are present since the first generation and are not altered with the successive breedings.

Methylation of cytosines within CpG dinucleotides is a feature of vertebrate DNA. The precise role of DNA methylation is unknown to date, although it has been implicated in several processes relating to transcriptional regulation. One approach to study DNA methylation is the characterization of proteins that bind specifically to methylated DNA. One such family of proteins is the methyl-CpG binding domain (MBD) containing family and MBD1 is a member of this family. MBD1 is implicated in transcriptional repression and various mechanisms by which it might bring about gene silencing have been proposed. These are mainly based on studies reporting interactions between MBD1 and various proteins that regulate chromatin structure. Also MBD1 function can be modified by PIAS proteins, which stimulate its conjugation to SUMO (small ubiquitinlike modifier).The original aim of this work was to address two questions about MBD1: (1) Does MBD1 form part of a stable complex with other factors, and if so, what are the identities of the other components? Purification of MBD1 revealed the presence of no stably bound interacting proteins. However, some evidence indicates MBD1 may interact with itself and form dimers, a finding which impacts on many aspects of the function of MBD1. Also a proteomics screen for transient interaction partners identified candidate binding partners for MBD1 and the related protein MeCP2, which may throw light on the function of these proteins. (2) Are there any activities which regulate MBD1 function by the removal of SUMO from this protein? No activities capable of removing SUMO from native MBD1 were found but it was demonstrated that this modification leads to the destabilization of MBD1 in vitro. The relevance of this finding in vivo is yet to be determined.

Post-translational protein modifications (PTM) vastly increase the complexity and functional diversity of the proteome, to precisely regulate crucial cellular processes. The plant immune system is composed of complex signalling networks that are influenced by various PTMs. Activation of plant immunity is associated with a rapid burst of nitric oxide (NO), which can covalently modify cysteine thiols within target proteins by a process termed S-nitrosylation to form S-nitrosothiols (SNOs), constituting a redox-based PTM. Another key PTM involved in plant immunity is SUMOylation, an essential mechanism involving the conjugation of the small ubiquitin-like modifier (SUMO) peptide to lysine residues within target proteins. Although the targets and mechanisms of S-nitrosylation and SUMOylation are becoming evident, how these key PTMs are themselves regulated remains obscure. Work presented in this thesis reveals that during plant immune signalling, the sole Arabidopsis thaliana SUMO conjugating enzyme, SUMO CONJUGATING ENZYME 1 (SCE1), is S-nitrosylated at a highly conserved, but previously uncharacterized cysteine. S-nitrosylation of SCE1 was shown to inhibit its SUMO conjugating activity in vitro and mutational analysis revealed that the site of this modification, Cys139, is not required for enzyme activity but rather constitutes a redox-sensitive inhibitory switch. Generation and characterization of transgenic Arabidopsis plants overexpressing both wild-type and mutant forms of SCE1 revealed that Cys139 is required for efficient immunity against bacterial pathogens. Furthermore, after immune activation, S-nitrosylation of this residue inhibits global SUMOylation of proteins. These results provide evidence of a novel means of crosstalk between S-nitrosylation and SUMOylation in the context of plant immunity. The abundant cellular antioxidant, glutathione (GSH), is S-nitrosylated to form S-nitrosoglutathione (GSNO), which is thought to constitute a stable reservoir of NO bioactivity. In Arabidopsis, GSNO levels are controlled by the enzyme S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which indirectly influences the levels of protein SNOs. In this study, transgenic plants overexpressing FLAG-epitope tagged GSNOR1 were generated in various mutant backgrounds, including nitric oxide overproducer 1 (nox1), to further investigate the roles of GSNOR1 and NO in plant immunity. It was shown that ectopic GSNOR1 expression completely recovers developmental and disease susceptibility phenotypes of gsnor1, but not nox1 mutant plants, highlighting in vivo differences between accumulation of GSNO and free NO. Surprisingly, elevated NO levels in nox1 plants promote S-nitrosylation of GSNOR1, inhibiting its enzymatic activity. This suggests a previously unreported means by which NO might regulate its own bioavailability. Further work in this study revealed that recombinant GSNOR1 can be SUMOylated in vitro, which appeared to increase its enzymatic activity. Several potential SUMO modification sites were identified within GSNOR1 and mutational analysis revealed that at least one of these, Lys191, is SUMOylated. Co-immunoprecipitation experiments revealed that transgenic GSNOR1 might be SUMOylated in vivo, although the site(s) and biological function of SUMOylation were not identified. Nonetheless, these results reveal another possible layer of interplay between S-nitrosylation and SUMOylation.

Thesis (Ph.D. (Pharmacy))--North-West University, Potchefstroom Campus, 2008.

Die Spinozerebelläre Ataxie Typ 3 (SCA3) oder Machado-Joseph-Krankheit (MJD) gehört zur Gruppe der neurodegenerativen Polyglutaminerkrankungen (PolyQ-Erkrankungen) und ist die häufigste autosomal-dominante zerebelläre Ataxie weltweit. Ein in der Länge hochvariabler Polyglutaminabschnitt ist vermutlich die Ursache für die Toxizität der ansonsten nicht verwandten Proteine, welche die PolyQ-Erkrankungen verursachen. Abgesehen von dem verlängerten Polyglutaminbereich scheinen die physiologische Funktion und der zelluläre Kontext dieser Proteine und ihrer Interaktionspartner entscheidend für die spezifische Pathogenese und den Krankheitsverlauf zu sein. Diese Arbeit soll dazu beitragen, genetische Interaktoren zu identifizieren, welche die PolyQ-Toxizität verstärken oder vermindern, um somit die molekularen Krankheitsmechanismen zu entschlüsseln, die durch die Trinukleotid-Wiederholungen ausgelöst werden.  Dafür wurde ein humanes, von Ataxin-3 abgeleitetes Transgen in den Facettenaugen von Drosophila exprimiert.  Die daraus resultierende Degeneration der Photorezeptoren induziert einen Raue-Augen-Phänotyp (Rough Eye Phenotype, REP) in adulten Fliegen. Um genetische Modifikatoren des REP zu identifizieren, wurde die Expression bestimmter Gene (Fliegengene mit einem humanen Ortholog, insgesamt ca. 7.500) augenspezifisch per RNAi vermindert. Mögliche Veränderungen im beobachteten REP sind dann höchstwahrscheinlich auf den RNAi-vermittelten Knockdown der Genexpression zurückzuführen. Damit wären die stummgeschalteten Kandidatengene zur Modifizierung der PolyQ-induzierten Neurotoxizität fähig.  Die auf diese Weise identifizierten Genprodukte sind in verschiedene biologische Prozesse involviert und stehen stellvertretend für unterschiedlichste molekulare Funktionen. Für eine Auswahl von Kandidatengenen wurden zusätzliche Untersuchungen angestellt, um die Art und das Ausmaß der Interaktionen zu bestimmen. Dabei wurden neue Modifikatorengene analysiert, welche z. B. in die Methylierung von tRNA oder den Sphingolipid-Metabolismus involviert sind. Diese Ergebnisse können neue Erkenntnisse bei der Aufklärung der Pathogenese der MJD und anderer PolyQ-Erkrankungen hervorbringen und gleichzeitig zum Verständnis der Rolle von Ataxin-3 und seinen Modulatorproteinen beitragen.

The main objective of the thesis was to compare two methods of calculating adjusted attributable fractions and deaths as applied to smoking exposure and four health outcomes, lung cancer, ischaemic heart disease, chronic obstructive pulmonary disease, and cerebrovascular disease, for Canadians 30 years or older in the year 2005. An additional objective was to calculate variance estimates for the evaluation of precision. Such estimates have not been published for Canada to date. Attributable fractions were calculated using the fully adjusted method and the partial adjustment method. This method requires confounder strata specific (stratified) estimates of relative risk, along with accompanying estimates of variance. These estimates have not previously been published, and were derived from the Cancer Prevention Study II cohort. Estimates of the prevalence of smoking in Canada were obtained from the Canadian Community Health Survey 2005. Variance estimates were calculated using a Monte Carlo simulation. The fully adjusted method produced smaller attributable fractions in each of the eight disease-sex-specific categories than the partially adjusted method. This suggests an upwards bias when using the partial adjustment method in the attributable fraction for the relationship between cigarette smoking and cause-specific mortality in Canadian men and women. Summed across both sexes and the four smoking related causes of death, the number of deaths attributable to smoking was estimated to be 25,684 using the fully adjusted method and 28,466 using the partial adjustment method, an upward bias of over ten percent, or 2,782 deaths. It is desirable, theoretically, to use methods which can fully adjust for the effect of confounding and effect modification. Given the large datasets required and access to original data, using these methods may not be feasible for some who would wish to do so.

Thesis (Ph.D. (Pharmacy))--North-West University, Potchefstroom Campus, 2008.

Thesis (Ph.D. (Pharmacy))--North-West University, Potchefstroom Campus, 2008.

The main objective of the thesis was to compare two methods of calculating adjusted attributable fractions and deaths as applied to smoking exposure and four health outcomes, lung cancer, ischaemic heart disease, chronic obstructive pulmonary disease, and cerebrovascular disease, for Canadians 30 years or older in the year 2005. An additional objective was to calculate variance estimates for the evaluation of precision. Such estimates have not been published for Canada to date. Attributable fractions were calculated using the fully adjusted method and the partial adjustment method. This method requires confounder strata specific (stratified) estimates of relative risk, along with accompanying estimates of variance. These estimates have not previously been published, and were derived from the Cancer Prevention Study II cohort. Estimates of the prevalence of smoking in Canada were obtained from the Canadian Community Health Survey 2005. Variance estimates were calculated using a Monte Carlo simulation. The fully adjusted method produced smaller attributable fractions in each of the eight disease-sex-specific categories than the partially adjusted method. This suggests an upwards bias when using the partial adjustment method in the attributable fraction for the relationship between cigarette smoking and cause-specific mortality in Canadian men and women. Summed across both sexes and the four smoking related causes of death, the number of deaths attributable to smoking was estimated to be 25,684 using the fully adjusted method and 28,466 using the partial adjustment method, an upward bias of over ten percent, or 2,782 deaths. It is desirable, theoretically, to use methods which can fully adjust for the effect of confounding and effect modification. Given the large datasets required and access to original data, using these methods may not be feasible for some who would wish to do so.