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Thesis submitted in fulfilment of the requirements for the degree Masters of Technology: Radiography in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology / BACKGROUND: Diabetic patients are at increased risk of cardiovascular events and stroke, and its prevention is therefore the desired goal. In the arsenal of available techniques, ultrasound plays a vital role in primary healthcare. It is reliable, cost-effective and a noninvasive diagnostic tool that may prove beneficial for screening individuals at risk of cardiovascular disease (CVD) and stroke in SA. OBJECTIVE: To determine the interrelationships between carotid ultrasound findings with glycaemia status and contributing risk factors of atherosclerosis in the selected population. METHODS: Initially blinded by the glycaemia status, blood results, contributing risks and patient demographics, both carotid arteries were evaluated with duplex ultrasound (DUS), during July 2010 – July 2011. Using graphs, figures, frequency tables, means and standard deviations for the selected study population, univariate, multivariate and stepwise regression analysis was done to determine the association between ultrasound findings and risk factors for atherosclerosis. The hypothesis tested in this study was to determine if there is an increased incidence of carotid artery intima-media thickening (CIMT), plaque formation and stenosis in patients diagnosed with T2DM and hyperglycaemia in a very specific sub-population of mixedancestory, residing in Bellville South Africa (BSA). RESULTS: Of 534 subjects, 375 were of mixed ancestry and ≥35yrs of age, which met the inclusion criteria for the carotid ultrasound substudy. The glycaemic status for each individual was established, and 44% (165/375) were diagnosed hyperglycaemic, of which 66.7% (110/165) were diabetic (T2DM) and 33.3% (55/165) were pre-diabetic (Pre-DM). Majority (56%:265/375) had a normal glycaemic status. The ultrasound measurement of the carotid wall thicknesses (Mean Rt. and Lt. CIMT) revealed a statistically significant rise from normal glycaemia status to DM status for both the males (p = 0.0115*; p = 0.0259*) and females (p < 0.0001**; p < 0.0001**) respectively. In terms of plaques and internal carotid artery (ICA) stenosis (124/375), when grouped into normal and hyperglycaemic sub-groups, indicated plaque presence and some form of narrowing. A <50% stenotic ratio noted in 61% (76/124) of the hyperglycaemic group, that was 1.6 times higher than those with normal glycaemia (48/124). Predisposing factors demonstrated significantly higher levels in the females than in the males. The univariate multiple regression analysis after adjusted R² of 0.3247 for all independent variables (predisposing /contributing risk factor markers) of age (yrs.), SBP (mmHg), hs-CRP (mg/L), S-Cotinine (ng/mL) and LDL (mmol/L) showed statistically significant positive associations with dependent variable of the mean carotid wall thickness (p < 0.0001**, p< 0.0001**, p = 0.0033*, p = 0.0409* and p = 0.0044)* respectively. Statistically significant positive differences and standard error (SE), for every unit of change (1yr.) of age (yrs.), as a contributing factor for herosclerosis, there was a change in the mean carotid wall thickness as predicted according to this model. The total contribution of independent risk factors to CIMT ultrasound measurements were calculated as 34.5% (Adjusted R² = 0.3247). In the multivariate stepwise regression analysis, the independent variables of age (p< 0.0001) ** and systolic blood pressure (p < 0.0001) ** showed the strongest positive association with carotid wall thickeness changes. The hs-CRP (mg/L) inflammatory markers (p = 0.0014)* and LDL (mmol/L) (p = 0.0208)* were the 2nd and 3rd highest positive associated contributory risk factors for carotid artery wall thickening. The hip circumference (p = 0.0008)* and waist circumference (p = 0.0 555) + risk factors related to obesity was significant and approached significance, respectively, with the predicted increase of carotid artery wall thickening. CONCLUSION: Subjects diagnosed with T2DM and hyperglycaemia had increased levels of CIMT, plaques and carotid artery stenosis, compared to those subjects without T2DM. Age and systolic blood pressure, inflammatory (raised hs-CRP) and LDL cholesterol changes, and central (truncal) waist circumference adiposity, were positively associated with increased carotid intima media thickness. Smoking (S-Cotinine) and gender also reflected a direct relationship with CIMT changes. The hip circumference adiposity and diastolic blood pressure measurements were not directly associated with an increase in CIMT, which are in keeping with hypertension and obesity formulas. These findings confirm the association of thickened CIMT, plaques and stenosis with ‘unhealthy’ T2DM subjects at higher risk of CVD and stroke. The total contribution of independent risk factors to CIMT measurements were calculated as 34.5% (Adjusted R² =0.3247). The gathered information, discussion of results, and concluding statements thereby supports the recommendation of carotid artery ultrasound evaluation, for screening and diagnosis in primary health care, for ‘flagging’ high risk individuals at risk of stroke, so that lifestyle changes and appropriate management is early adopted.

Orientador: Eloiza Helena Tajara da Silva / Banca: Dorotéia Rossi Silva Souza / Banca: Fábio César Gozzo / Banca: Victor Evangelista de Faria Ferraz / Banca: Elaine Sbroggio de Oliveira Rodini / Resumo: A Neurofibromatose Tipo 1 (NF1) é uma doença autossômica dominante causada por mutações no gene NF1, responsável pela síntese da proteína neurofibromina. Muitos estudos publicados sobre NF1 têm focado as alterações desse gene e de seu produto em indivíduos afetados, mas as análises de expressão protéica são escassas. No presente estudo, nós investigamos diferenças quantitativas e qualitativas da expressão de proteínas entre amostras de neurofibroma e pele adjacente histologicamente normal, utilizando abordagem proteômica. As proteínas de neurofibroma e pele normal foram separadas por eletroforese bidimensional (2-DE) e identificadas por peptide mass fingerprinting, utilizando espectrometria de massas por dessorção e ionização a laser auxiliada por matriz com base no tempo de vôo (MALDI-TOF). Cinco proteínas foram identificadas: a caspase 14 e a proteína de choque térmico 27/HSP 27, que exibiram expressão reduzida em neurofibromas; a imunoglobulina, a flavina redutase e a proteína de ligação a fosfatidiletanolamina/PEBP, com expressão elevada em neurofibromas. Do nosso conhecimento, este é o primeiro relato de análise comparativa de neurofibromas e pele normal de pacientes com neurofibromatose tipo 1. Das proteínas identificadas, a HSP27 e a PEBP estão conectadas com as vias de sinalização celular p21ras ou cAMP, também relacionadas com a atuação da neurofibromina. A caspase 14 não exibe um elo conhecido com essas cascatas e tal fato pode abrir novos caminhos para o estudo da neurofibromatose. Estudos adicionais ainda são necessários para elucidar o papel dessas proteínas no desenvolvimento da neurofibromatose. Nosso estudo é um passo inicial na descoberta de mecanismos moleculares desta doença e mostra o valor da utilização da análise proteômica na identificação de novos parceiros da neurofibromina relacionados com o desenvolvimento da NF1. / Abstract: Neurofibromatosis Type 1 (NF1) is a common autosomal dominant disorder caused by mutations in the NF1 gene. Many of the studies published on NF1 have focused attention on the gene level, but protein expression analyses are scarce. In the present study, we investigated quantitative and qualitative differences in neurofibroma and histologically normal surrounding skin protein expression of NF1 patients, using a proteomic approach. Proteins from neurofibroma and normal skin were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Five proteins were identified: caspase 14 and heat shock protein 27 kDa protein/HSP 27 (downregulated in neurofibroma), immunoglobulin, flavin reductase and phosphatidylethanolamine binding protein/PEBP (upregulated in neurofibroma). To our knowledge, this is the first report of a comparative analysis of neurofibromas and normal skin from neurofibromatosis type 1 patients. Of the proteins identified, HSP27 and PEBP have a connection with p21ras or cAMP signaling. Caspase 14 has no known link with these pathways and may open a new avenue for studying neurofibromatosis. Further studies are still needed to elucidate the actual roles of the differentially expressed proteins. Our work is an initial step toward uncovering the molecular mechanism of this disease and shows the value of using proteomic analysis to identify novel partners of neurofibromin related to the development of NF1. / Doutor

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Les complexes PRC1 et PRC2 contrôlent l’expression génique via l’organisation de la structure de la chromatine. Ce contrôle se fait par l’ajout de la marque H2AK119ub1 par le PRC1 et l’ajout de la marque H3K27me3 par le PRC2. Cette étude s’attache à étudier le rôle de la protéine Pcgf1 (membre du complexe PRC1) et de la protéine Ezh2 (membre du complexe PRC2) lors du développement du poisson-zèbre. Les gènes sont inactivés par TALEN. Le complexe PRC1 est formé par différentes protéines dont les Pcgf. Il existe de nombreux homologues Pcgf qui ont des fonctions distinctes. Cette étude s’intéresse au rôle de la protéine Pcgf1 lors du développement du poisson-zèbre. Les individus pcgf1-/- sont viables et fertiles. Cependant, leur développement précoce est retardé et les adultes montrent des signes de vieillissement accéléré. Ce mutant est le premier modèle de vertébré qui met en évidence le rôle de Pcgf1 dans la prolifération cellulaire lors du développement et son association au vieillissement. La protéine Ezh2 est impliquée dans le devenir cellulaire et la différenciation. Les embryons se développent normalement puis les larves meurent à 12 jours post-fécondation. De façon intéressante, les embryons de poisson-zèbre peuvent gastruler en l’absence d’Ezh2, contrairement au modèle murin. Les organes sont correctement mis en place à 5 jours post-fécondation. Les larves présentent un défaut de maintien de la paroi du bulbe intestinal. La protéine Ezh2 est importante pour le maintien du pancréas exocrine. L’absence d’Ezh2 cause une augmentation importante du nombre de cellules apoptotiques. Ezh2 est essentiel lors de la régénération de la nageoire caudale. / PCR1 and PRC2 are complexes that control gene expression via chromatin structure reorganization. This expression regulation is maintained by adding epigentics marks H2AK119ub1 by the PRC1 and adding of H3K27me3 by the PRC2. The study devotes to study the role of the protein Pcgf1 (part of the PRC1 complex) and of the Ezh2 protein (part of the PRC2 complex) during the zebrafish development. The PRC1 complex is formed by different proteins including Pcgf proteins. There are several Pcgf homologs that have different functions. The study reveals that some Pcgf proteins have a different expression during caudal fin regeneration and development. We are interested in Pcgf1 protein during the zebrafish development. The pcgf1 gene was inactivated by using TALEN. The fish pcgf1-/- are viable and fertile. However, the early development is delayed and adults show signs of accelerated aging. This mutant is the first vertebrate model showing the role of Pcgf1 in cells proliferation during development and aging. Ezh2 protein is involved in cell-fate decisions and differenciation. Inactivation of ezh2 gene by TALEN reveals the essential role of Ezh2 during development. Indeed, at the beginning embryos develop normally then larvae die at 12 days post-fertilization. Interestingly, zebrafish embryo can gastrulate without Ezh2. This contradicts with observations in mouse model. The organs are properly formed at 5 days postfertilization. Larvae show defects in the intestinal bulb wall. Ezh2 is important for exocrine pancreas maintenance. The absence of Ezh2 causes an increase in apoptic cells. Ezh2 is essential during caudale fin regeneration.

Glycogen storage disease type II (GSDII) is an autosomal recessive lysosomal storage disorder caused by acid alpha-1,4-glucosidase (GAA) deficiency. This study aimed to provide an in-depth description of a late-onset GSDII (LO-GSDII) cohort (n=36) and assess potential genotype-phenotype correlation. We performed a clinical record-based study, some patients (n= 19) were also followed prospectively. Phenotypes were highly variable. We focused our clinical assessment onrespiratory failure, as it is the most frequent cause of death in LO-GSDII. In addition to standard spirometric measures, in a subgroup of patients (n = 10) we utilized a new tool, optoelectronic plethysmography (OEP), to investigate the pathophysiology of respiratory muscle impairment.The GAA gene was sequenced in every patient, and pathogenic mutations were identified inall of them. Almost all (35/36) patients carried the same mutation on one allele, IVS1-32-13T>G, which was in compound heterozygosity with a variety of other GAA mutations. To investigate genotype-phenotype correlation, we divided the patient cohort in two groups, according to the severity of the mutation on the second allele. The respiratory function study focused on diaphragmatic weakness. According to the change in forced vital capacity in supine position (ΔFVC), we defined patients with ΔFVC>25% ashaving diaphragmatic weakness (DW) and those with ΔFVC<25% as without diaphragmatic weakness (noDW). We measured pulmonary function and chest wall volumes using OEP inboth groups. We found a good correlation between the supine abdominal contribution to tidal volume (%VAB) and ΔFVC. Patients showed reduced chest wall and abdominal inspiratory capacity and low abdominal expiratory reserve volume. In terms of genotype-phenotype correlation, we counted more subjects in the group with severe second mutations (n=21) who had severe motor disability and respiratory dysfunction. However, this finding remains preliminary because differences were not significant, likely because of small sample size. Finally, in two smaller substudies, we investigated the occurrence of urinary and fecal incontinence in LO-GSDII, and reported a possibly non-fortuitous association of LO-GSDII and hydromyelia in two individuals. Overall, this work 1) provided new insight into genotype-phenotype correlation in GSDII, suggesting that it is of complex nature; 2) refined the analysis of respiratory muscle impairment and showed the utility of OEP for respiratory assessment in this neuromuscular disorder, and possibly in others as well; 3) indicated some so far little studied phenotypic features of LO-GSD-II that deserve further investigation. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished