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von Willebrand Disease (VWD) is the most common inherited bleeding disorder in humans, resulting from quantitative or qualitative deficiencies of von Willebrand factor (VWF). Type 3 VWD is the rarest and most severe form of the disease. This thesis characterizes the phenotype-genotype correlations of a cohort of Canadian type 3 VWD patients and their family members. Three main findings are highlighted: 1) 50% of families showed evidence of co-dominant inheritance as opposed to recessive, 2) 42% of mutations identified were located in the VWF propeptide region (VWFpp), 3) index cases (IC) with mutations in the VWFpp had a more severe bleeding diatheses than IC with mutations elsewhere. We investigated two of the identified VWFpp mutations (ex4-5del and Cys633Arg) to elucidate their molecular mechanisms using two cellular models. Patient-derived blood outgrowth endothelial cells (BOEC) are ideal for studying the underlying molecular mechanism of VWF mutations as they represent the native vascular endothelium. BOEC were isolated from type 3 VWD IC and family members with the mutations of interest. A heterologous cellular system was also used to study the VWF mutations in vitro. The VWFpp mutations caused impaired VWF secretion, defective multimerization, qualitative and quantitative defects in Weibel-Palade body (WPB) formation, and resulted in VWF retention within the endoplasmic reticulum. We attempted to restore secretion and multimerization by co-transfecting each mutant with the wild-type VWF propeptide (VWFpp), which was unsuccessful. Additionally, we investigated a third mutation, c.8419_8422dupTCCC, which is unique to the Canadian VWD population and is found at a high frequency in a specific geographic population. While we hypothesized that this mutation would disrupt dimerization due to its location in the C-terminal cysteine knot (CK) domain of VWF we did not find this to be true. The results presented within this thesis provide new insight into the genetics and pathobiology of type 3 VWD, the functional contribution of the VWFpp to type 3 VWD and highlight the utility of BOEC as a cellular model for evaluating the pathogenic mechanisms of VWF mutations. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-10-17 21:15:37.685

Kawasaki disease (KD) is a multisystem vasculitis that results in localized coronary artery elastin breakdown and aneurysm formation. It is the leading cause of acquired heart disease of children in North America. Despite conventional treatment, a significant proportion of patients continue to develop coronary sequelae. The mechanisms of arterial aneurysm formation in KD are not known. Using a murine model of KD, Lactobacillus casei cell wall extract-induced coronary arteritis, the processes leading to coronary aneurysm formation were examined. Vessel damage occurred as a result of the increased enzymatic activity of the elastase, matrix metalloproteinase (MMP)-9. MMP-9 protein and activity levels were elevated in the heart post-disease induction. Expression and activity were specific for and localized to inflamed coronary arteries. The pro-inflammatory cytokine, tumour necrosis factor (TNF)-α, was required for increasing local MMP-9 expression. Importantly, MMP-9-deficient animals had a significantly reduced incidence of elastin breakdown. Furthermore, in a cohort of KD patients, serum MMP-9 did not correlate with coronary outcome, highlighting the importance of local expression of this elastase. Intravenous immunoglobulin (IVIG) and aspirin/salicylate are therapeutic agents in current use for the treatment of KD, though their exact mechanisms of action in KD are not known. The biologic effects of IVIG and salicylate on critical stages of disease development were examined. IVIG and salicylate had differential effects on TNF-α expression, with therapeutic concentrations of IVIG inhibiting, and salicylate inducing, TNF-α expression leading to an indirect modulation of MMP-9 expression. Interestingly, TNF-α expression and MMP-9 activity were both directly inhibited by the metal-chelating drug doxycycline. Treatment of affected mice with doxycycline significantly improved coronary outcome. Inhibiting both the inflammatory response as well as the downstream effects of inflammation were of therapeutic value in this model of KD. These results taken together demonstrate the importance of MMP-9 in the pathogenesis of coronary artery aneurysms in KD. Targeting MMP activity holds the promise of transforming KD from the leading cause of acquired heart disease to a self-limited febrile illness.

Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, characterized by deposition of lipoproteins in the arterial wall. Atherosclerotic plaques form preferentially in distinct regions of the vasculature such as branch points, curvatures and bifurcations, suggesting that local hemodynamic forces may contribute to disease susceptibility. Shear stress imparted on endothelial cells (ECs) by the flowing blood has been shown to modulate gene expression and remodelling of the artery. In this thesis, an in vitro model was established to recreate the contrasting environments found in atherosclerosis-prone and atherosclerosis-resistant regions of the vasculature to demonstrate a direct causal-relationship between shear stress and expression of endothelial nitric oxide synthase (eNOS) and p65 in ECs. In vitro assessment of cell shape and expression patterns of these anti- and atherogenic genes demonstrated that shear stress can induce cell morphology and gene expression patterns that are similar to ECs in atherosclerosis-prone and atherosclerosis-resistant regions of the mouse vasculature. Regulation of eNOS transcription by shear stress was demonstrated using a transgenic mouse model and in vitro heterogeneous nuclear RNA (hnRNA) quantification. Similar to ECs in atherosclerosis-prone regions, epithelial cells lining the small intestine lumen express high levels of p65. To investigate the effects of p65 expression levels on constitutive and tumour necrosis factor α (TNFα)-induced nuclear factor-κB (NF-κB) signalling, p65 expression was suppressed in HeLa cells by RNA interference. Lower p65 expression resulted in reduced TNFα-induced expression of NF-κB target genes, including many subunits of inhibitor of nuclear factor κB (IκB), demonstrating modulation of NF-κB priming by p65 expression levels. Suppression of p65 also affected constitutive expression levels of IκB, and resulted in re-setting of the NF-κB/IκB equilibrium. Experiments using inhibitors of canonical NF-κB signalling found that basal expression of NF-κB components is independent of nuclear factor κB kinase β (IKKβ) activity and proteasome-mediated degradation of IκBα. Together, these studies elucidate the mechanism of flow-mediated gene regulation and the effect of resulting changes in p65 expression on NF-κB signalling.

With superior longevity, approximately 5 times that of compact fluorescents (CFLs), and high efficacy, around 1.5 times that of CFLs, LEDs are now attracting vast attention from both academic and industrial sectors. Unfortunately, current power supply drivers for LEDs have the following drawbacks: (1) for a two-stage configuration, the power factor correction (PFC) circuit can help LEDs achieve good operating performance but contain too many components and are large in size, have low efficiency and relatively high cost; (2) a single-stage configuration can perform well in PFC and efficiency, however reliability issues occur due to the use of the electrolytic capacitor. In this thesis, the theoretical analysis and implementation of two high power factor, soft-switched, electrolytic-capacitor-less LED drivers are presented. The two drivers solve the aforementioned issues while minimizing its size and cost. The detailed theoretical analysis illustrates the advantages of the presented circuits and provides insight into their design and operation. The simulated and experimental implementations verified the performance of both circuits, which achieve a high power factor, indicating that the drivers have good operating performance. Elimination of the electrolytic capacitors improves the LED drivers’ reliability. In addition, with the help of soft-switching capability, high efficiency is achieved. Simulation and experimental results are presented to support all merits of the two circuits. / Thesis (Master, Electrical & Computer Engineering) -- Queen's University, 2013-04-30 13:22:28.471

The APOBEC3 (Apo3) family of proteins are single stranded (ss) DNA cytosine deaminases (C → U). They are grouped into two different structural groups, the single catalytic domain Apo3 enzymes (Apo3A, Apo3C, and Apo3H) and the double catalytic domain Apo3 enzymes (Apo3B, Apo3D, Apo3F, and Apo3G). Apo3G has been implicated in protection from HIV proliferation by becoming encapsidated into budding HIV virions and subsequently mutationally inactivating the synthesized provirus. This largely occurs in the absence of HIV viral infectivity factor (Vif) which mediates the ubiquitination and degradation of Apo3G. Apo3G is a processive enzyme, able to catalyze numerous deaminations in a 5'CCC motif in a single interaction with a substrate. There is a paucity of biochemical data on other Apo3 family members. We performed basic biochemical assays that determined the relative specific activities, processivity, cytosine motif preferences, and binding affinities for DNA, of Apo3A and Apo3H using synthetic DNA substrates in deamination assays. We found Apo3A to be an enzyme with low processivity and Apo3H to be a highly processive enzyme; both of which deaminate a 5'TC motif. Using a reconstituted HIV replication assay we assessed if processivity is needed for efficient restriction of HIV. We were able to demonstrate that each, Apo3G, Apo3A, and Apo3H were able to catalyze deaminations during in vitro reverse transcription. The mutation profile of both Apo3A and Apo3H showed that the 5'TC motif preference was less effective compared to Apo3G in triggering missense and nonsense mutations in the HIV protease active site coding sequence. Nuclear DNA can become deaminated by the related Apo3 family member activation-induced deaminase (AID), when it is present in the nucleus of activated B cells. Apo3A and Apo3H are located in the nucleus but the extent of the damage they cause has only recently been investigated. Here we used an in vitro transcription assay to determine the efficiency of Apo3A and Apo3H deamination during transcription and found that, like AID, they are highly capable of causing deaminations during transcription. Taken together, the results presented here demonstrate that processivity is not necessary for an Apo3 enzyme to catalyze deaminations during HIV reverse transcription and that Apo3A and Apo3H can catalyze deaminations during DNA transcription that could damage host genomic DNA. These results imply a potential cost for maintaining nuclear deaminases.

Personality tests are widely used in South Africa. The application of personality assessment techniques for clinical and personnel decisions has been a major activity for psychologists. All main personality models have ken developed in a Western context: the question therefore arises whether these models are adequate and sufficient for South Africa. There is a need to develop personality tests that are based on South African cultures. In South Africa the continuous use of Western-based personality tests raise a challenge. The challenge also lies with the current legislation with regard to the use of psychological tests. The challenge is to construct an inventory suited to the local needs while retaining the standards of validity and reliability expected of established assessment instruments. Our socially diverse society and its wide implications for the cultural dynamics of personality evaluations that we find in South Africa warrant further research. A qualitative research design was used in this study, including interviewing as a data gathering method. A total of 120 Tshivenda speaking people from the Thohoyandou district in Limpopo province and Pretoria in Gauteng province were interviewed. A total of 4 722 personality descriptors with a view to Tshivenda speaking people were obtained from the participants, and then translated into English. Content analysis was used to analyse, interpret and reduce these descriptors to a total of 150 personality characteristics which are the most important perspectives of personality for the Tshivenda speaking individuals. The personality characteristics were divided into eight categories, namely interpersonal relatedness, sociability, conscientiousness, emotionality, meanness, intellect, dominance and a category for other traits. The interpersonal relatedness factor in the Vhavenda personality characteristics could be regarded as a cultural factor. The Tshivenda speaking people are also sociable with a preference for companionship, social skills and numerous friendships. They also have a strong sense of purpose and high inspiration levels. The Tshivenda speaking people also experience emotions and feelings related to situations that they face. Intellect characteristics could also be extrapolated from the personality characteristics of the Vhavenda people. The findings of this study were compared to the five factor model, and evidence was found for extraversion and conscientiousness in particular. Very few characteristics of openness on the five factor model correspond to that of the Tshivenda speaking people, except in the cases of a few personality characteristics that were labelled under the category of intellect. Recommendations for future research are made. / Thesis (M.A. (Industrial Psychology))--North-West University, Potchefstroom Campus, 2007.

Aim The aim of this study was to investigate the most commonly used approaches to measure individual genome-wide heterozygosity (IGWH) and to investigate whether IGWH can be considered as a health risk factor or a protective factor in humans. Methods This study was based on two samples from isolated communities of Croatian Adriatic islands, with a total of 1,930 adult examinees from Islands of Vis (N=986) and Korcula (N=944). Examinees were genotyped with a total of 302,662 single nucleotide polymorphisms. Heterozygosity was estimated using five commonly calculated methods. Results Correlation coefficients between different heterozygosity methods were generally in the range of 0.7-0.8. A worsening in some phenotypic traits, including cholesterol and triglycerides as well as increased odds for osteoporosis and metabolic syndrome was recorded in cases of IGWH reduction. Nevertheless, in these cases heterozygosity explained a relatively low amount of variance, generally in range of 0.4-0.6% of total trait variance. Conclusion However, these results were significant in Vis Island sample, while in the replication sample, Korcula Island, most of the associations were not significant, possibly due to the overall lower amount of inbreeding and higher heterozygosity in Korcula Island sample. The results warrant further research in order to provide more information on the extent and importance of individual genome-wide heterozygosity, which might have an important role in communities which experience consanguinity on a greater scale. Two main shortcomings of the study include possible lack of power to detect inbreeding depression and the need to replicate the results in other populations.

The transcription factor Interferon Regulatory Factor-1 (IRF-1) has been demonstrated to suppress tumour growth through the regulation of many anti-oncogenic genes. Pro- and anti-apoptotic factors, cell cycle control genes, DNA damage response genes and prometastatic factors are all under the control of IRF-1, which effects both transcriptional activation and repression. In addition to these cell autonomous tumour suppressor activities, IRF-1 is also a key regulator of the immune system and, as such, mediates immune surveillance of tumours. Numerous studies have confirmed that loss or mis-regulation of IRF-1 is a key factor in several different types of cancer. Despite strong evidence for the crucial role of IRF-1 in cancer, and frequent assertions that this protein warrants further investigation as a drug target, very little is known about its regulation. Furthermore, since recent studies have linked upregulation of IRF-1 to the development of autoimmune diseases, it is particularly important that drugs be able to decouple autoimmune and anti-cancer functions of IRF-1 to avoid harmful side effects. This thesis describes how phosphorylation of IRF-1 in its regulatory C-terminal Mf1 domain modulates transactivatory and tumour suppressor activity. Phosphospecific antibodies were developed as tools to study the C-terminal phosphorylation. Using these, it was shown that treatment of cells with Interferon-γ(IFN-γ) not only causes accumulation of IRF-1 protein, but also results in phosphorylation of IRF-1 at two sites in the C-terminal Mf1 domain. Phosphomimetic mutants demonstrated that these phosphorylations enhanced the transactivatory activity of IRF-1 at various promoters, but did not affect repressor activity. Gel shift assays revealed that dual phosphorylation of IRF-1 (IRF-1 D/D) promoted DNAbinding and suggested this was through increased interaction with the cofactor/histone acetylase p300 which induces a conformational change in IRF-1, favouring DNA-binding. Acetylation by p300 appears to be important although it is not yet clear whether this directly or indirectly affects IRF-1 activity. Since the tumour suppressor activity of IRF-1 is of particular interest, the effect of phosphorylation was examined in clonogenic and invasion assays. IRF-1 D/D more efficiently suppressed colony formation in both anchorage dependent and independent assays, and may improve inhibition of invasion in Transwell assays. Thus, cell treatment with the therapeutic agent IFN-γ nduces phosphorylation of IRF-1, resulting in enhanced DNA binding of IRF-1 through improved p300 binding. In cells the outcome is more effective tumour suppression and inhibition of metastasis.

Background: Support for the four factor construct validity of the third edition of the Wechsler Adult Intelligence Scale (WAIS-III) has been found in clinical and non clinical populations but some studies question whether more complex models consistent with the concepts of fluid and crystallised intelligence provide a better explanation of the data. The WAIS-III is frequently used in the diagnosis of learning disability, however, previous exploratory factor analysis of data from a population with low IQ did not support the explicit four factor structure of the WAIS-III. Method: A confirmatory factor analysis of the WAIS-III was carried out on data from people with severe and significant learning disability and people with borderline IQ (IQ = 70-79). Results: The data from the borderline IQ sample and the sample with significant learning disability showed at best a weak fit to the explicit four factor models and more complex five or six factor models. However fit of the data from the sample with severe learning disability was poor for all models. Discussion: The findings show little support for the explicit four factor construct validity of the WAIS-III for people with borderline IQ or significant or severe intellectual impairment. Some support is found for the direction taken by the new Wechsler children’s and adult scales (WISC-IV & WAIS-IV) in aligning interpretation of the scales more closely to concepts such as fluid and crystallised theory. The research also suggests the cut-off point of IQ 70 is not reflective of an actual difference in cognitive profile as measured by the WAIS-III. Limitations of this study and implications for further research are also discussed.