Blood vessels are critical for the delivery of oxygen and nutrients to all cells in the body. To properly function, blood vessels and their primary components must develop and mature into a healthy network, capable of dynamic alterations to meet new needs of the body. The early genetic and molecular programs that "push" the vasculature to develop are the same programs that reactivate when there are normal changes to the body such as injury, muscle growth or decline, or aging; and when pathologies arise like cancer, stroke, and diabetes. Therefore, it is crucial to understand how the vasculature develops into a healthy system by studying all components as they mature. Endothelial cells that comprise the vessels themselves are joined by specialized partner cells called pericytes that help guide and mature vessel growth. Pericytes lie elongated along endothelial cells and have multiple points of contact with the endothelium. In this position, pericytes assist in cell-cell communication and even blood flow regulation in the microvasculature. To study the relationship between endothelial cells and pericytes during development, we observed vascular morphology in three and four dimensions, as well as the genetic and molecular mechanisms underlying how these cells are recruited and interact in several experimental models. Thus, to thoroughly analyze the morphology of these vessels, we developed a rigorous methodology using a MATLAB program to determine the colocalization and coverage of pericytes associated with vessels in large image sets. After developing analytical methods to investigate all the components of the blood vessel wall, we expanded our investigation of how pericytes and other aspects of microvasculature develop in animal models, specifically a more commonly used murine model for vascular development and for treatment of human diseases. Our findings of vascular development in mice suggest that there are important differences in how human and mouse brain blood vessels form. Therefore, studies using mice must be carefully designed to account for these discrepancies. Additionally, research into why human and mouse neurovascular development and maturation are different can aid in the development of improved experimental models to better treat human pathologies. / Doctor of Philosophy / Blood vessels have the crucial job of delivering oxygen and nutrients to all the cells in the body. To perform this duty, blood vessels- and the components that make them- must develop and mature into a healthy network, capable of altering itself to meet new needs of the body. The early programs that “push” the vessel system to develop are the same programs that reactivate when there are normal changes to the body such as injury, muscle growth or decline, or aging; and when abnormal diseases arise like cancer, stroke, and diabetes. Therefore, it is critical to understand how blood vessels develop into healthy systems by studying all of their components as they mature. Endothelial cells that comprise the vessels themselves are joined by specialized partner cells called pericytes that help guide and mature vessel growth. Pericytes lie elongated along endothelial cells and have multiple points of contact with the endothelium. In this position, pericytes assist in cell-cell communication and even blood flow regulation in smaller vessels called capillaries. To study the relationship between endothelial cells and pericytes during development, we observed vascular anatomy in three and four dimensions, as well as mechanisms underlying how these cells come together and interact in several experimental models. Thus, to thoroughly analyze the morphology of these vessels, we developed a rigorous methodology using a MATLAB program to determine the colocalization and coverage of pericytes associated with vessels in large image sets. After developing analytical method to investigate all the components of the blood vessel wall, we expanded our investigation of how pericytes and other aspects of blood vessels develop in animal models, specifically a more commonly used animal model for vascular development and for treatment of human diseases. Our findings of vascular development in mice suggest that there are important differences in how human and mouse brain blood vessels form. Therefore, studies using mice must be carefully designed to account for these discrepancies. Additionally, research into why human and mouse neurovascular development and maturation are different can aid in the development of improved experimental models to better treat human illness and injury.

Effector proteins are exported to the interior of host cells by numerous plant pathogens. Effector proteins have been well characterized in bacteria. However, the mechanisms through which these effectors promote virulence are largely unknown. Bioinformatic analysis of genome sequences from oomycete pathogens Phytophthora sojae, P. ramorum, P. infestans and Hyaloperonospora arabidopsidis (Hpa) have led to the identification of a large number of candidate effector genes. These effector genes have characteristic motifs (signal peptide, RxLR and dEER) that target the effectors into plant cells. Although these effector genes are very diverse, certain genes are conserved between P. sojae and H. arabidopsidis, suggesting that they play important roles in pathogenicity. The goal of my first project was to characterize a pair of conserved effector candidates from Hpa and P. sojae. We hypothesized that these effectors have important conserved roles with regard to infection. We found that the Hpa effector was expressed early during the course of infection of Arabidopsis and triggered an ecotype-specific defense response in Arabidopsis, suggesting that it was recognized by host surveillance proteins. Both the effectors from Hpa and P. sojae respectively could suppress immunity triggered by pathogen associated molecular patterns (PTI) and by effectors (ETI) in planta. They also enhanced bacterial virulence in Arabidopsis when delivered by the Type III secretion system. Similar results were seen with experiments with transgenic Arabidopsis expressing the effectors. My second project showed that a different Hpa effector protein, HaRxL10, targets the Jasmonate-Zim Domain (JAZ) proteins that repressed responses to the phytohormone jasmonic acid (JA). This manipulation activates a regulatory cascade that reduces accumulation of a second phytohormone, salicylic acid (SA) and thereby attenuates immunity. This virulence mechanism is functionally equivalent to but mechanistically distinct from activation of JA-SA crosstalk by the bacterial JA mimic coronatine. These results reveal a new mechanism underpinning oomycete virulence and demonstrate that the JA-SA crosstalk is an Achilles\' heel that is manipulated by unrelated pathogens through distinct mechanisms. / Ph. D.

No / BACKGROUND: A Full Thickness Macular Hole (FTMH) is often associated with vitreomacular traction, and this can be asymmetric with vitreomacular traction on one side of the hole but not the other. In cross-section, the elevated retinal rim around a developed FTMH is seen as a drawbridge elevation, and this drawbridge elevation may be used as a measure of morphological change. Examination of the drawbridge elevation of the retinal rim in FTMH with asymmetric vitreomacular traction may help to clarify the role of vitreomacular traction in the development of FTMH. METHOD: Cases of FTMH were identified with an initial OCT scan showing vitreomacular traction on one side of the hole only and that had a follow-up OCT scan showing progression of the hole. A tangent to the retinal surface at a distance of 700 microns from the axis of the hole was used as a marker of the drawbridge elevation of the retinal rim around the macular hole. Comparisons of the drawbridge elevation and change in drawbridge elevation between the sides with and without initial vitreomacular traction were made. RESULTS: There was no significant difference between the drawbridge elevation, or change in drawbridge elevation, on the side of the hole with initial vitreomacular traction compared to the side without initial traction. CONCLUSION: There is some intrinsic mechanism within the retina to link the morphological changes on the two sides of a FTMH. A bistable hypothesis of FTMH formation and closure is postulated to explain this linkage.

No / The inner retinal complex is a well-defined layer in spectral-domain OCT scans of the retina. The central edge of this layer at the fovea provides anatomical landmarks that can be observed in serial OCT scans of developing full-thickness macular holes (FTMH). Measurement of the movement of these points may clarify the mechanism of FTMH formation. This is a retrospective study of primary FTMH that had a sequence of two OCT scans showing progression of the hole. Measurements were made of the dimensions of the hole, including measurements using the central edge of the inner retinal complex (CEIRC) as markers. The inner retinal separation (distance between the CEIRC across the centre of the fovea) and the Height-IRS (average height of CEIRC above the retinal pigment epithelium) were measured. Eighteen cases were identified in 17 patients. The average increase in the base diameter (368 microns) and the average increase in minimum linear dimension (187 microns) were much larger than the average increase in the inner retinal separation (73 microns). The average increase in Height-IRS was 103 microns. The tangential separation of the outer retina to produce the macular hole is much larger than the tangential separation of the inner retinal layers. A model based on the histology of the Muller cells at the fovea is proposed to explain the findings of this study.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The etiology of anxiety disorders remains incompletely understood. Clear evidence for a genetic component has been proposed; however, there is also an increasing focus on environmental factors and the interaction between these and the genetic components that may mediate (anxiety) disorder pathogenesis. No single gene or genetic component has been explicitly identified as being involved in the development of anxiety disorders. This is most likely due to a number of reasons, which include, for example, the heterogeneity of anxiety disorders, the contribution of environmental factors and methodological limitations (e.g. small sample size) of research studies. Until now, genetic association studies usually focused on one particular psychiatric disorder at a time. However, with the difficulty in identifying susceptibility genes and/or loci in heterogeneous disorders like obsessive-compulsive disorder and other conditions in the anxiety spectrum, it is perhaps timely to consider multivariate genetics and epidemiological studies in a number of disorders sharing a core characteristic – such as anxiety. In addition to genetic underpinnings, a number of environmental variables have also been identified as risk factors for pathological anxiety, including adverse life events like childhood physical and sexual abuse. The hypothesis for this project is that a pre-existing genetic vulnerability (or genetic risk) interacts with the impact of adverse life events to result in the development of one or more anxiety disorder(s). Considering phenotypic overlap amongst the anxiety disorders, it is likely that diverse networks of genes and/ or interacting pathways are responsible for the phenotypic manifestations observed. Sprague Dawley rats exhibiting behaviours indicative of anxiety in the context of environmental stressors (maternal separation and restraint stress) were used as model for the identification of novel susceptibility genes for anxiety disorders in humans. The striatum has previously been implicated as a candidate in the brain architecture of anxiety pathogenicity, and is also a site exhibiting a high degree of synaptic plasticity. The synaptic plasticity pathway was investigated using the dorsal striatum of the rat brain and several genes were identified to be aberrantly expressed in “anxious” rats relative to controls (Mmp9, Bdnf, Ntf4, Egr2, Egr4, Grm2 and Arc). In humans, it was found that the severity of early adversity was significantly and positively associated with the presence of an anxiety disorder in adulthood. When the human homologues of the susceptibility candidate genes that were identified using the animal model were screened in a human cohort of patients with obsessive-compulsive disorder (OCD), panic disorder (PD) or social anxiety disorder (SAD) (relative to controls), five single nucleotide polymorphisms (SNPs) were found to be significantly associated with these conditions. Four of these SNPs were also found to significantly interact with the severity of childhood trauma. Haplotype analysis of variants within the identified susceptibility candidates revealed novel haplotype associations, four of which are located in the MMP9 gene. Notably, this the first study to link these particular mutations in the MMP9 gene with anxiety disorders and this finding is consistent with previous work suggesting that MMP9 is involved in conditions like cardiovascular disease and cancer which have been associated with increased prevalence of anxiety disorders. In conclusion, this project yielded important findings pertaining to the etiology of anxiety disorders. The use of a combined anxiety disorders cohort (OCD, PD and SAD) may suggest that the associations found here may hold true for anxiety disorders in general and not only for a particular clinically delineated condition. Childhood trauma was confirmed as an increased susceptibility risk for anxiety disorders. Also, this research contributed several novel susceptibility genes (MMP9, EGR2, EGR4, NTF4, and ARC), five significant SNP associations, four significant SNP-environment interactions and five haplotype associations (within MMP9 and BDNF) as candidates for anxiety pathogenicity. The identified polymorphisms and haplotypes were demonstrated to be associated with susceptibility to anxiety disorders in a gene-environment correlation and gene-environment interaction. / AFRIKAANSE OPSOMMING: Die oorsake van angssteurings word steeds nie volledig verstaan nie. Daar is duidelike bewyse vir 'n genetiese komponent, maar daar is ook toenemende fokus op omgewingsfaktore en die interaksie tussen hierdie omgewingsfaktore en genetiese komponente by angssteurings. Geen enkele geen of genetiese komponent is al geïdentifiseer as diè wat betrokke is by die ontwikkeling van angssteurings nie. Dit is waarskynlik weens 'n aantal redes, wat byvoorbeeld, die heterogeneïteit van angssteurings, die bydrae van omgewingsfaktore en metodologiese beperkings (bv. klein steekproef) van die navorsingstudies, insluit. Verder het genetiese assosiasiestudies tot nou toe gewoonlik net op een spesifieke psigiatriese versteuring op 'n slag gefokus. Maar, gegewe die uitdaging om vatbaarheidsgene en / of loci in heterogene steurings soos obsessief – kompulsiewe steuring (OKV) en ander toestande op die angsspektrum te identifiseer, is dit tyd om genetiese en kliniese studies in ‘n aantal steurings - met ‘n oorvleuende kern-element soos angs -, gesamentlik te oorweeg. Bykomend tot die genetiese boustene, is ‘n aantal omgewingsveranderlikes soos traumatiese lewenservarings tydens die kinderjare as risikofaktore vir patologiese angs geidentifiseer. Die hipotese vir hierdie projek is dat daar 'n interaksie tussen genetiese kwesbaarheid (of genetiese risiko) en traumatiese lewensevarings is en dat dit tot die ontwikkeling van 'n / veelvoudige angssteuring(s) kan lei. Inaggenome die fenotipiese oorvleueling tussen die angssteurings, is dit waarskynlik dat diverse netwerke van gene en / of interaktiewe geen-paaie vir die manifestasie van hierdie toestande verantwoordelik is. Sprague Dawley-rotte met gedragswyses aanduidend van angs, in die konteks van omgewingstressore (d.i. skeiding van die ma-rot en bedwang-stres [restraint stress]), is as model gebruik vir die identifisering van nuwe vatbaarheidsgene vir angssteurings in mense. Die striatum is voorheen as ‘n kandidaat in die brein-argitektuur van patologiese angs voorgehou, en is ook ‘n plek met ‘n hoë mate van sinaptiese plastisiteit. Die sinaptiese plastisiteit is ondersoek deur te fokus op die dorsale striatum van die rotbrein en daar is verskeie gene gevind wat anders is in “angstige” rotte in vergelyking met kontroles (Mmp9, Bdnf, Ntf4, Egr2, Egr4, Grm2 en Arc). In mense is daar gevind dat die ernstigheidsgraad van vroeë trauma beduidend en positief met die teenwoordigheid van ‘n angssteuring tydens volwassenheid verband hou. Toe die menslike ekwivalente van die vatbaarheidsgene wat met die dieremodel geïdentifiseer is in ‘n mens-kohort met obsessief-kompulsiewe steuring (OKS), panieksteuring (PS) en sosiale angssteuring (SAS) ondersoek is, is gevind dat daar 5 enkele nukleotied polimorfismes (ENPs) is wat met die toestande verband hou. Daar is ook gevind dat vier van hierdie ENPs beduidend verband hou met die ernstigheidsgraad van trauma tydens die kinderjare. Haplotipe analise van variante binne die geïdentifiseerde vatbaarheidsgene het op nuwe haplotipe assosiasies – waarvan 4 op die MMP9-geen geleë is – gedui. Hierdie is dus die eerste studie wat gevind het dat dié spesifieke mutasies van die MMP9-geen met angssteurings verband hou. Hierdie bevinding strook met vorige werk wat daarop dui dat die MMP9-geen by toestande soos kardiovaskulêre siekte en kanker wat ook met verhoogde voorkoms van angssteurings verband hou, betrokke is. Ter afsluiting kan ons sê dat hierdie projek belangrike bevindinge oor die oorsake van angssteurings gemaak het. Die gebruik van ‘n gekombineerde angssteurings-kohort (OKS. PS en SAS) kan moontlik suggereer dat die assosiasies wat ons hier gevind het, waar is vir alle angssteurings en nie net vir ‘n spesifieke afgebakende toestand nie. Traumatiese ervarings tydens die kinderjare is ook bevestig as ‘n risiko vir die ontwikkeling van angssteurings. Hierdie navorsing het ook verskeie nuwe vatbaarheidsgene (MMP9, EGR2, EGR4, NTF4, en ARC), 5 beduidende ENP assosiasies, 4 beduidende ENP-omgewings-interaksies en 5 haplotipe assosiasies (by MMP9 en BDNF) geïdentifiseer as moontlike kandidate wat ‘n rol speel by die ontstaan van patologiese angs. Daar is ook gevind dat die geïdentifiseerde polimorfismes en haplotipes met vatbaarheid vir angssteurings in ‘n geen-omgewing- korrelasie en geen-omgewing- interaksie verband hou. Stellenbosch University http://scholar.sun.ac.za

Le sujet de ma thèse était de décrire sur le plan moléculaire le processus d'embryogenèse somatique chez la vigne. Pour cela, les étapes-clés d'entrée et de sortie du cycle d'embryogenèse secondaire ont été caractérisées par l'analyse de l'expression de quelques gènes impliqués dans le développement ou la défense, en particulier les gènes PR10.Grâce à l'exploitation de la séquence complète du génome de Vitis vinifèra disponible sur le site du Genoscope, j'ai pu caractériser exhaustivement la famille multigènique des PR10. Celle-ci est composée de 17 séquences disposées en tandem et formant un cluster compact sur le chromosome 5, dont 3 pseudogènes et au moins 13 séquences transcrites. L'expression de 10 de ces gènes a d'abord été analysée par RT-PCR semi-quantitative dans différents organes de la plante et dans des tissus traités au 2,4-D. Elle suggère une diversification fonctionnelle marquée. De plus, le niveau d'expression de plusieurs gènes PR10 est élevé dans les cals embryogènes, suggérant qu'ils pourraient jouer un rôle lors de l'embryogenèse somatique. L'étude de l'expression des gènes PR10 par RT-PCR quantitative en temps réel dans différents tissus ayant montré une capacité embryogénique variable lorsqu'ils sont soumis à un traitement par le 2,4-D met en évidence que le niveau d'expression varie entre les gènes et selon les tissus. L'expression de certains gènes est fortement induite par le 2,4-D dans les tissus à capacité embryogénique et seulement faiblement dans les tissus ne donnant jamais d'embryons somatiques, ce qui suggère fortement que ceux-ci pourraient être des marqueurs de la capacité embryogénique chez la vigne. / The objective of my work was to analyse the somatic embryogenesis process of Vitis vinifera at a molecular scale. Thus, the expression of genes implied in development or defence, especially PR10 genes, was monitored during the key-steps of entrance and exit of secondary somatic embryogenesis. The complete sequence of the Vitis vinifera genome available on the Genoscope website allowed the exhaustive characterization of the PR10 multigene family, which is constituted by 17 sequences localised on a tandem array on the chromosome 5. Among these 17 sequences, 3 are pseudogenesand at !east 13 are transcribed sequences. The expression of 10 PR10 genes was first monitored in various grapevine tissues and in tissues after 2,4-D treatment using semi-quantitative RT-PCR. The results suggest a strong functional diversification. Moreover, the expression of several PR10 genes is high in embryogenic calli, suggesting that these genes could intervene in somatic embryogenesis. The expression of PR10 genes was also monitored in tissues showing different somatic embryogenic capabilities under 2,4-D treatment using quantitative RT-PCR. The results show that regulation of PR10 genes is dependent of the gene and tissue considered. Moreover, the expression of some genes is highly induced by 2,4-D treatment in tissues having embryogenic capability, white it is only weakly induced in tissues having no embryogenic capability, suggesting that these gene could be markers of embryogenic capability in grapevine.

[Truncated abstract] Asthma genetic studies have identified many genes that contribute to the pathogenesis of asthma and related variables. Members of the secretoglobin family appear to play an important role in controlling airway inflammation but they have received relatively little attention in asthma genetic research. In this thesis, I have investigated the genes of two members of the secretoglobin family (16 kDa Clara cell secretory protein (CC16) and secretoglobin 3A2 (SCGB3A2)) that are expressed at high levels in the airways and are important anti-inflammatory agents. The overall aim of these studies was to investigate the genetic variability of the CC16 and SCGB3A2 genes and their influence on airway inflammatory disease. The main hypothesis was that genetic variability in the genes for CC16 and SCGB3A2 exert an influence on airway inflammatory disease. Three populations were investigated: (1) a paediatric case control population (n=99), (2) an unselected birth cohort followed longitudinally at ages 1 month (n=244), six (n=123) and 11 years (n=195) and (3) an unselected Aboriginal Australian population (n=251). The case-control population was screened for novel DNA sequence variants in the CC16 promoter and the SCGB3A2 5’UTR and exons. No novel sequence variants were identified in the CC16 promoter and two were identified in the SCGB3A2 5’UTR (G- 811A and G-205A). A single nucleotide polymorphism previously identified in the CC16 gene (A38G) and the two polymorphisms identified in the SCGB3A2 gene were genotyped in both unselected populations. Genotype/phenotype associations were identified with adjustment for potential confounders such as age, gender, height and maternal tobacco smoking, where appropriate. This was due to the contribution of these factors to the aetiology of asthma, atopy and related phenotypes. All three polymorphism frequencies were significantly different between these two ethnically diverse populations

The Australian HIV Observational Database (AHOD) is a collaboration of hospitals, sexual health clinics and specialist general practices throughout Australia, established in April 1999. Core data variables collected include demographic data, immunological and virological markers, AIDS diagnosis, antiretroviral and prophylactic treatment and cause of death. The first electronic data transfer occurred in September 1999 followed by six monthly data transfers thereafter. All analyses included in this thesis are based on patients recruited to AHOD by March 2004. By March 2004, 2329 patients had been recruited to AHOD from 27 sites throughout Australia. Of these, 352 (15%) patients were recruited from non-metropolitan clinics. The majority of patients were male (94%), and infected with HIV through male homosexual contact (73%). Almost 90% of AHOD patients are antiretroviral treatment experience, and the majority of patients are receiving triple therapy as mandated by standard of care guidelines in Australia. Antiretroviral treatment use has changed in Australia reflecting changes in the availability of new treatment strategies and agents. The crude mortality rate was 1.58 per 100 person years, and of the 105 deaths, more than half died from HIV-unrelated deaths. The prevalence of HBV and HCV in AHOD was 4.8% and 10.9%, respectively. HIV disease progression in the era of highly active antiretroviral treatment (HAART) among AHOD patients is consistent with what has been reported in developed countries. Common factors associated with HIV disease progression were low CD4 cell count, high viral load and prior treatment with mono or double therapy at the time of commencing HAART. This was demonstrated in AHOD in terms of long-term CD4 cell response, the rate of changing combination antiretroviral therapy and factors predicting death. HBV and HCV coinfection is also relatively common in AHOD, similar to other developed country cohorts. Coinfection does not appear to be serious impediments to the treatment of HIV infected patients. However, HIV disease outcome following HAART does appear to be adversely affected by HIV/HCV coinfection but not in terms of HIV/HBV coinfection. Patients attending non-metropolitan sites were found to be similar to those attending metropolitan sites in terms of both immunological response and survival.

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Atherosclerosis is a common degenerative disease in which the clinical manifestations are often through stroke or myocardial infarction. Some of the established risk factors for atherosclerosis include elevated plasma low-density lipoprotein (LDL)-cholesterol levels, obesity, diabetes mellitus (DM) and cigarette smoking. Of the risk factors, an elevation in plasma LDL is one of the most established and the most researched. This is partly a consequence of the deposition of cholesterol within arterial intima being a crucial step in the progression of atherosclerosis, combined with the finding that LDL particles are a major transporter of cholesterol in circulation. Recently there is increasing evidence showing a role of the other major transporter of cholesterol in circulation, chylomicron remnants, in the progression of atherosclerosis. The notion of atherosclerosis as a postprandial phenomenon has been further substantiated by the emergence of evidence showing a direct role of chylomicron remnants in arterial cholesterol deposition. Based on evidence that chylomicron remnants are proatherogenic, the suggestion arises that accumulation of postprandial lipoproteins in plasma may add another dimension of risk to the development of coronary artery disease (CAD). This thesis tests the general hypothesis that individuals with or at high risk of CAD have postprandial dyslipidaemia and that this metabolic abnormality is correctable with a class of lipid-lowering drugs called statins. To test the hypothesis, clinical studies were conducted in normolipidaemic CAD patients, heterozygous familial hypercholesterolaemia (FH) and postmenopausal women with type 2 DM. Determination of postprandial dyslipidaemia by comparison with control populations were conducted initially in each patient group (Studies 1, 3 and 5), followed by intervention studies investigating possible modulation of the dyslipidaemia with a statin (Studies 2, 4 and 6). Six observation statements based on case-control comparisons of postprandial lipaemia in patients with or at risk of CAD and the effects of statins on postprandial dyslipidaemia in the patient groups were derived from the general hypothesis. The observation statements were examined in the individual studies described below. Postprandial lipoprotein metabolism was assessed using a number of methods. For comparison of postprandial lipaemia in Studies 1 and 2, a classic oral fat challenge was utilised. As markers of chylomicrons and chylomicron remnants, retinyl palmitate and triglyceride were measured postprandially as well as apolipoprotein (apo) B48 concentrations, a specific marker of intestinal lipoproteins. ApoB48 was also measured in the fasting state and found to predict the postprandial responses of retinyl palmitate, triglyceride and apoB48. This suggested that fasting measurement of apoB48 could be used as a simple indicator of postprandial dyslipidaemia. Consequently for Studies 3 - 6, fasting apoB48 measurements were used as primary markers of postprandial dyslipidaemia. Other markers for chylomicrons and their remnants utilised were fasting plasma concentrations of remnant-like particle-cholesterol (RLP-C) and apoC-III. As well as these static markers, chylomicron remnant catabolism was measured using a stable isotope breath test. The breath test involves the intravenous injection of a chylomicron remnant-like emulsion labelled with ¹³C-oleate and measurement of enriched ¹³CO2 in expired breath by isotope ratio mass spectrometry. The fractional catabolic rate (FCR) of the injected emulsion was subsequently calculated using multi-compartmental modeling (SAAM II). The studies are presented in this thesis as published and unpublished works. In Study 1, postprandial lipoprotein metabolism was compared between 18 normolipidaemic CAD patients (cholesterol 4.54 ± 0.12 mmol/L, triglyceride 1.09 ± 0.16) with 13 asymptomatic healthy controls using an oral fat challenge. Normolipidaemic CAD patients had higher postprandial area-under-curve (AUC) for triglyceride (+34%, p=0.019), retinyl palmitate (+74%, p=0.032) and apoB48 (+36%, p<0.001). Fasting apoB48 was also higher (+41%, p=0.001) and found to correlate significantly with AUC of triglyceride (p=0.017), retinyl palmitate (p=0.001) and apoB48 (p<0.001). The data suggest that normolipidaemic CAD patients have increased concentrations of intestinal lipoproteins in the fasting and postprandial state. In addition to these findings, significant correlations of fasting apoB48 with postprandial markers (p<0.02) suggests the fasting marker to be a simpler surrogate marker for the degree of total postprandial lipaemia. Study 2 investigated the effect of atorvastatin treatment on postprandial dyslipidaemia found in the 18 near-normolipidaemic CAD patients from Study 1. The trial was conducted in a randomised, placebo-controlled design, using oral fat challenges before and after 12-weeks atorvastatin/placebo treatment. Compared with the placebo group, atorvastatin decreased the total postprandial AUC for iii triglyceride (-22%, p=0.05) and apoB48 (-34%, p=0.013). Fasting markers of apoB48 (-35%, p=0.019) and RLP-C (-36%, p=0.032) also decreased significantly. Atorvastatin was also found to increase LDL-receptor activity by +218% (p<0.001) as reflected in binding studies. The data suggest atorvastatin reduces the fasting levels of intestinal lipoproteins as well as total postprandial lipaemia, but without acute dynamic changes in postprandial lipaemia. The reduction in fasting and total postprandial lipoprotein levels could be partly attributed to an increase in LDL-receptor mediated removal from circulation. In Study 3, postprandial lipaemia was compared in 15 heterozygous FH patients with 15 healthy controls. FH patients had higher fasting concentrations of apoB48 (+56%, p<0.001) and RLP-C (+48%, p=0.003). The elevation in these fasting markers of chylomicrons and their remnants suggests FH patients have postprandial dyslipidaemia due to an accumulation of these particles in plasma. Study 4 examined the effects of long- (> 6 months) and short-term (4 weeks) simvastatin treatment on modulating postprandial dyslipidaemia found in the 15 FH patients from Study 3. Short- and long-term simvastatin treatment decreased the fasting concentrations of apoB48 (-29% and 15% respectively, p<0.05) and RLP-C (both -38%, p<0.001), but did not significantly alter the FCR of the injected chylomicron remnant-like emulsion. The data suggest that in heterozygous FH both long- and short-term simvastatin treatments decrease the fasting markers of postprandial lipoproteins by mechanisms that may not be mediated via processes differentiated by the 13CO2 breath test. This implies that the effect on postprandial lipaemia may be from a decrease in production and/or a possible increase in catabolism of triglyceride-rich lipoproteins (TRLs). In Study 5, postprandial lipaemia was compared in 24 postmenopausal women age and body mass index matched with 14 postmenopausal women with type 2 DM. Postmenopausal diabetic women were found to have higher fasting concentrations of apoB48 (+21%, p=0.021) and apoC-III (+16%, p=0.042) as well as lower FCR of the chylomicron remnant-like emulsion (-50%, p<0.001). The data suggest that postmenopausal diabetic women have postprandial dyslipidaemia, and that this is due to delayed catabolism of chylomicron remnants. Study 6 was an hypothesis-generating exercise examining the effects of 4-weeks pravastatin treatment on postprandial dyslipidaemia found in 7 postmenopausal women with type 2 DM from Study 5. Although plasma LDL-cholesterol was reduced (-19%, p=0.028), there were no significant effects found on fasting apoB48 concentrations (-12%, p=0.116) or the FCR of the chylomicron remnant-like emulsion (+38%, p=0.345). A larger sample size of patients and/or treatment with a more potent statin at a dosage known to affect chylomicron remnant metabolism would be required to demonstrate a significant reduction in postprandial dyslipidaemia in postmenopausal women with type 2 DM. The results of the above mentioned studies combined support the general hypothesis that postprandial dyslipidaemia is a feature of patients with or at risk of CAD. This defect may be demonstrated using fasting apoB48 as an indicator of the degree of postprandial lipaemia. Postprandial dyslipidaemia may reflect a reduction in catabolism, as suggested with the breath test in type 2 DM, and/or an over overproduction of chylomicrons. Both these mechanisms would also increase competition for lipolysis and clearance pathways between hepatically and intestinally-derived lipoproteins. The exact mechanisms by which postprandial dyslipidaemia occurs are yet to be determined. Statins appear to improve defective postprandial lipaemia in patients with or at risk of CAD, which is in agreement with the general hypothesis. The effectiveness of a statin is dependant on their potency in inhibiting cholesterol biosynthesis and increasing receptor mediated clearance of LDL and chylomicron remnants. The studies conducted in this thesis show that postprandial dyslipidaemia can be reduced by statins but not to the extent demonstrated in controls. However, the demonstrated reduction in fasting and total postprandial lipaemia translates to a lowering in overall arterial exposure to circulating proatherogenic particles. The elevation in fasting and postprandial levels of proatherogenic chylomicron remnants found in the patient groups described in this thesis indicates another dimension to their risk of coronary disease. The reductions in the overall levels of proatherogenic particles in patients with or at high CAD risk, infers a possible reduction in the risk of coronary disease in these patients.

Indiana University-Purdue University Indianapolis (IUPUI) / Progression of Chlamydia into upper reproductive tract epithelium and the induction of subsequent immune responses to infection are major contributors to Chlamydia-induced pathogenesis of the genital tract. We reported that C. muridarum infection of the oviduct epithelial cells (OEs) secrete IFN-β in a TLR3 dependent manner. However, we showed that the C. muridarum infected TLR3-deficient OEs were still able to secrete minimal amounts of IFN-β into the supernatants, which is suggestive that there are other signaling pathways that contribute to Chlamydia-induced IFN-β synthesis in these cells. Previous studies describing the activation of the JAK/STAT signaling pathway during Chlamydia infection of cervical epithelial cells proposes a putative role for STAT1 in the synthesis of type I IFNs during Chlamydia infection. The present study investigated the role of STAT1 in Chlamydia-induced IFN-β production in OEs. OEs were infected with Chlamydia muridarum and analyzed at 24 hours by RT-PCR and western blot to determine STAT1 expression. STAT (-/-) OEs were infected and IFN-β production measured by ELISA. Quantitative real-time PCR analyses were performed at 6 and 16 hour post-infection to elucidate the mechanisms involved in IFN-β production during infection. Fluorescent microscopy was used to observe changes in Chlamydia replication. STAT1 activation and expression were significantly increased in wild-type (WT) OEs upon infection. TLR3 (-/-) OEs showed diminished STAT1 protein activation and expression. Augmented STAT1 protein expression corresponded to STAT1 mRNA levels. ELISA analyses revealed significantly less IFN-β production in infected STAT1 (-/-) OEs compared to WT OEs. Quantitative real-time PCR data showed that gene expression of IFN-β and of type I IFN signaling components were significantly increased during late stage Chlamydia infection, dependent on STAT1. Temporal regulation and increases in expression of IFN-α subtypes during infection were STAT1-dependent. Our results implicate STAT1-mediated signaling as a contributor to the C. muridarum-induced synthesis of IFN-β and other type I IFNs in OEs. We previously described a major role for TLR3 in the early-stage Chlamydia-induced synthesis of IFN-β in OEs; the results from this study suggest a role for STAT1 in the synthesis of type I IFNs that occurs during early and late stages of infection.