No description available.

No description available.

No description available.

No description available.

Successful prevention of mother-to-child HIV transmission (PMTCT) programs have reduced the risk of infant HIV infection in South Africa from 8% in 2008 to an estimated 1.4% in 2015, resulting in an increasing population of HIV-exposed uninfected (HEU) children. However, the long-term effects of HIV and antiretroviral therapy (ART) exposure on the developing brain is not well understood. While HEU children perform better than their counterparts living with HIV, they continue to demonstrate greater neurodevelopmental delay than HIV-unexposed uninfected (HUU) children. As a result, neuroimaging studies have looked at the developing brain in this population, however there is little consensus about typical exposure related effects. In addition, it is unclear whether previously reported exposure-related results are directly related to in utero exposure to HIV, or indirectly via family and/or environmental factors. Research focused on newborns allows one to eliminate possible contributions from other factors, clarifying the influence of ART and HIV exposure on the developing brain. This dissertation employs neuroimaging and neurocognitive data in a well-characterized infant cohort to better understand the influence of maternal HIV infection on the uninfected brain. HEU infants were exposed to ART in utero between 3 and 9 months, allowing for the study of potential ART exposure effects of as well as HIV exposure. This dissertation will identify HIV and ART exposure effects on brain structure. In addition, the relationship between neonate brain structural outcomes and cognitive abilities at 9-12 months will be determined to identify potential functional consequences of early structural abnormalities. Chapter two presents an analysis of manually traced subcortical volumes in 120 unexposed uninfected (HUU) and exposed uninfected (HEU) neonates. HEU neonates demonstrated significantly reduced mean caudate volumes bilaterally and left mean putamen volumes relative to HUU neonates. Further analysis revealed the observed differences in basal nuclei volumes were related to duration of ART in utero. Infants exposed to ART throughout pregnancy had similar caudate and putamen volumes compared to their HU counterparts. While infants exposed to ART post conception (from 3 - 8 months in utero) had significantly smaller mean caudate volumes bilaterally, and a trending smaller left putamen volume compared to HUU infants. Chapter three examines the potential functional consequences of HIV/ART volumetric reductions. We modelled manually traced neonatal subcortical volumes with neuropsychological outcomes at 9 - 12 months. Among HUU infants, bilateral pallidum volumes predicted neuropsychological measures across all domains. All volumes, with the exception of bilateral thalamus and vermis, predicted the general quotient score in HUU infants. In contrast, among the HEU infants, volumes did not relate to neuropsychological outcomes with the exception of the caudate, putamen and vermis predicting locomotion scores in the preconception group. While no HIV exposure differences were present in neuropsychological domains, HEU infants recruit alternative subcortical structures compared to typically developing unexposed infants. Chapter four presents a DTI-tractographic analysis of white matter connections between subcortical structures manually traced. HEU demonstrate white matter alterations in two tracts - higher FA between right putamen and left thalamus and higher MD between caudate and thalamus on the right hemisphere. The WM alterations observed in HEU appear to be from roles of both HIV and ART exposure. In contrast to ART dependent subcortical grey matter reductions, the observed white matter alterations are independent of maternal treatment initiation. In addition, we also find associations between unaltered white matter connections and both maternal immune health and ART duration during pregnancy. These results suggest white matter is influenced to varying degrees by HIV and ART exposure, as well as maternal health in pregnancy. Chapter five looks at the possible functional consequences of the reported alterations in white matter integrity. We modelled white matter connections between manually traced neonatal subcortical volumes with neuropsychological outcomes at 9 - 12 months. Similar to chapter 3, within HUU infants, we observed a number of white matter connections predictive of neuropsychological outcomes across all domains. And almost no white matter tracts predicted neuropsychological measures in HEU infants. These results again point to HEU infants recruiting different pathways to perform basic tasks. In conclusion, the results documented in this thesis points to the influence of HIV exposure, ART duration and maternal immune health on fetal brain development. However, these factors impact grey and white matter differently. ART initiated pre-conception was protective of caudate volumes but did not protect two white matter connections, the WM tract between right thalamus and right caudate, and WM that between left thalamus and right putamen. Within HUU neonates, basal ganglia and cerebellar volumes and white matter connections predicted neuropsychological outcomes in late infancy. However, HEU infants did not demonstrate the same associations suggesting they utilize alternate pathways from their HUU peers. While there were no exposure related differences across neuropsychological domains, the long-term functional consequences of altered structural recruitment is unknown. Finally, this thesis adds to the body of literature that early ART in pregnancy is neuroprotective, and that HIV exposure related structural alterations are evident as early as 2 - 4 weeks after birth.

Background: Diabetes mellitus with uncontrolled hyperglycaemia is a major cause of cardiovascular complications and mortality. The developing foetal heart in-utero is particularly susceptible to hyperglycaemia through pathological remodelling, which results in life-long structural abnormalities such as cardiomyopathy and electrical defects like arrhythmias. However, the underlying mechanisms and potential therapeutic drug targets remain unclear. In this study, a cardiac developmental cellular model was used to study hyperglycaemia-induced remodelling. Methods: Mouse embryonic stem cells (mESCs) were differentiated into pulsatile, cardiac-like cells via embryoid body (EB) formation and cultured under baseline- or high glucose conditions. A Ca2+ -sensitive fluorescent dye Fluo-4 was used to measure calcium transients and a voltage-sensitive dye di-4-ANEPPS was used to record action potentials. Cellular biomarkers were detected using immunocytochemistry, confocal microscopy, and Western blotting as well as terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) and 5-ethynyl-2-deoxyuridine (EdU) assay. Results: Undifferentiated mESCs were positive for pluripotent transcription factors Nanog and Oct3/4, whereas the cardiac differentiated mESCs were positive for cardiac proteins troponin T, α-actinin 2, connexin 43, sarco-endoplasmic reticulum calcium ATPase 2 (SERCA 2) and α- and β-myosin heavy chain. Hyperglycaemia decreased the number of beating EBs, their beating rate, and their amplitude of contraction. It also decreased the calcium transient amplitude and the contractile response to ryanodine receptor stimulation by caffeine but did not alter the SERCA 2 expression. The amplitude and duration of action potentials in beating EBs were not altered by hyperglycaemia. However, structural changes included a decrease in EB size and expression of myofilament proteins, α-actinin and α- and β-myosin heavy chain and a disruption of the striated organization of the myofilaments. Hyperglycaemia increased the proportion of TUNEL-positive cells and the expression of the pro-apoptotic marker cytochrome c and decreased the anti-apoptotic protein Bcell lymphoma 2 but did not alter the mitochondrial staining with Mitotracker. It also increased the oxidative stress marker nitrotyrosine but did not alter the extent of EdU nuclear staining nor the expression of the receptor of advanced glycation end-product. The antioxidant n-acetyl cysteine decreased the fraction of hyperglycaemia-induced TUNEL-positive cells and improved the α-actinin striated pattern. Conclusion: Hyperglycaemia suppressed the cardiac differentiation and contractile activity of mESCs as well as disrupted the cardiac myofilament organisation and expression. These effects of hyperglycaemia were likely mediated by mitochondrial-dependent apoptosis triggered by oxidative stress as well as by the abnormalities in calcium signalling. These results have potential clinical implications in foetal diabetic cardiac disease and add novel insights into the mechanistic factors that represent new therapeutic drug targets in the developing foetal heart.

As the feminist movement in Africa continues to question and dismantle long-held religious and cultural beliefs and practices, this has influenced critical debates on the validity of their co-existence with human rights norms and standards on violence against women and discrimination. This dissertation aims to critically interrogate the relationship between culture, violence against women (VAW), and women's rights in Africa. Specifically, it delves into the cultural practice of bride price (also known as Lobola) to understand whether the practice is a cause and/or consequence of VAW in family relations. This dissertation hypothesises that Lobola is both a cause and consequence of VAW and should be explicitly identified as a form of VAW, a human rights violation. This dissertation adopts a theoretical analysis using Heise's Integrated Ecological Framework (Heise's framework) on VAW, which provides a valuable tool to analyse and deconstruct the systemic causes and influences of VAW. Heise's framework is adopted to analyse how certain cultural practices at the macro systemic level may relate to and influence VAW practices. To conduct this analysis using Heise's framework, this dissertation uses available literature on previously conducted focus group discussions on experiences, opinions and perceptions of Lobola by both young people and adults. The theoretical analysis highlights a positive correlation between Lobola and the violence women face in marriage and upon divorce. However, the research also establishes that the practice of Lobola itself does not present as a form of violence against women - but because of the perceptions, beliefs, and power systems it creates, the practice becomes an aggravator of VAW. The results from the analysis show that Lobola creates rigid gender roles, skewed beliefs of entitlement and ownership of women's lives and bodies, and asymmetrical power relations that influence VAW. Since the theoretical analysis proved that Lobola aggravates the violence women face in marriage and upon divorce, the dissertation also carries out a legal analysis to understand the position of harmful practices and the legal consequences using international human rights norms and standards on women's rights and VAW. In line with fulfilling its mandate, the United Nations Committee on the Elimination of all Forms of Discrimination against Women (CEDAW Committee), after receiving four state periodic reports from Kenya, Zambia, Zimbabwe and Uganda between 2010 and 2012, in its concluding observations expressed MBWPRE001-Pretty Mubaiwa concern over the persistence of harmful traditional and cultural practices that affect women. In each instance, the Committee reiterated that Lobola is a harmful practice that aggravates discrimination against women. These concluding observations ushered in an expanded narrative on harmful traditional and cultural practices that disproportionately affect women and thus promote gender-based discrimination. Using these four case studies, this dissertation, therefore, looks at these countries' customary and civil laws to understand the legislative landscape around traditional and cultural practices. The findings include that in all four states, Lobola is required to recognise and register a customary marriage. This means that legally, Lobola is recognised as a legitimate requirement for a marriage to be recognised and registered. Additionally, an appraisal of the standard-setting frameworks protecting the right of women to be free from violence and discrimination shows that, even though there are legally binding standards and mechanisms at the regional and international levels that are mandated to protect these rights, there are normative gaps that continue to impact the protection of women from violence negatively. The CEDAW Committee has called Lobola a harmful practice. However, as the principal women's rights body within the UN system, the Committee has failed to take further steps towards standard-setting or follow-up with states to continue encouraging the elimination of this practice. This gap limits the scope for women to pursue justice when they experience multiple and intersecting forms of violence in general. This dissertation thus concludes that Lobola should be specifically recognised as a form of VAW within the realm of harmful traditional practice. It is inconsistent with the current international standards on equality, non-discrimination, and violence against women.

The anatomy of the orbital region is of great importance for many highly specialised clinical disciplines such as ophthalmology, maxillofacial surgery, and neurosurgery. The main source of arterial blood supply to the orbital region is by the ophthalmic artery, a branch of the internal carotid artery, and to a lesser extent by the anastomotic patterns which are formed through the external carotid artery. A range of arterial variations which may be developmental in origin, or which may develop due to pathologies later in life, may affect the ophthalmic artery in terms of its origin, course, and branching. If clinicians are not aware of the variations occurring in this region, the eye of the patient may be at risk of injury during invasive procedures, which may lead to partial or complete visual loss. Up until the present time, there have been only a few cadaveric studies that revealed some of the variant patterns and the overall frequencies of the recorded anastomotic patterns for the orbital blood supply. Whilst the anatomical variations are known, the frequencies of variations in the population are not. Furthermore, no published data exists regarding the variations in the orbital blood supply in a South African population. Therefore, the aim of this study was to investigate the orbital vascular supply within the South Africans of different age groups, to document and describe any variations in anastomotic patterns and record their frequencies. The current study was conducted through dissections of bodies in the Department of Human Biology, University of Cape Town, and patients' angiograms from Groote Schuur Hospital. The angiograms included data obtained from other hospitals within the Cape Town area and were reviewed retrospectively. The dissection sample included six full term fetuses and 63 adults, and the angiograms accounted for 870 individuals. The ophthalmic artery was studied from the point of origin from the internal carotid artery and its course in relation to the optic nerve, and both sides were compared to note any similarities or differences. Statistical analyses were performed to record the frequencies of the patterns of variations and to note whether there were any associations between sex, age, sidedness, and these variations. The results revealed statistically significant associations between age and sex for the patterns of variation. Several variations were noted in the current study. Among the novel findings were those in the origin of the ophthalmic artery from the internal carotid artery, whereby a lateral and inferior origin were recorded in both samples (dissected bodies and angiograms). In addition, it was noted that the ophthalmic artery may take origin from the A2 segment of the anterior cerebral artery, which is also a novel finding. This study, therefore, adds significantly to the current body of knowledge regarding the patterns of arterial supply to the ophthalmic region in a South African sample.

Background: Predisposition to ACL rupture is multifactorial, resulting from a complex interplay of intrinsic and extrinsic risk factors. Variation in the genome is now considered a key intrinsic risk factor, but the majority of currently implicated loci have been identified through case-control genetic association studies, which are limited by a candidate gene approach and insufficient statistical power. The primary aim of this thesis was to use a whole genome sequencing (WGS) approach within the context of a twin family study to identify novel or previously implicated genetic loci contributing to ACL rupture predisposition (Chapter 2). Additionally, this research aimed to explore prioritised genetic polymorphisms previously associated with ACL rupture and functioning in key biological pathways implicated through the WGS analyses, independently and as a collective, with ACL rupture predisposition in a large combined ACL rupture dataset (Chapter 3 and 4). Methods: The complete genomes of all family members in two unrelated families, each with affected twins were sequenced. Variants with potential loss of function effect were prioritised, and explored for probable biological function in the ACL rupture risk pathway. Furthermore, identity by descent analysis (IBD) was performed to identify potential disease causing mutations, on chromosomal regions shared between family members, and across families. Enriched biological pathway analyses were further explored to prioritise potential candidate genes. Two biological networks were prioritised which highlighted the angiogenesis and proteoglycan family of proteins. Specific polymorphisms within previously investigated candidate genes were further explored in case-control genetic association studies conducted in a large collective data set, including participants from three independent (Sweden, Poland and Australia) cohorts, combined with previously published South African and Polish data. The anterior cruciate ligament (ACL) rupture group included individuals diagnosed with a clinical diagnosis of an ACL rupture based on physical examination, and confirmed by either magnetic resonance imaging or arthroscopy. Only ACL ruptures resulting from a non-contact mechanism of injury were included. The control group comprised individuals of similar age to cases with no prior history of ACL injury or other ligament and tendon injuries, and participating in regular sporting activity, which was similar to cases. Participant samples were genotyped for single nucleotide polymorphisms in the VEGFA (rs699947 C/A rs1570360 G/A, rs2010963 G/C) and KDR (rs2071559 A/G, rs1870377 T/A) genes (Sweden CON: 116 ACL: 95; Poland CON: 149 ACL: 127 and Australia CON: 83 ACL: 342). Additionally, in the ACAN (rs2351491 C/T, rs1042631 T/C, rs1516797 T/G), DCN (rs516115 T/C) and BGN (rs1126499 C/T, rs1042103 G/A) genes (Sweden and Poland). Haplotype analyses were explored (VEGFA, KDR, ACAN and BGN) using the individual genotype data. In addition, inferred allele interactions were presented for VEGFA-KDR, ACAN-BGN ACAN-DCN, BGN-DCN, and VEGFA-DCN as a proxy for gene-gene interactions within the discrete angiogenesis and proteoglycan gene families, and between genes as a proxy for pathway interactions. For association studies, frequencies were calculated for the genotype, allele, inferred haplotypes and allele interactions, and the distributions compared between the control and ACL rupture participants. The statistical programs in R were used for all the analyses, and a p value < 0.05 was accepted to be significant. Results: The WGS analyses highlighted six candidate genetic loci in three genes (COL12A1, CATSPER2, and KCNJ12) with predicted loss of function effects in all affected and unaffected family members within the two studied families. Of the three genes, polymorphisms within COL12A1 were previously associated with ACL rupture predisposition, while CATSPER2 and KCNJ12 are two novel genetic loci with no known previous association with predisposition to ACL rupture. The IBD analyses identified several regions shared in each independent family, of which a segment including a long intergenic non-protein coding RNA (lincRNA) LINC01250 gene in the telomeric region of chromosome 2p25.3 was shared between affected twins in both families, and an affected brother. Furthermore, several functional partners were highlighted. Genetic association analyses of the prioritised polymorphisms in a combined cohort identified an independent association of the VEGFA rs2010963 CC genotype and C allele with increased risk (genotype p = 0.0001, FDR p = 0.001, OR 2.16, 95% CI: 1.47-3.19; allele p = 0.0006, FDR p = 0.003, OR 1.29, 95% CI: 1.11-1.49). Furthermore, the association of the VEGFA A-A-G and A-G-G inferred haplotypes (rs699947 A/C-rs1570360 G/Ars2010963 G/C) with reduced risk (p = 0.010, haplo.score: -2.58, OR: 0.85, 95% CI: 0.69-1.05; A-G-G: p = 0.036, haplo.score: -2.09, OR: 0.81, 95% CI: 0.64-1.02) of ACL rupture. Moreover, a reduced interval (rs1570360 G/A-rs2010963 G/C) revealed an association of the VEGFA -GG and -A-G inferred haplotypes with reduced risk (-G-G: p = 0.031, haplo.score: -2.15, OR: 1.00 and -A-G: p = 0.024, haplo.score: -2.25, OR: 0.98, 95% CI: 0.82-1.18) and the -G-C inferred haplotype with increased risk p = 0.012, haplo.score: 2.50, OR: 1.18, 95% CI: 0.99- 1.40). The KDR genotype and haplotype analyses illustrated that it is highly unlikely that the investigated KDR polymorphisms are associated with modulating ACL rupture risk. Inferred allele interactions noted a significant association of the VEGFA (rs699947 A/C, rs2010963 G/C) - KDR (rs2071559 A/G, rs1870377 T/A) A-G-A-A (p = 0.005, OR: 0.51, 95% CI: 0.30- 0.87) and A-G-G-A (p = 0.018, OR: 0.93, 95% CI: 0.54-1.60) combinations with reduced ACL rupture risk. Further, a significant association of the VEGFA (rs699947 C/A, rs1570360 G/A, rs20109630 G/C) - DCN (rs516115 T/C) A-G-G-T (p = 0.010, OR: 0.53, 95% CI: 0.30-0.91), A-A-G-C (p = 0.010, OR: 0.42, 95% CI: 0.21-0.81) and A-A-G-T (p = 0.046, OR: 0.77, CI: 0.49-1.2) allele combinations with reduced risk was noted for male participants in the collective cohort. No independent or haplotype associations with ACL rupture risk were noted for any of the investigated proteoglycan polymorphisms, in the collective cohort. Conclusion: Collectively, this work has expanded current knowledge on the genetic regions contributing to ACL rupture predisposition, and further highlights the polygenic nature of multifactorial phenotypes. Employing whole genome sequencing in a twin family context, together with a pathway based approach, novel and previously implicated genetic loci were identified towards the aims of the thesis. The catalogue of candidate in silico mutations and modifier genes that clustered in pathophysiological pathways important in ACL rupture, and with implications for therapeutic intervention were identified, and need to be interrogated. Of particular interest are the novel CATSPER2, KCNJ12 and LINC01250 genetic loci. Furthermore, additional evidence to support the implication of the VEGFA gene in modulating ACL rupture risk is provided, and highlighted is the potential collaboration of members within the angiogenesis and proteoglycan gene family in modulating risk. The studies in Chapter 3 and 4 suggest genetic association studies in single populations are less informative, and instead larger collective cohorts with increased statistical power should be employed. Further to that, rather than investigating single polymorphisms, larger regions of the genome should be explored to determine the potential interacting components contributing to musculoskeletal injury risk. Going forward, characterisation of the functional biological effect of implicated loci may assist in unravelling the underlying mechanisms altering tissue homeostasis, and subsequently an individual's capacity for healing and adaptive response.

The use of photomedicine in ancient civilizations dates back 4000 years ago but it wasn't until the beginning of the 20th century that photodynamic therapy was discovered by man. The “trinity” of photodynamic therapy (PDT) comprises a photosensitizer, light and molecular oxygen. Following cellular uptake of the photosensitizer, its activation by light produces reactive oxygen species in the presence of oxygen. The resulting cytotoxic oxidative stress elicits cancer cell death by various mechanisms including apoptosis, necrosis and autophagy. Hypericin, an extract from St John's Wort, is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. South Africa has the second highest incidence of malignant melanoma skin cancer in the world; a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericinPDT in an in vitro tissue culture model. This investigation was three-fold. Firstly, the susceptibility of melanoma cells to the treatment was determined using cell viability assays. We found a dose of 3 µM light-activated hypericin was effective in reducing cell viability to 50 % or less than the control, for all melanoma cells employed in this study. We therefore used this killing-dose for further experiments. Next, hypericin uptake and its specific association with intracellular organelles was characterized using organelle-specific fluorescent-fusion proteins and dyes, in conjunction with the red fluorescent nature of hypericin and visualization by live confocal fluorescent microscopy. The intracellular localization of a photosensitizer directly influences its cytotoxic action and is thus crucial for effective cell death induction. Hypericin was taken up by all melanoma cells and co-localized with lysosomes and variably with melanosomes, the pigment producing organelles. No co-localization with the cell membrane, mitochondria, endoplasmic reticulum or nucleus was found. Investigating intracellular hypericin after treatment revealed a time-dependent decrease in all melanoma cells. Finally, melanoma cell death mechanisms were elucidated in response to the killing-dose of lightactivated hypericin. Ultrastructural examination of the cells with transmission electron microscopy 2 revealed extensive cytoplasmic vacuolisation, at 4 hours after treatment. In pigmented melanoma cells, the treatment furthermore induced the formation of glycogen aggregations. Fluorescent activated cell sorting analyses revealed a time-dependent increase in phosphatidylserine exposure, indicating apoptosis, in conjunction with a loss of cell membrane integrity, indicating necrosis, in all melanoma cells. An initial early necrotic population was found which decreased with time after treatment, whereas the late apoptotic/necrotic population increased. Minimal early apoptotic populations were found in all cell lines. In addition, melanoma cells showed a decrease in cellular size accompanied by an increase in granularity/pigmentation after treatment. Western blot analyses of proteins involved in specific cell death cascades furthermore verified the induction of apoptosis in melanoma cells by hypericin-PDT. The extrinsic apoptotic cascade was initiated in unpigmented A375 melanoma cells at 24 hours after treatment, mediated by activation of the suicidal proteases caspase 8 and caspase 3. Intrinsic apoptosis was found in pigmented UCT Mel-1 cells at 4 and 7 hours, mediated by activation of caspase 3 and cleavage of poly(ADP-ribose)polymerase 1 (PARP1). Induction of apoptosis by cleavage of PARP1 was furthermore evident in 501mel cells at 7 hours after treatment; however this cleavage was not mediated by caspase 3. Apoptosis inducing factor was found in its vital form in all melanoma cells, indicating that caspase-independent apoptosis or regulated necrosis by parthanatos were not induced by hypericin-PDT. In summary, this study demonstrated the effectiveness of hypericin-PDT in killing both unpigmented and pigmented melanoma cells by the induction of apoptosis. Further investigations into the exact mechanisms of the cell death response, including the observed loss of cell membrane integrity and the involvement of lysosomes and melanosomes are interesting avenues to explore in future studies. Translation of hypericin-PDT into a three-dimensional skin model with melanoma invasion is of particular interest, to further simulate the natural environment of this aggressive cancer and thereby enable the identification of enhanced treatment options.