Electronic Theses and Dissertations (PhDs)

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Characterising skeletopathy in an animal model of Type 2 diabetes
(2024) Dlamini, Gcwalisile Frances
Type two diabetes (T2D) is a chronic, progressive heterogonous syndrome with a genetic and environmental origin. It is now recognized as an epidemic with a high morbidity and mortality rate. The endocrinology of type 2 diabetes (T2D) and its predisposing factors have been studied extensively, while diabetic skeletopathy has received negligible research. Previous studies report that fractures in T2D vary with specific sub regions in bones, therefore prompting our study to focus mainly on the femoral head and neck as well as the humerus head. Femoral neck fractures are the commonest, followed by the proximal femur, distal radius and proximal humerus. Susceptibility to fracture is a sequelae of poor bone remodeling. Poor bone remodeling is established at molecular and cellular levels. It depends on the activity of osteoblasts, osteocytes and osteoclasts, which are under the influence of TGF-β1, a pro-osteogenic cytokine, together with BMP3, an anti-osteogenic cytokine.T2D induced bone marrow adipocity and the accumulation of AGEs in cortical bone have also been implicated in increasing susceptibility to fracture. It is still unclear how T2D affects molecular and cellular elements that culminate in weaker bones observed in diabetic patients. In addition, it is debatable if T2D affects the skeleton at disease onset or later in the disease. Therefore, this study aimed to characterize T2D induced skeletopathy and related it to age, in the Zucker Diabetic Sprague Dawley (ZDSD) rat, using the femur and humerus. This study initially confirmed the diabetic state by monitoring animal weights, fasting blood glucose levels, and fasting oral glucose tolerance tests (OGTTs) every fortnight. Then triglyceride levels and quantified serum levels of osteoregulatory hormones such as insulin and osteocalcin were monitored. To assess oxidative stress, Malondialdehyde (MDA) serum levels were also determined by ELISA. Once diabetes was successfully induced, rats were grouped according to strain and age at termination. Termination age was at 20 weeks and 28 weeks . The Sprague Dawley (SD) rats were v the controls, while the Zucker Diabetic Sprague Dawley rats (ZDSD) were the experimental groups. These were designated as SD20WK (n=8) and ZDSD20WK (n=7) respectively. Another batch was designated as SD28WK (n=8), and ZDSD (n=15) that were terminated at 28 weeks of age. The latter were further divided into moderate diabetes (ZDSD28WK-MOD) (n=9) and severe diabetes (ZDSD28WK-SVD) groups (n=6). Bilateral humeri and femora were harvested then fixed in 10% buffered formalin. Right proximal femora and humeri were scanned using a 3D-μCT scanner (Nikon XTH 225L) to analyse trabecular morphometric parameters, cortical bone area and medullary canal area. Biomechanical strength was analyzed by three point bending tests using a universal tensile tester. Left proximal femora and humeri were processed for histology. Some sections were stained with Haematoxylin and Eosin (H&E) to assess normal histologic morphology and adipocyte quantification. Remnant sections were immunolabelled using the anti-TRAP and anti-ALP antibodies for osteocyte and osteoblast quantification respectively, to assess osteolysis and osteogenesis. Immunolocalization of AGEs, TGF-β1 and BMP3 was also conducted to investigate their role in diabetic skeletopathy. We found that diabetes affected osteoblastogenesis as measured by ALP positive cells and bone marrow adipocytes. TRAP positive osteocytes numbers were increased in the presence of T2D, suggesting an increased osteolysis. There was reduced TGFB1 expression with increased BMP3 expression. The number of AGEs immuno-positive cells as well as its extracellular expression was increased. Our finding suggest that osteoblast and osteocyte numbers are regulated by TGFβ1 and BMP3 in both bones, under the influence of AGEs. Our findings from osteometry, 3-point bending tests and Micro CT support that diabetes weakens bone. The diabetic effect results in lighter, shorter hollow bones that perform poorly under loading, as well as exhibit unfavourable trabeculae microarchitecture. Our findings confirm that T2D causes increased fragility in the proximal femur and humerus as well the mid-diaphysis. These perturbations occur early and late in the disease, and they are also exacerbated by the presence of hyperglycemia. vi We conclude that the ZDSD rat can be used as a translational model for diabetic skeletop
Effect of boophone disticha on the behaviour and hippocampal neuroanatomy in a BALB/c mouse model
(2024) Xhakaza, Nkosiphendule Khuthazelani
Depression is one of the most common neuropsychiatric disorders and is associated with dysfunction of the neuroendocrine system and alterations in specific brain proteins. Boophone disticha (BD) is an indigenous psychoactive bulb that belongs to the Amaryllidacae family, which is widely used in Southern Africa to treat depression, with scientific evidence of potent antidepressant-like effects. The present study examined the antidepressant effects of BD and its mechanisms of action by measuring some behavioural parameters in the elevated plus maze, light dark box, open field forced swimming, brain content of corticosterone, brain derived neurotropic factor (BDNF), and neuroblast differentiation in the hippocampus of Balb/c mice exposed to the five-day repeated forced swim stress (5dRFSS) and 28 days chronic restraint stress. Male Balb/c mice were subjected to the 5dRFSS and 28 days chronic restraint protocols to induce depressivelike behaviour (decreased swimming, increased floating, decreased open arm entry, decreased time spent in the open arms and decreased head dips in the elevated plus maze test, increased time in dark box in the light dark box test, reduced frequency of rearing and increased time on the sides of the open field in the open field test), and treated with distilled water, fluoxetine and BD. Three weeks Boophone disticha treatment (10mg/kg/p.o) significantly attenuated both the 5dRFSS and chronic restraint-induced behavioural abnormalities and the elevated brain tissue corticosterone levels observed in stressed mice. Additionally, 5dRFSS exposure significantly decreased the number of neuroblasts in the hippocampus and BDNF levels in the brain of Balb/c mice, while fluoxetine and BD treatment attenuated these changes. In the chronic restraint stressed mice, similar effects of BD treatment were observed after 21 days of treatment, however, the levels of corticosterone were not different in control and stressed animals, probably due to habituation to stress. In both 5dRFSS and chronic restraint stress, the antidepressant effects of BD were comparable to those of fluoxetine, but unlike fluoxetine, BD did not show any anxiogenic effects, suggesting better pharmacological functions. It is important to note that in chronic restraint stress mice, it appeared that animals seemed to have habituated to stressful conditions, demonstrated in part by brain tissue levels of corticosterone that were not elevated in stressed animals treated with distilled water. However, BDNF levels remained significantly low in stressed animals treated with distilled water, suggesting that the effect of chronic stress in this parameter were not reversed when animals habituated. In conclusion, our study shows that BD exerted antidepressant-like effects in both 5dRFSS and chronic restraint stress mice, mediated in part by normalizing brain corticosterone and BDNF levels. Due to some degree of habituation in chronic stress model, caution should be exercised when evaluation effects of treatment in different parameters to evaluate antistress effects of tested agents, particularly levels of corticosterone. Furthermore, the persistent low levels of BDNF suggest that habituation of animals to chronic stress is due to normalising levels of corticosterone but not BDNF. The above occurrence could suggest that recovery from chronic stress without antidepressant treatment could alleviate other behavioural symptoms but not cognitive impairment which is influenced in part by BDNF levels.
Evaluation of antioxidant properties and neuroprotective effects of methanolic leaf extract of combretum molle in D-galactose-induced aging model of Sprague Dawley rats
(2024) Fasemore, Thandi Mamorapelo Dorothy
Several physical and biochemical changes in the body occur because of the biological process of aging. As part of natural aging, the brain encounters morphological and functional changes that affect dendritic trees and synapses, neurotransmission, circulation, and metabolism. The brain's high metabolism, elevated levels of lipids, and inadequate antioxidant defences make it susceptible to oxidative stress. A reducing sugar called D-galactose (D-gal) causes a significant build-up of reactive oxygen species (ROS). Combretum molle (C.molle) is a plant rich in compounds that scavenge free radicals and is frequently used to cure a variety of human illnesses in African traditional medicine. This study investigated the potential impact of C.molle on rat brain aging brought on by D-galactose. Fifty adult male Sprague Dawley rats were treated for 90 days and were composed of 5 groups (n=10) as follows: I) Control group received saline and distilled water, II) C.molle only group received intragastric gavage of C.molle (500 mg/kg), III) D-gal only group received a subcutaneous injection of D-galactose (150 mg/kg), IV) CMD 90 group received D-galactose and C.molle simultaneously for 90 days, V) CMD 45 group received D-galactose for the first 45 days and C. molle for the remaining 45 days. The animals underwent behavioral evaluation post-treatment for a further period of 7 days twice a day. The rat’s cognitive function was evaluated through Novel object recognition and object location tests. The C.molle ’s neuroprotection was evaluated through levels of acetylcholinesterase (AchE), Acetylcholine (Ach), Brain Derived Neurotropic Factor (BDNF), and Tumor Necrosis Factor (TNF) alpha including the effects on adult neurogenesis through Ki-67 and doublecortin (DCX) immunohistochemistry. The oxidative stress level was measured through the evaluation of lipid peroxidation marker malondialdehyde (MDA), glutathione peroxidase (GSH-Px), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) activity. The C.molle significantly attenuated the effects of D-galactose-induced changes in the hippocampus and cortex, ranging from cognitive capacity, and oxidative stress by increasing GSH, BDNF, Ach, GSH-Px, CAT, and SOD activity. Additionally, C. molle caused a decrease in the levels of MDA, TNF alpha, and AchE activity, and ameliorated reduced cell proliferation and neuroblast differentiation brought about by D-galactose.
In vitro and in silico characterization of the anticholinesterase activity of select terpenoids against anopheles vectors
(2024) Rants’o, Thankhoe Abram
Malaria is a life-threatening plasmodial disease that is transmitted by female Anopheles mosquitoes. Major African malaria vectors include Anopheles arabiensis, An. funestus, An. gambiae and An. coluzzii. Malaria vector control programs have shown effectiveness in reducing the Anopheles populations. The main insecticide classes used in these interventions include pyrethroids, organochlorines, organophosphates, carbamates, and neonicotinoids. Nevertheless, the development of Anopheles resistance to these insecticide classes has greatly reduced the effectiveness of these interventions. A common resistance mechanism is through rapid detoxification of insecticides by overexpressed P450 monooxygenases. Although acetylcholinesterase (AChE) is a valid target in Anopheles vector, current anticholinesterase insecticides suffer from resistance and low selectivity between insect and mammal AChE targets. This indicates the urgent need to discover novel AChE inhibitors with higher affinity to Anopheles AChE compared to the mammal target, and less prone to resistance caused by the overexpressed monooxygenases. Identification of novel AChE inhibitors from natural sources and their potential to kill Anopheles during all its different life stages, presents a cost-effective approach. This PhD study aimed to identify such novel AChE inhibitors from essential oil sources and assess them for consistent activity against Anopheles species with hyperactive P450 monooxygenases. In this study, molecular differences between Anopheles and human AChEs were identified showing the opportunity to develop selective Anopheles AChE inhibitors. A novel approach was used to integrate the in silico and in vitro assays in assessing the Anopheles AChE inhibitory potential of select terpenoids and coupled these to the in vivo assays against different life stages of Anopheles. The terpenoids, farnesol, (-)-α-bisabolol, cisnerolidol, trans-nerolidol, and methyleugenol were identified as potent Anopheles AChE inhibitors and larvicidal agents with moderate adulticidal effects. Farnesol and (-)-α-bisabolol also displayed pupicidal activity, while methyleugenol inhibited the hatching of Anopheles eggs. Generally, farnesol and (-)-α-bisabolol were highly active across the Anopheles species, except in the strain with P450-based metabolic resistance. In contrast, the efficacy of cisnerolidol, trans-nerolidol, and methyleugenol was not affected by this resistance mechanism. This research suggests that cis-nerolidol, trans-nerolidol, and methyleugenol are potential candidates for further development as anticholinesterase bioinsecticides.
Post-mortem interval estimation and insect succession patterns in the tropical climate of nigeria
(2022) Etoniru, Izuchukwu Stanley
Post-mortem interval (PMI) estimation is the first step in the identification of badly decomposed remains. Apart from identifying the victim, obtaining the PMI is an important aspect in investigation into the cause and manner of death, and helps to narrow down the number of suspects. The ongoing armed conflict in Nigeria which has lingered over a decade has left a large burden of human remains. These remains are mostly left in the fields where the attacks occurred for fear of further attacks, especially in cases of terrorism. They are, therefore, badly decomposed at the time they are recovered, and identification becomes more difficult in a country that has very few forensic scientists. Law enforcement agencies usually resort to mass burials without identification. The aim of this study was to assess decomposition rates in southern Nigeria and to derive formulae for PMI estimation using the quantitative variables Accumulated Degree Days (ADD) and total body score (TBS), and to obtain the arthropod succession pattern during decomposition using a pig model. To achieve this aim, a longitudinal examination of quantitative variables, TBS and ADD, was conducted over a period of 14 months. This period included both the dry and wet seasons. Scatter plots between TBS and PMI, and TBS and ADD were used to show decomposition patterns. Arthropod succession patterns were also observed during the study for each carcass. Decomposition was found to progress rapidly, and desiccation was a frequent occurrence during decomposition. There were marked differences in decomposition patterns between the seasons, with the wet season exhibiting a more rapid decomposition. Linear regression formulae for ADD and PMI, and 95% confidence interval charts for TBS for ADD were derived. The arthropods arrived very early on the pig cadavers. There was more arthropod abundance and species richness in the wet season than in the dry season. There were also some arthropods that were observed only in the wet season. A combination of these formulae and insect activity will lead to a more precise PMI estimation in Nigeria and regions with similar climate. The data on insect succession developed from this study will serve as a reference for forensic researchers

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Browsing Electronic Theses and Dissertations (PhDs) by SDG "SDG-3: Good health and well-being"

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