Copper is an extremely toxic metal in biological systems and thus, its availability to the system, must be effectively and efficiently controlled. Copper is vital for life, as it is essential for critical enzymes in biological systems. It is free copper in the biological systems that is toxic, as free copper induces free radical generation, which disrupts lipid membranes, interacts with DNA causing mutations, and eventually leads to cell death. Wilson’s disease is a inherited copper disease, which results in hepatolenticular disease. Copper is unable to be excreted, and thus accumulates, eventually spilling over into the bloodstream from the liver, and “poisons” the patient. The Wilson’s disease patient leads a short life, due to neurological and hepatological problems. There is no cure for Wilson’s disease, only chelation therapy using potent chelators such as penicillamine and EDTA. Zinc, in high doses, can be used to compete with copper absorption. This has proved to be the only successful therapy at present. This study investigates the possible use of melatonin as a copper binder/chelator. Melatonin has been shown to interact with copper in vitro. By binding/chelating to copper, melatonin may inhibit copper-induced free radical generation, and thus prevent copper from interacting with DNA to cause mutations and act as a cytotoxin. In vivo studies on copper (2mg/kg) administered for 2-weeks and 6-weeks were carried out on Wistar rats. The potential of melatonin (12mg/kg) to prevent copper-induced cellular damage was investigated. The results indicate that melatonin does not protect the lipid membranes from copper-induced lipid peroxidation. In vitro investigations using 1mM, 5mM and 10mM copper and 5mM melatonin, show that melatonin prevents copper-induced lipid peroxidation at a copper concentration of 1mM (p<0.001). The 5mM and 10mM copper induces less lipid peroxidation, compared to the 1mM copper. It has been reported that metal ions, antioxidants and chelating agents can influence peroxide decomposition during the assay. Melatonin (5mM) administration does not significantly prevent copper-induced lipid peroxidation at 5mM and 10mM copper. It is possible that due to melatonin’s relatively low concentration, it is unable to inhibit lipid peroxidation induced by the copper. The chemical nature of the interaction between melatonin and copper was also investigated, using NMR, IR and electrochemistry techniques. The NMR and IR techniques show that melatonin coordinates with Cu²⁺ and not Cu¹⁺, at the carbonyl group of melatonin. The electrochemistry experiments using cyclic voltammetry and adsorptive stripping voltammetry, show that melatonin forms a strong bond with Cu¹⁺. Cu²⁺ prefers binding to oxygen, and that is clearly seen in the NMR and IR. Cu¹⁺ prefers binding to nitrogen and then oxygen, and this is seen in the electrochemistry, as Cu¹⁺ is forced to bind through one of the nitrogens on the melatonin. Previously, it has been shown that melatonin binds/chelates with Cu²⁺. Histochemical investigations show that copper administration for 2-weeks and 6-weeks, causes extensive mitochondrial damage in liver and kidney’s proximal convoluted tubule epithelium cells. Melatonin (12mg/kg) co-administration with copper for 2-weeks and 6-weeks did not significantly protect the mitochondria from copper-induced damage. Copper-specific stains (rhodanine, silver sulphide and rubeanic acid) were used to stain liver, brain and kidney tissue samples. Rhodanine and silver sulphide were equally sensitive in staining copper in the 2-week samples, but not at all in the 6-week samples. This could not be explained. Rubeanic acid was ineffective in all samples tested. Thus, it appears that specific copper stains cannot be used in making a definitive diagnosis in cases of copper overload, and that specific copper stains do not always correlate with a high concentration of copper present in tissues. Pineal organ culture was used to determine the effect of copper administration on pineal indole synthesis. Exogenous (³H) tryptophan was administered to the pineal organ cultures, and the level of (³H) pineal indoles synthesised, were measured. Pineals from 2-week and 6-week copper/melatonin treated animals exhibited paradoxical 5- methoxytryptophol (ML) levels, as compared to the 2-week and 6-week copper treated animals. The 2-week copper/melatonin administered animals, showed a decrease in the ML level (p<0.01), and the copper/melatonin administered for 6-weeks, showed an increase in the ML levels (p<0.01). This indicates that melatonin interacts with the HIOMT enzyme. Pineals from 6-week copper/melatonin treated animals, as compared to the 6-week copper treated animals, showed an increase in N-acetylserotonin levels. This indicates that melatonin prevents the inhibition of the NAT enzyme. The final experiment was to determine in vitro, the effect of Cu²⁺ and Cu¹⁺ administration, on mitochondrial electron transport chain. Rat liver homogenate was incubated with and solutions of Cu²⁺ (10mM) and Cu¹⁺ (10mM) and melatonin (10mM). Cu²⁺ administration caused an inhibition of the electron transport at t=0 and t=60, whereas Cu¹⁺ administration at t=0 caused an inhibition of electron transport, but at t=60, Cu¹⁺ administration stimulated electron transport. Melatonin administered with Cu²⁺, resulted in an inhibition of the electron transport chain at t=0 and t=60. The findings of this study indicate that melatonin might have a potentially beneficial effect in copper overloading, by binding/chelating copper.

M.Phil. (Optometry) / Despite many years of research, diabetic retinopathy (DR), and diabetic macular edema (DME) remain difficult to diagnose, prevent, and treat. The complicated nature of the disease, the limited information on DR and DME and the increasing prevalence of diabetes mellitus (DM) in South Africa, provided motivation for this study. To the best of my knowledge, this is the first study in our country to identify demographic, medical and visual aspects ofDR and DME collectively. A further incentive was the availability in optometry of recently developed computer software based upon multivariate statistics, which provided a unique opportunity to analyze, for example, tri-variate contrast sensitivity acuities using stereo-pair scatter plots. All refractive status measurements were also analyzed and compared with the same method. Together, the results from this study provide a broader clinical and research perceptive on DR and DME. In this cross-sectional study, 202 diabetic patients at the Helen Joseph Hospital in Johannesburg were recruited. Demographic variables included age, gender, race, age of diagnosis, duration of DM, and social habits. Medical variables included systemic conditions present, blood pressures, body mass index (BMI), lipid profiles, glycerated haemoglobin (HbAlc), and other available biochemical data (for example cholesterol, urea and creatinine levels). Visual variables included, distance, pinhole and near visual acuities, contrast sensitivity acuities, refractive status measured with autorefraction, colour vision, Amsler grid, intraocular pressures (lOP), and fundus photography. Administration of the Impact of Visuallmpainnent (IVI) questionnaire provided new information concerning the restrictions in daily living participation caused by DR or DME. The predominant characteristics of the study population consisted of Type 1DM among female Coloured subjects. Approximately 66% of all subjects had also been diagnosed with hypertension. The mean age ofthe subjects was 52 (± 14) years, age of diagnosis 41 (± 13) years, and duration ofDM 10.8 (± 9.7) years. Mean blood pressures (136/81 ± 20.5/11 mmHg) and glycated haemoglobin (HbAlc, 9.9 ± 3.4%) values were slightly higher than the recommended control levels (BP= 120/80 mmHg and HbAlc = 6 to 7%).

Parkinson’s disease, one of the most common neurodegenerative disorders associated with ageing, is characterised by abnormal and profound loss of nigrostriatal dopaminergic neurons. The cause of Parkinson’s disease is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate oxidative damage and mitochondrial impairment, particularly at the level of complex I enzyme. Recently, rotenone, a commonly used organic pesticide and a classical inhibitor of mitochondrial complex I has been reported to reproduce the specific features of Parkinson’s disease in rodents. The mitochondrial respiratory chain is one of the most important sites of reactive oxygen species production under physiological conditions. Toxic free radicals have been implicated in a variety of neurodegenerative diseases as well as ageing itself. Melatonin, a secretory product of the pineal gland is a multifaceted free radical scavenger and natural antioxidant. In the present study, the neuroprotective effects of melatonin against the environmental neurotoxin, rotenone was investigated. Initial studies showed that inhibition of mitochondrial complex I enzyme by rotenone induced superoxide radical generation. Melatonin, administered to the rat in vivo and in vitro was able to offer neuroprotection by curtailing the production of superoxide radicals induced by rotenone. Mitochondria, being the major target of rotenone, the effects of melatonin were investigated at the mitochondrial level. Melatonin was able to increase the electron transport chain activity thus preventing the respiratory inhibition by rotenone. The pineal hormone also counteracted the action of rotenone on complex I enzyme. These results suggest melatonin’s ability to potentially limit the free radical generation and thereby modulate the mitochondrial functions. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. Melatonin also offered significant protection in vivo and in vitro against rotenone induced lipid peroxidation. Since iron plays a major role in oxidative damage and in the progression of Parkinson’s disease, the effect of melatonin on both rotenone and iron induced lipid peroxidation was investigated, the results of which show that melatonin affords protection and this was suggested to be due to its interaction with the rotenone-iron complex that might have formed. Electrochemical studies were further used to characterise the interactions between melatonin, rotenone and iron (III). Melatonin was shown to bind with iron and thus reducing their toxicity. Histological studies were undertaken to assess the effects of melatonin on rotenone induced toxicity on the dopaminergic neurons in the rat brain. Rotenone treated brains showed extensive neuronal damage whereas with melatonin less damage was observed. Rotenone induces apoptosis via reactive oxygen species production and apoptotic cell death has been identified in PD brains. Furthermore, the apoptotic cell death was detected and quantified by the TUNEL staining. Rotenone treated sections showed signs of apoptosis whereas with melatonin, less apoptotic damage was observed. The findings of this study indicate that the neurohormone, melatonin may protect against rotenone-induced neurodegeneration. Since melatonin production falls substantially during ageing, the loss of this antioxidant is theorized to be instrumental in the degenerative processes associated with advanced age. Considering how devastating diseases such as Parkinson’s disease, are to a patient and the patient’s families, the discovery of protective agents are a matter of urgency. Further investigations using the pesticide model will help to determine the involvement of environmental exposure in the pathogenesis of human diseases as well as to test therapeutic strategies for the treatment of such diseases.

The hippocampus, located in the medial temporal lobe, is an important region of the brain responsible for the formation of memory. Thus, any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include the neurotoxin, Quinolinic acid. Quinolinic acid (QUIN) is a neurotoxic metabolite of the tryptophan-kynurenine pathway and is an endogenous glutamate agonist that selectively injures and kills vulnerable neurons via the activation of the NMDA class of excitatory amino acid receptors. Estrogen is a female hormone that is responsible for reproduction. However, in the last decade estrogen has been shown to exhibit a wide range of actions on the brain, including neuroprotection. Estrogen has been shown to exhibit intrinsic antioxidant activity and protects cultured neurons against oxidative cell death. This is achieved by estrogen’s ability to scavenge free radicals, which is dependent on the presence of the hydroxyl group at the C3 position on the A ring of the steroid molecule. Numerous studies have shown that estrogen protects neurons against various toxic substances and may play a role in delaying the onset of neurodegenerative diseases, such as Alzheimer’s disease. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. The study aims to elucidate and further characterise the mechanism behind estrogen’s neuroprotection, using QUIN as a model of neurotoxicity. Initial studies confirm estrogen’s ability to scavenge potent free radicals. In addition, the results show that estrogen forms an interaction with iron (II) and also acts at the NMDA receptor as an agonist. Both mechanisms reduce the ability of QUIN to cause damage to neurons, since QUIN-induced toxicity is dependent on the activation of the NMDA receptor and the formation of a complex with iron (II) to induce lipid peroxidation. Heat shock proteins, especially Hsp 70 play a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. Estrogen has been shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that estrogen helps to protect against cellular protein damage induced by any form of stress the cell may encounter. The discovery of neuroprotective agents, such as estrogen, is becoming important as accumulating evidence indicates a protective role in vivo. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders. Considering how devastating diseases, such as Alzheimer’s disease, are to a patient and the patient’s families, the discovery of new protective agents are a matter of urgency.

An increasing number of studies show that nutritional antioxidants such as vitamin E and polyphenols are capable of blocking neuronal death in vitro and may have therapeutic properties in animal models of neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. In the present study, the neuroprotective ability of one such polyphenolic antioxidant, curcumin, was investigated. Curcumin is the yellow curry spice derived from turmeric, and is widely used as a dietary component and herbal medicine in India. Most neurological disorders are postulated to have an oxidative or excitototoxic basis. Thus the effects of curcumin on oxidative stress in the rat brain were investigated. Curcumin, administered to the rat in vivo and in vitro, was able to exert protective effects on oxidative damage in the brain, induced by cyanide, a mitochondrial inhibitor. Curcumin also offered protection against quinolinic acid induced lipid peroxidation, and this protection was extended to lipid peroxidation induced by metals such as lead and cadmium in the rat brain. Experiments conducted on the pineal gland revealed an increased production of the neuroprotective hormone melatonin in presence of curcumin in vivo. The hippocampus is functionally related to vital behaviour and intellectual activities and is known to be a primary target for neuronal degeneration in the brains of patients with Alzheimer’s disease. Histological studies were undertaken to assess the effects of curcumin on lead induced toxicity on the rat hippocampus, the results of which show that curcumin affords significant protection to the hippocampus of the lead treated rats. This study also sought to elucidate possible mechanisms by which curcumin exerts its neuroprotective capabilities. Curcumin was found to inhibit the action of cyanide on the mitochondrial electron transport chain, one of the most common sources of free radicals. Electrochemical, UV/VIS and Infrared spectroscopy were used to characterise interactions between curcumin and the metals lead, cadmium, iron (II) and iron (III). Curcumin was shown to directly chelate these metals with the formation and isolation of two new curcumin complexes with lead, and one complex each with cadmium and iron (III). These results suggest chelation of toxic metals as a mechanism of neuroprotection afforded by curcumin. The need for neuroprotective agents is urgent considering the rapid rise in the elderly population and the proportionate increase in neurological disorders. The findings of this study indicate that curcumin, a well-established dietary antioxidant, is capable of playing a bigger role in neuroprotection, which needs to be further explored and exploited.

Global cerebral ischemia is an established animal model mimicking the effects of cardiac arrest in humans. It is characterized by selective neuronal damage in the hippocampus and significant behavioural and cognitive impairments. In this light, novel therapeutic compounds with numerous physiological targets as well as neuroprotective capabilities and the capacity to lessen residual cognitive deficits pose as great candidates in the treatment of ischemic pathology. The current thesis investigates the possible therapeutic properties of resveratrol (3, 4, 5´trihydroxystilbene), a naturally occurring phytoalexin present in the skin of grapes, against cerebral ischemia-induced neuronal degeneration and cognitive impairments, as well as elaborate on possible mechanisms of action of the compound in male Wistar rats. In Article 1, neuronal density assessment and behavioural testing following chronic pretreatment with resveratrol at two doses (1 and 10 mg/kg) revealed that the compound has important neuroprotective properties at short and long post-ischemic intervals. Despite comparable neuronal protection, the two resveratrol doses showed distinct behavioural effects, highlighting independent actions of the polyphenol on discrete physiological systems mediating cellular survival and behavioural recovery. Articles 2 and 3 investigated possible mechanisms of action of the polyphenol that have not yet been explored with regards to cerebral ischemia. Specifically, Article 2 demonstrated that resveratrol influences markers of plasticity in both ischemic and control animals as well as promotes angiogenesis in the hippocampal region postischemia. Further elaborating on documented effects attributing non-neuronal mechanisms of action of resveratrol in reducing glial activation postischemia, Article 3 highlighted important regulatory effects of resveratrol on mediating glial type-1 glutamate transporter expression at a short reperfusion interval. These findings support the notion of multiple biological targets by resveratrol and highlight its potential role in attenuating forebrain ischemia-induced neuronal degeneration through multiple physiological targets, while cautioning against possible dose-related effects on behaviour and in healthy controls.

Objective: Physical exercise decreases disability and pain associated with chronic articular cartilage degradation. However, understanding of the pathology is lacking. In this study, the levels of 17 biomarkers of inflammation and cartilage degradation were measured in synovial fluid (SF) before and after a 30-minute run in able-bodied and previously-injured individuals. Materials & Methods: Four able-bodied recreational runners (3 men and 1 woman: 24 ± 2 years, 68 ± 7 kg, and 173 ± 9 cm) and 4 recreational runners who had undergone a unilateral anterior cruciate ligament reconstruction (ACLr) (2 men and 2 women: 23 ± 1 years, 71 ± 6 kg, and 175 ± 4 cm) were recruited to participate in this study. Using a saline-assisted method, SF was aspirated before and after both a 30-minute unloading and 30-minute exercise session. Samples were corrected for blood contamination and analyzed for 15 cytokines and 2 matrix metalloproteinases (MMPs). Mixed model analyses were used to determine the main effects of session, case/control status, pre/post aspirations, and the interactions between case/control status and pre/post aspirations. Results: Blood protein contamination was calculated and accounted for in 15 of 32 synovial fluid samples. Granulocyte colony stimulating factor (GCSF) was the only detectable cytokine of the 15 analyzed. No statistical differences were found in GCSF concentrations between pretreatment and posttreatment aspirations (p = 0.45), ACLr and able-bodied control groups (p = 0.60), or unloading and exercise sessions (p = 0.96). MMP-13 was undetectable. No statistical differences were found in MMP-3 between pretreatment and posttreatment aspirations (p = 0.15), ACLr and able-bodied control groups (p = 0.85), or unloading and exercise sessions (p = 0.14).Conclusions: Two (GCSF and MMP-3) of the 17 measured biomarkers were detectable. There were no significant differences in either GCSF or MMP-3 due to a 30-minute run or 30-minute unloading period in either the able-bodied or ACLr participants. Further, there were no significant differences between biomarker concentrations and case-control status. A novel method of controlling for blood contamination in synovial fluid samples was implemented.

In this thesis, I use a range of techniques in computational neuroscience, communication theory, and electrophysiology to characterize functional changes that occur in early stages of retinal degeneration in the mouse. At post natal day 14, retinal ganglion cells in the rd1 mouse exhibit peculiar differences from age matched controls: an increased latency of responses to the onset of a light stimulus, decreased spike count in response to stimulus onset, increased spontaneous firing activity, and a decrease in information transmission. I propose this is due to an up-regulation of OFF bipolar cell excitation, a critical factor in functional changes seen in rd1, and use innovative techniques to discover findings that support these claims.

Both proliferative diabetic retinopathy and exudative age-related macular degeneration are major causes of blindness which are caused by growth of defective, leaky and tortuous blood vessels in the retina. Hypoxia is implicated in triggering both of these diseases and results in induction of HIF-1alpha transcription factor in addition to the angiogenic factor VEGF. Müller cells are the major glial cell in the retina and they contribute to neovascularization in hypoxic regions of the retina through eliciting secretion of growth factors, cytokines and angiogenic factors. As Müller cells span the breadth of the retina they can secrete angiostatic factors as well as neuroprotective trophic factors, the Müller cell is a valuable cell type for targeting by potential new gene therapies. The current investigation tests the hypoxia responsiveness of an AAV vector containing a hybrid hypoxia response element together with a GFAP promoter, and this vector encodes the angiostatic protein decorin, a well characterized multi-receptor tyrosine kinase inhibitor. Decorin may have advantages over other key angiostatic factors such as endostatin or angiostatin by virtue of its multiple anti-angiogenic signaling modalities. We employed Q-RT-PCR to evaluate the cell specificity and hypoxia responsiveness of an AAV-Vector termed AAV-REG-Decorin containing a hybrid HRE and GFAP promoter driving expression of the decorin transgene. The vector also contains a silencer element between the HRE and the GFAP domains to enable low basal expression in normoxia as well as high level inducibility in hypoxia. AAV-REGDecorin was found to elicit high level expression of decorin mRNA in hypoxia with greater than 9 – fold induction of the transgene in hypoxic conditions in astrocytes by comparison to normoxic astrocytes. AAV-REG-Decorin showed low levels of transgene expression by comparison to the positive control vector AAV-CMV -decorin containing the ubiquitously active CMV-promoter. The expression levels of decorin mRNA from AAV-REG-Decorin and from AAV-GFAP-Decorin were low in the PC12 neuronal cell model and in the ARPE19 line of retinal pigment epithelial cells with respect to those of AAV-CMV-decorin and no induction of Decorin mRNA was found with AAV-REGDecorin in these two control cell lines. Our novel gene therapy vector will serve as a platform for testing efficacy in rodent disease models (OIR and laser induced choroidal neovascularization) for assessment of the benefits of tightly regulated antiangiogenic gene therapy eliciting decorin transgene expression, both in terms of timing and the cellular source of production, during the progression of the retinal pathophysiology. / Includes bibliography. / Dissertation (PhD)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

C1QTNF5 is a 25kDa short chain collagen of unknown function which is mutated in late-onset retinal macular degeneration (L-ORMD). L-ORMD is an autosomal dominant disease characterised by sub-retinal pigment epithelial deposits leading to photoreceptor death and visual loss and shows several similarities to age-related macular degeneration (AMD). A Tyr402His polymorphism in complement factor H (CFH), a regulatory protein in the innate immune system, has been associated with increased risk of AMD. C1QTNF5 and CFH are both expressed and secreted by the retinal pigment epithelium (RPE) which supports photoreceptors and is responsible for phagocytosis of shed rod photoreceptor outer segments (ROS). The properties of the normal C1QTNF5 and disease-associated Ser163Arg mutation were examined in detail, including protein characterisation, cellular processing and function. Recombinant wild type and mutant C1QTNF5 were produced and their multimerisation and solubility functions compared. Both proteins were found to be soluble and to form similar multimeric species which were resistant to reducing conditions, as seen in other short chain collagens. Due to the similarities between LORMD and AMD, a proposed interaction between C1QTNF5 and CFH was investigated. CFH is composed of 20 short consensus repeats (SCR) and interactions were confirmed between C1QTNF5 and both CFH and SCR modules 7-8 and 19-20. CFH showed a greater affinity for mutant C1QTNF5 compared with wild type on the basis of surface plasmon resonance assays. Stably transfected RPE-derived cell lines were created which expressed either wild type or mutant C1QTNF5. Both proteins were found to be secreted and showed similar cellular processing with no evidence of aggregation or retention of the mutant protein within the endoplasmic reticulum. In order to investigate C1QTNF5 function, phagocytosis of ROS by the stably transfected cell lines was carried out. Cells expressing wild type C1QTNF5 showed greater ROS phagocytosis compared with mutant C1QTNF5-expressing or untransfected cells. Addition of anti-C1QTNF5 antibody increased ROS phagocytosis further. In summary, it is proposed that wild type and mutant C1QTNF5 are secreted by the RPE where they interact with CFH. C1QTNF5 is also shown to have a role in ROS phagocytosis, with mutation in C1QTNF5 affecting phagocytosis efficiency, which may contribute to sub-RPE deposit formation. The results suggest that CFH may also be involved in this process, suggesting a common pathogenic pathway between L-ORMD and AMD.