T-cell Intracellular Antigen-1 (TIA1) is a multi-domain RNA-binding protein involved in stress granule formation and implicated in neurodegenerative diseases. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids, and a C-terminal intrinsically disordered prion-related domain (PRD), which plays a role in promoting liquid-liquid phase separation. Motivated by our previous findings indicating that RRMs 2 and 3 exhibit a well-ordered structure in the oligomeric full-length form, whereas RRM1 and PRD demonstrate a propensity for phase separation, the present work in this dissertation aims to investigate the functional competence of the oligomeric state and its binding capabilities. Moreover, the study explores the effects of ligand binding on oligomerization dynamics and potential alterations in protein conformation primarily using solid-state NMR methods. The NMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at RRM2, but also weakly at RRM3, and Zn2+ binds primarily to RRM3. The binding of Zn2+ and DNA was reversible and without the formation of amyloid fibrils. The addition of Zn2+ caused the TIA1:DNA complexes to collapse, indicating that Zn2+ may play a regulatory role by shifting the nucleic acid binding off RRM3 and onto RRM2 by occupying various “half” binding sites on RRM3 and introducing a mesh of crosslinks in the supramolecular complex. Furthermore, this dissertation presents an investigation into the interdomain interactions between RRM2 and RRM3, facilitated by the successful preparation of segmentally labeled protein samples using the trans-splicing approach. The results confirm the hypothesis that Zn2+ can bring RRM2 and RRM3 closer together by crosslinking different monomers, as evidenced by the observation of enhanced NMR signals from heteronuclear correlations around the Zn2+ binding sites. In conclusion, studying the structure of full-length TIA1 oligomers is expected to reveal the mechanisms by which an RNA regulatory protein assembles and binds to its biologically relevant ligands while preserving a highly ordered oligomeric structure.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / The overall objective of this thesis is to provide additional data to assist clinicians and experimental neurologists alike in the quest for better understanding, more accurately diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD). The general theme of the thesis is the interaction between certain environmental stimuli, including the exposure to adverse events during early central nervous system (CNS) development and the manifestation of elements of neurodegeneration, whether by means of neurochemical changes or expressed as a dysfunctional voluntary motor system. The first chapter provides a general introduction to the research theme of the thesis. This includes, in particular, a discussion on current understanding concerning the etiology and clinical profile of PD, the relative contribution made by genetic factors compared to environmental ones, and current treatment strategies for treating the disease. Mention is also made of the failure of these therapeutic applications for reversing or protecting against the disease, due to the side-effects associated with them. The material covered in chapter 1 provides the basis for the more complete discussion concerning these various aspects, contained in the chapters to follow. The overall aim was also to characterise the effects of commonly used toxin-induced animal models of PD, and the extent of vulnerability that the CNS displays towards them. The destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points along the nigrostriatal tract is used extensively to model PD pathology in rats and is an established animal model of the disease. However, mature or even aged animals are mainly used in these studies, while the effects that the toxin might have on the developing CNS remain unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum. Although both lesion types were used, the MFB model is considered a more accurate portrayal of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of the disease. The different lesions’ effects on motor function were determined by observing animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of a cylindrical enclosure. Following the final behavioral assessment, the concentration of dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using 4 HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment, there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic terminals that may be sufficient for maintaining relatively high extracellular and synaptic concentrations of DA. However, since substantial changes in motor-function were observed, it is suggested that the capacity of the remaining dopaminergic neurons to respond to increased functional demands may be limited. In addition, the behavioral results indicate that the distinct indices relating to different functional deficits depend on the lesioning of anatomically distinct structures along the nigrostrial tract. It has long been known that far fewer women are diagnosed with PD than men are. This seeming protection offered to females against degenerative disease of the CNS may relate to estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United States alone, the aim of chapter 2 was to examine the evidence for a possible relationship between PD and the female reproductive hormone estrogen. A review of the current literature available on the topic was performed by consulting Medline, and by performing a search of the case-reports contained within the World Health Organization’s (WHO) International Drug Monitoring database, for possible PD-related symptoms that may arise from estrogen replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen protects women from obtaining the disease, or at least some features of it. Intensive research efforts are called for, with sufficient power to establish the relationship between ERT and the onset and development of parkinsonism. Chapter 3 reports on the results obtained from an experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14 days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6- OHDA the female rats were less impaired than males in making adjustment steps in response to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender differences in cell survival factors and/or other promoters of neuroplasticity may have contributed to the beneficial outcome seen in the females. For example, nerve growth factor (NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It is unclear whether gonadal steroids are involved, and, if so, whether female hormones are protective or whether male hormones are prodegenerative. Determining the mechanisms for the improved outcome seen in the young female rats may lead to potential treatment strategies against PD. 5 Many studies have shown that early life stress may lead to impaired brain development, and may be a risk factor for developing psychiatric diseases, including clinical depression. However, few studies have investigated the impact that early stress may have on the onset and development of neurodegenerative disorders such as PD. The study reported on in chapter 5 conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6- OHDA. The combined effects of these models on motor deficits and brain protein levels were investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion with a battery of tests that are sensitive to the degree of DA loss sustained. The results show that animals that had been subjected to MS display poorer performance in the vibrissae and single-limb akinesia test compared to non-MS control animals (that had also been subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH staining in MS rats compared to non-MS ones. The results from this study therefore suggest that exposure to adverse experiences during the early stages of life may contribute towards making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring during mature stages of life. Therefore, taken together, early exposure to stress may predispose an individual towards the onset and development of neurodegenerative disease, which especially becomes a threat during the later stages of adult life. Moreover, within the framework of these characteristics, the capacity of a widely-used pharmacological agent (statins) was tested for possible future therapeutic application in PD (chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests that it may associate with defective activity of complex I of the mitochondrial electron transport chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased oxygen free radical production, depressed antioxidant enzyme activities, and greater susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for lowering cholesterol levels in patients who display elevated concentrations of low-density lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2- 6 Dimensional electrophoresis (2-DE) with subsequent identification of the spots using electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was performed and the results BLAST-searched using bio-informatics tools for naming the identified peptides. The motor test results indicate that while unilateral rotenone causes behavioral asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly classified according to their cellular function into 6 categories, with the majority involved in energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated techniques and non-biased ability to quantify proteins, provides a methodology with which to study the changes in neurons associated with neurodegeneration. As an emerging tool for establishing disease-associated protein profiles, it also generates a greater understanding as to how these proteins interact and undergo post-translational modifications. Furthermore, due to the advances made in bioInformatics, insight is created concerning their functional characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped that the application of this technology will lead towards a presymptomatic diagnosis of PD, and the identification of risk factors and new therapeutic targets at which pharmacological intervention can be aimed. The final chapter (chapter 8) provides a retrospective look at the academic work that had been performed for the purpose of this thesis, recaps on the main findings, and also highlights certain aspects of the project and provides relevant suggestions for future research. Lastly, the appendix provides a detailed overview of the methods followed for the experiments described in this thesis. It provides not only a comprehensive description of the techniques that had been followed, but provides information concerning the care taken with the animals (i.e. post-surgery) in order to control for the potential influence of experimental variables on the results.

Hepcidin is a well-known iron-regulatory hormone that plays a key role in maintaining peripheral iron homeostasis. The presence and wide-spread distribution of hepcidin in the brain suggests that this peptide may also be an important player in brain iron homeostasis. In this study, we tested the hypothesis that hepcidin exerts an important role in the regulation of brain iron content, which might benefit iron-associated NDs such as PD. We also examined the hypothesis that hepcidin could control iron transport processes via regulating iron transport proteins in the brain cells, thus maintaining brain iron homeostasis. / In conclusion, the results of the present study implied that hepcidin plays an important role in maintaining brain iron homeostasis. Hepcidin is beneficial for PD and this effect is related to its iron-regulatory effect via inhibiting iron accumulation in the substantia nigra. Hepcidin effectively controls iron uptake and release through regulating iron transport proteins expressions in the brain, which would contribute to brain iron homeostasis. Therefore, manipulation of hepcidin level in the brain has a potential to be developed into a novel preventive approach for the iron-associated NDs such as PD. / In the second part, we investigated the effect of hepcidin on the processes of iron uptake and release in the cultured brain cells including neurons, astrocytes and brain vascular endothelial cells (BVECs). The expressions of iron uptake proteins such as transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) as well as the iron exporter ferroportin 1 (Fpn1) were also observed. We found that hepcidin reduced both iron uptake and release via decreasing iron transport proteins expressions in these brain cells, which would contribute to its iron regulatory effect. Finally, we further explored the mechanisms underlying the regulatory effect of hepcidin on the iron transporters in the last part, and found that the action of hepcidin in reducing TfR1 expression is a direct and cAMP-PKA (Cyclic Adenosine 3', 5'-monophosphate/ Protein Kinase-A) pathway-dependent event. / Iron is a transition trace metal essential for mammalian cellular and tissue viability. It also plays important roles in the central nervous system (CNS), including embryonic brain development, myelination, and neurotransmitters synthesis. However, abnormal iron accumulation has been demonstrated in a number of neurodegenerative diseases (NDs) such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases (HD). Currently very little is known about the mechanisms involved in brain iron homeostasis and therefore it is not known why and how iron is abnormally increased in the brain. However, given the essential role that excess iron plays in the pathological processes in the NDs, to suppress the accumulated iron is expected to be an effective strategy to prevent and treat these NDs. / To investigate whether hepcidin could benefit iron-associated NDs including PD and whether this beneficial role is related to its iron-regulatory function in the brain, in the first part of study, we investigated the effects of hepcidin on the 6-hydroxydopamine (6-OHDA) in vitro and in vivo PD models. We found that in primary cultured mesencephalic (MES) neurons, hepcidin overexpression via adenovirus-hepcidin (Ad-hepcidin) infection prevented 6-OHDA-induced increase in cellular iron content and protected the MES neurons. In the 6-OHDA model of PD in vivo, overexpression of hepcidin in the substantia nigra via Ad-hepcidin intranigral injection significantly prevented iron accumulation and dopaminergic neurons loss in the pars compacta of substantia nigra (SNc). These effects were accompanied by a marked improvement in motor performance of the PD animals. These findings indicate that hepcidin could benefit iron-associated NDs such as PD and via its iron-regulatory role in the brain. / Du, Fang. / Adviser: Ya Ke. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lam Wun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLDGEMENT --- p.iii / TABLE OF CONTENT --- p.iv / ABBREVIATIONS --- p.x / LISTS OF TABLES --- p.xi / LISTS OF FIGURES --- p.xii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegeneration and triplet repeat diseases --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Polyglutamine nuclear inclusions --- p.4 / Chapter 1.3.1 --- Kinetics of polyglutamine nuclear inclusion formation --- p.4 / Chapter 1.3.2 --- Roles of protein inclusions in neurodegeneration --- p.7 / Chapter 1.4 --- Polyglutamine pathogenic pathways --- p.8 / Chapter 1.4.1 --- Protein depletion theory --- p.9 / Chapter 1.4.2 --- Induction of apoptotic pathways --- p.13 / Chapter 1.5 --- Previous study on NI proteins --- p.14 / Chapter 1.6 --- Drosophila model for studying polyglutamine diseases --- p.15 / Chapter 1.6.1 --- Drosophila model for studying human diseases --- p.15 / Chapter 1.6.2 --- GAL4/UAS gene expression system --- p.15 / Chapter 1.6.3 --- Drosophila polyglutamine models --- p.17 / Chapter 1.7 --- Objectives of the study --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila genetics --- p.22 / Chapter 2.1.1 --- Drosophila culture --- p.22 / Chapter 2.1.2 --- GAL4/UAS gene expression system --- p.22 / Chapter 2.1.3 --- Eye phenotypic analysis --- p.25 / Chapter 2.1.4 --- Polyglutamine fly models --- p.25 / Chapter 2.1.5 --- Generation and characterization of GFP-polyglutamine transgenic fly models --- p.25 / Chapter 2.2 --- Proteomic identification of nuclear inclusion proteins --- p.26 / Chapter 2.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.26 / Chapter 2.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.27 / Chapter 2.2.2.1 --- Approach overview --- p.27 / Chapter 2.2.2.2 --- Sample preparation for two-dimensional gel electrophoresis --- p.27 / Chapter 2.2.2.3 --- Two-dimensional gel electrophoresis --- p.29 / Chapter 2.2.2.4 --- Polyacrylamide gel staining --- p.31 / Chapter 2.2.2.5 --- Computer analysis of 2D patterns --- p.31 / Chapter 2.2.2.6 --- In-gel trypsin digestion --- p.32 / Chapter 2.2.2.7 --- Mass spectrometric analysis --- p.33 / Chapter 2.2.3 --- Detection of NIs by flow cytometry --- p.34 / Chapter 2.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.34 / Chapter 2.3.1 --- Sample preparation for SDS-PAGE --- p.34 / Chapter 2.3.2 --- SDS-PAGE --- p.35 / Chapter 2.4 --- Immunodetection --- p.36 / Chapter 2.4.1 --- Electroblotting --- p.36 / Chapter 2.4.2 --- Western blotting --- p.36 / Chapter 2.4.3 --- Filter trap assay --- p.37 / Chapter 2.5 --- Sav antibody production --- p.38 / Chapter 2.5.1 --- Sav peptide synthesis --- p.38 / Chapter 2.5.2 --- Rabbit immunization --- p.38 / Chapter 2.6 --- Cryosectioning and immunostaining of adult fly heads --- p.39 / Chapter 2.7 --- Alcohol dehydrogenase assay --- p.40 / Chapter 2.8 --- Semi-quantitative reverse transcription- Polymerase Chain Reaction --- p.41 / Chapter 2.8.1 --- Total RNA preparation from fly heads --- p.41 / Chapter 2.8.2 --- Reverse transcription- Polymerase Chain Reaction (RT-PCR) --- p.41 / Chapter 2.9 --- Reagents and buffers --- p.42 / Chapter 3. --- RESULTS / Chapter 3.1 --- Transgenic polyglutamine fly models --- p.48 / Chapter 3.1.1 --- Characteristics of MJD polyglutamine fly model --- p.48 / Chapter 3.1.1.1 --- Overexpression of expanded truncated human MJD proteins in Drosophila causes eye degeneration --- p.49 / Chapter 3.1.1.2 --- Overexpression of expanded truncated human MJD proteins in Drosophila results in nuclear inclusion formation --- p.49 / Chapter 3.1.1.3 --- Formic acid dissolves fly polyglutamine nuclear inclusions --- p.51 / Chapter 3.1.1.3.1 --- Formic acid dissolves fly polyglutamine NIs as shown by Western blot analysis --- p.51 / Chapter 3.1.1.3.2 --- Formic acid dissolves fly polyglutamine NIs as shown by filter trap assay --- p.53 / Chapter 3.1.2 --- Summary --- p.55 / Chapter 3.2 --- Proteomic identification of nuclear inclusion (NI) proteins --- p.56 / Chapter 3.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.56 / Chapter 3.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.63 / Chapter 3.2.2.1 --- Two-dimensional gels showing differential protein spots as potential NI proteins --- p.63 / Chapter 3.2.2.2 --- NI protein candidates identified by the 2D approach --- p.75 / Chapter 3.2.3 --- Study of polyglutamine NI proteins by flow cytometry analysis --- p.90 / Chapter 3.2.3.1 --- Detection of fly polyglutamine NIs by flow cytometry --- p.90 / Chapter 3.2.3.2 --- Characterization of a new GFP-polyglutamine fly model --- p.92 / Chapter 3.3 --- Characterization of the nuclear inclusion protein candidates --- p.96 / Chapter 3.3.1 --- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) --- p.96 / Chapter 3.3.1.1 --- Confirmation of GAPDH as a NI protein --- p.97 / Chapter 3.3.1.2 --- Discussion --- p.97 / Chapter 3.3.2 --- Receptor of activated protein kinase C (RACK1) --- p.99 / Chapter 3.3.2.1 --- Confirmation of RACK1 as a NI protein --- p.99 / Chapter 3.3.2.1.1 --- Colocalization of RACK1 with NIs --- p.99 / Chapter 3.3.2.1.2 --- Formic Acid extracts RACK1 from NIs --- p.101 / Chapter 3.3.2.2 --- Reduction of soluble RACK1 protein level in polyglutamine fly --- p.101 / Chapter 3.3.2.2.1 --- Soluble RACK1 protein level reduced in polyglutamine fly --- p.101 / Chapter 3.3.2.2.2 --- RACK1 transcript level remains unchanged in polyglutamine fly --- p.103 / Chapter 3.3.2.3 --- Overexpression of RACK 1 partially suppresses polyglutamine degeneration --- p.105 / Chapter 3.3.2.4 --- Discussion --- p.107 / Chapter 3.3.3 --- Warts (Wts) --- p.111 / Chapter 3.3.3.1 --- Overexpression of Wts partially suppresses polyglutamine degeneration --- p.111 / Chapter 3.3.3.2 --- Wts mutant slightly enhances polyglutamine degeneration --- p.113 / Chapter 3.3.3.3 --- Genetic analysis of Warts pathway in polyglutamine pathogenesis --- p.113 / Chapter 3.3.3.3.1 --- Overexpression of Salvador partially suppresses polyglutamine degeneration --- p.116 / Chapter 3.3.3.3.2 --- Hpo mutant slightly enhances polyglutamine degeneration --- p.119 / Chapter 3.3.3.3.3 --- Overexpression of DIAP1 partially suppresses polyglutamine degeneration --- p.119 / Chapter 3.3.3.4 --- Discussion --- p.121 / Chapter 3.3.4 --- Alcohol dehydrogenase (Adh) --- p.122 / Chapter 3.3.4.1 --- Adh activity is reduced in polyglutamine flies --- p.122 / Chapter 3.3.4.2 --- Overexpression of Hsp70 partially restores the reduced Adh activity in polyglutamine flies --- p.122 / Chapter 3.3.4.3 --- Discussion --- p.125 / Chapter 3.3.5 --- Genetic analysis of other NI protein candidates --- p.127 / Chapter 3.3.5.1 --- Overexpression of CG7920 protein partially suppresses polyglutamine degeneration --- p.127 / Chapter 3.3.5.2 --- Pten dsRNA slightly enhances polyglutamine degeneration --- p.129 / Chapter 3.3.6 --- Summary --- p.131 / Chapter 4. --- DISSCUSSION / Chapter 4.1 --- Protein depletion theory --- p.133 / Chapter 4.2 --- Comparison of different approaches for identification of NI proteins --- p.134 / Chapter 4.3 --- Long-term significance --- p.136 / Chapter 4.4 --- Future studies --- p.137 / Chapter 4.4.1 --- Characterization of other NI protein candidates --- p.137 / Chapter 4.4.2 --- Study of NI proteins by an alternative approach --- p.137 / Chapter 4.4.3 --- Study of NI proteins using other polyglutamine fly models --- p.137 / Chapter 5. --- CONCLUSION --- p.139 / Chapter 6. --- REFERENCES --- p.140

Hybrid neural-microelectronic systems, systems composed of biological neural networks and neuronal models, have great potential for the treatment of neural injury and disease. The utility of such systems will be ultimately determined by the ability of the engineered component to correctly replicate the function of biological neural networks. These models can take the form of mechanistic models, which reproduce neural function by describing the physiologic mechanisms that produce neural activity, and empirical models, which reproduce neural function through more simplified mathematical expressions. We present our research into the role of model complexity in creating robust and flexible behaviors in hybrid systems. Beginning with a complex mechanistic model of a leech heartbeat interneuron, we create a series of three systematically reduced models that incorporate both mechanistic and empirical components. We then evaluate the robustness of these models to parameter variation, and assess the flexibility of the models activities. The modeling studies are validated by incorporating both mechanistic and semi-empirical models in hybrid systems with a living leech heartbeat interneuron. Our results indicate that model complexity serves to increase both the robustness of the system and the ability of the system to produce flexible outputs.

The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.

AIDS Dementia Complex (ADC) is a neurodegenerative disorder implicated in HIV-1 infection that is associated with elevated levels of the neurotoxin, quinolinic acid (QA) which causes a cascade of events to occur, leading to the production of reactive oxygen species (ROS), these being ultimately responsible for oxidative neurotoxicity. In clinical studies, Non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP) have been shown to potentially delay the progressive degeneration of neurons, thus reducing the frequency and neurological deficits associated with ADC. Despite these neuroprotective implications, there is still no biochemical data to demonstrate the mechanisms through which these agents offer neuroprotection. The present study aims to elucidate and further characterize the possible antioxidant and neuroprotective mechanisms of NVP and EFV in vitro and in vivo, using QA-induced neurotoxicity as a model. Research has demonstrated that antioxidants and metal chelators have the ability to offer neuroprotection against free radical induced injury and may be beneficial in the prevention or treatment of neurodegeneration. Hence the antioxidant and metal binding properties of these agents were investigated respectively. Inorganic studies, including the 1, 1-diphenyl-2 picrylhydrazyl (DPPH) assay, show that these agents readily scavenge free radicals in vitro, thus postulating the antioxidant property of these agents. The enhancement of superoxide radical generation and iron mediated Fenton reaction by QA is related to lipid peroxidation in biological systems, the extent of which was assayed using the nitroblue tetrazolium and thiobarbituric acid method respectively. Both agents significantly curtail QA-induced lipid peroxidation and potentially scavenge superoxide anions generated by cyanide in vitro. Furthermore, in vivo results demonstrate the ability of NVP and EFV to protect hippocampal neurons against lipid peroxidation induced by QA and superoxide radicals generated as a consequence thereof. The alleviation of QA-induced oxidative stress in vitro possibly occurs through the binding of iron (II) and / or iron (III), and this argument is further strengthened by the ability of EFV and not NVP to reduce iron (II)-induced lipid peroxidation in vitro directly. In addition the ferrozine and electrochemistry assay were used to measure the extent of iron (II) Fe[superscript 2+] and iron (III) Fe[superscript 3+] chelation activity. Both assays demonstrate that these agents bind iron (II) and iron (III), and prevent redox recycling of iron and subsequent complexation of Fe[superscript 2+] with QA which enhances neuronal damage. Both NNRTIs inhibit the endogenous biosynthesis of QA by inhibiting liver tryptophan 2, 3-dioxygenase activity in vivo and subsequently increasing hippocampal serotonin levels. Furthermore, these agents reduce the turnover of hippocampal serotonin to 5-hydroxyindole acetic acid. NVP and not EFV increase 5-hydroxyindole acetic acid and norepinephrine levels in the hippocampus. The results of the pineal indole metabolism study show that NVP increases the synthesis of melatonin, but decreases N-acetylserotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol levels. Furthermore, it shows that EFV decreases 5-hydroxyindole acetic acid and melatonin synthesis. Behavioural studies using a Morris water maze show that the post-treatment of rats with NVP and EFV significantly improves QA-induced spatial memory deficits in the hippocampus. This study therefore provides novel information regarding the neuroprotective mechanisms of NVP and EFV. These findings strengthen the argument that these NNRTIs not only have antiviral effects but possess potential neuroprotective properties, which may contribute to the effectiveness of these drugs in the treatment of ADC.

Accumulating evidence suggests that anti-inflammatory agents and antioxidants have neuroprotective properties and may be beneficial in the treatment of neurodegenerative disorders. In the present study, the possible neuroprotective properties of tolmetin, sulindac and turmeric were investigated. The antioxidant effects of tolmetin and sulindac were determined by inducing free radical generation with quinolinic acid (QA), cyanide or iron (II) in rat brain homogenates or primary hippocampal neurons. Tolmetin and sulindac significantly reduce lipid peroxidation and scavenge the superoxide anion. Metal binding studies were conducted to determine whether metal chelation is a possible mechanism through which these agents reduce QA and iron (II)-induced lipid peroxidation. UV/VIS, infrared spectroscopy as well as electrochemical studies show that both agents bind to iron (II) and/or iron (III). Histological examination of the hippocampus showed that pre-treatment of animals with tolmetin or sulindac offers protection against intrahippocampal injections of QA. These agents also attenuate QA-induced apoptosis and reduce the loss of neurons in the hippocampus. The co-incubation of primary hippocampal neurons with the NSAIDS also enhanced cell viability which is significantly reduced by QA. Behavioural studies using a water maze showed that the treatment of animals after QA-induced neurotoxicity reduces QA-induced spatial memory loss. Tolmetin and sulindac also reduced glutathione depletion and protein oxidation in rat hippocampus. Both NSAIDS inhibit liver tryptophan 2,3-dioxygenase activity in vitro and in vivo and subsequently increased hippocampal serotonin levels. However, both NSAIDS also reduce dopamine levels in rat striatum. Tolmetin but not sulindac increased the synthesis of melatonin by the pineal gland. The active components of turmeric known as the curcuminoids were separated using preparative thin layer chromatography (TLC). The purity was confirmed by TLC, NMR and mass spectrometry. The environmental toxin lead, induces lipid peroxidation and reduces primary hippocampal neuronal viability. The co-incubation of the neurons with the curcuminoids significantly reduces lead-induced lipid peroxidation and enhances neuronal cell viability in the presence of lead. Lead-induced spatial memory deficit is also attenuated with curcumin, demethoxycurcumin but not bisdemethoxycurcumin. The curcuminoids also reduce lead-induced hippocampal glutathione depletion and protein oxidation. Metal binding studies show that the curcuminoids bind to lead and is another possible mechanism through which the curcuminoids reduce lead-induced neurotoxicity. The findings of this study indicate a possible role of tolmetin, sulindac and turmeric in neurodegenerative disorders such as Alzheimer’s disease. However, tolmetin and sulindac reduce dopamine levels.

Homocysteine (Hcy) is a sulfur containing amino acid and is a potent neurotoxin. It has been shown that elevated levels of Hcy, termed hyperhomocysteinemia, plays a role in the pathologies of Alzheimer’s disease (AD) and age-related cognitive decline. Hcy is a glutamate agonist, which causes in increase in Ca[superscript (2+)] influx via the activation of NMDA class of excitatory amino acid receptors, which results in neuronal cell death and apoptosis. Estrogen and progesterone are female hormones that are responsible for reproduction and maternal behaviour. However, in the last decade, it is evident that both female hormones have neuroprotective properties in many animal models of neurodegeneration. Collectively, both estrogen and progesterone reduce the consequences of the oxidative stress by enhancing the antioxidant defence mechanisms, reducing excitotoxicity by altering glutamate receptor activity and reducing the damage caused by lipid peroxidation. However, the mechanisms by which estrogen and progesterone provide such neuroprotection probably depend on the type and concentration of hormone present. Moreover, numerous studies have shown that hormone replacement therapy (HRT, estrogen and progestins) or estrogen-only replacement therapy (ERT) may prevent or delay the onset of AD and improve cognition for women with AD. Clinical trials have also shown that women taking HRT may modify the effects of Hcy levels on cognitive functioning. Oxidative stress increases in the aging brain and thus has a powerful effect on enhanced susceptibility to neurodegenerative disease. The detection and measurement of lipid peroxidation and superoxide anion radicals in the brain tissue supports the involvement of free radical reactions in neurotoxicity and in neurodegenerative disorders. The hippocampus is an important region of the brain responsible for the formation of memory. However, agents that induce stress in this area have harmful effects and could lead to dementia. This study aims to investigate and clarify the neuroprotective effects of estrogen and progesterone, using Hcy-induced neurodegenerative models. The initial studies demonstrate that estrogen and progesterone have the ability to scavenge potent free radicals. Histological studies undertaken reveal that both estrogen and progesterone protect against Hcy-induced neuronal cell death. In addition, immunohistochemical investigations show that Hcy-induced apoptosis in the hippocampus can be inhibited by both estrogen and progesterone. However, estrogen also acts at the NMDA receptor as an agonist, while progesterone blocks at the NMDA receptor. These mechanisms reduce the ability of Hcy to cause damage to neurons, since Hcy-induced neurotoxicity is dependent on the overstimulation of the NMDA receptor. SOD and GPx are important enzymatic antioxidants which can react with ROS and neutralize them before these inflict damage in the brain. Hcy can increase oxidative stress by inhibiting expression and function of these antioxidants. However, it has been shown that the antioxidant abilities of both estrogen and progesterone can up-regulate the activities of SOD and GPx. These results provide further evidence that estrogen and progesterone act as antioxidants and are free radical scavengers. The discovery of neuroprotective agents is becoming important as accumulating evidence indicates the protective role of both estrogen and progesterone in Hcy-induced neurodegeneration. Thus further work in clinical trials is needed to examine whether reducing Hcy levels with HRT can become the treatment of neurodegenerative disorders, such as Alzheimer’s disease.

The imbalance in the levels of certain microRNAs (miRNAs) in Alzheimer’s disease (AD) brains promotes alterations in tau proteostasis and neurodegeneration. However, potential mechanisms governing how specific miRNAs are dysregulated in AD brains are still under investigation. Epitranscriptomics is a mode of post-transcriptional regulation that can control brain functions during development and adulthood. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases that has the ability to modify mammalian non-coding RNAs. Importantly, autosomal-recessive loss of function mutations in NSun2 have been associated with neurological abnormalities in humans. Here, we report that dysregulation of NSun2 can induce alterations in tau phosphorylation by modulating the levels of miR-125b, a main player in tau pathology. We were able to provide supporting evidence by utilizing several model systems such as Drosophila, human induced pluripotent stem cell (iPSC) derived neurons, rat primary neuronal cultures and mice. Our Western blot analysis not only shows that NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex, but also NSun2 protein expression levels are downregulated in post-mortem brain tissues from AD patients. Remarkably, we also found decreased NSun2 protein levels in AD mice and human cellular models. To prove these observed alterations were unique to AD, we further evaluated brain tissues from other tauopathies. Strikingly, NSun2 protein levels were similar between tauopathy cases and controls indicating that dysregulation of NSun2 might be unique to AD cases. Further, we investigated the pathological role of NSun2 by utilizing a well-established Drosophila melanogaster model of tau-induced toxicity. We found that a reduction of NSun2 protein levels exacerbated tau toxicity while overexpression of NSun2 partially abrogated toxicity proving bidirectionality. We used a lentiviral system to knock down NSun2 expression in iPSC derived neuronal cultures. Western blot analysis and immunofluorescence staining showed a significant change in tau phosphorylation levels. To investigate what could be triggering observed alterations in NSun2 levels, we performed experiments in rat primary hippocampal neurons. We found that the treatment with oligomeric amyloid-beta A caused a decrease in NSun2 protein levels and at the same time, increased tau phosphorylation levels in primary hippocampal neurons. Lastly, we performed RNA immunoprecipitation coupled with qPCR and histological analysis using NSun2 conditional knockout (KO) mice and observed that NSun2 deficiency promoted aberrant levels of m6A methylated miR-125b and tau hyperphosphorylation. Altogether, our study demonstrates that neuronal NSun2 deficiency in AD promotes neurodegeneration by altering tau phosphorylation and tau toxicity through an epitranscriptomic regulatory mechanism and highlights a potential novel therapeutic target.