Parkinson's disease (PD) is a neurodegenerative disorder with no known cure. This thesis explores the degenerative process in two neurotoxin-based models, the 6-hydroxydopamine and the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)/probenecid mouse models, to yield important information about the pathogenesis of PD. Neuronal survival patterns in Parkinsonian patients and animals are heterogeneous. More dopaminergic neurons are lost from the ventral tier of the substantia nigra (SN) than from the dorsal tier or the adjacent ventral tegmental area, possibly due to differential expression of the calcium-binding protein, calbindin D28K. Brain sections were processed for tyrosine hydroxylase (TH) and calbindin (CB) immunocytochemistry to distinguish the dopaminergic subpopulations. I show that more TH+/CB- and TH-/CB+ than TH+/CB+ neurons are lost in both models, suggesting that CB confers some degree of protection for dopaminergic neurons. With respect to connectivity, I show that both TH+ and CB+ neurons receive striatal and dorsal raphe inputs. I investigated the possibility of a progressive loss in midbrain neurons by prolonging the post-lesion survival period. In both models, there is an irreversible neuronal cell loss of TH+, CB+ and TH+/CB+ neurons but the effects of survival time and lesion treatments differ for the three neuronal types. The lesions also appear to be toxic to GABAergic neurons. I explore whether, once neurodegeneration has started, neurons can be rescued by pharmacological intervention. Salicylic acid appears both to reduce microglial activation and significantly improve TH+, but not CB+ or TH+/CB+ neuronal survival. PD appears multifactorial in origin and may involve complex interactions between genetic and environmental influences. I show that a xenobiotic-metabolising enzyme, arylamine N-acetyltransferase may fulfil a neuroprotective role in the SN by limiting the environmental risks. Taken together, this study provides a body of information on two different mouse PD models and highlights possible genetic predispositions to PD neuropathology.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: A simple and rapid scatter-based flow cytometric assay was developed to detect apoptosis in CD4+ and CD8+ T cells from a mixed population of cells. The assay was suitable for children. Apoptotic PBMCs were confirmed by morphologic assessment in clinical samples ex vivo and after overnight culture. The scatter-based assay was validated in a number of ways. Firstly, PBMCs were irradiated with 500 rads and cultured overnight to induce apoptosis. Thereafter, PBMCs were labeled with a CD4 MAb. CD4+ cells were sorted into apoptotic and viable populations by scatter characteristics (diminished forward and increased side scatter). Morphology was assessed by fluorescence microscopy. The majority of cells with apoptotic scatter characteristics had apoptotic morphology (chromatin condensation) (80.6%). Ninety-two percent of cells from the viable region had normal morphology. CD4+ T cell apoptosis measured by scatter was then correlated with the TdT assay for DNA fragmentation. Lastly, CD4+ T cell apoptosis by scatter and annexin V uptake were also shown to correlate. In the latter experiments, PBMC morphology and cell death by trypan blue uptake were studied simultaneously and confirmed the two flow cytometric assays. Apoptosis of CD4+ and CD8+ T cells has been shown in PBMCs from HIV infected adults analyzed after overnight culture. Since cell death may be an artifact of in vitro culture, and because there is little information on apoptosis in paediatric HIV disease, I undertook a cross-sectional analysis in PBMCs analyzed immediately ex vivo from HIV infected children and adults. Patients were studied in Denver, CO, USA. PBMCs from 21 children, 4 adolescents and 9 adults and seronegative age-matched controls were stained for CD4 and CD8 surface markers. Apoptotic cells were detected in a newly characterized flow cytometric assay by diminished forward and increased side scatter. For the scatter assay, PBMCs had been labeled initially by an indirect method involving an intermediary incubation in the presence of biotinylated MAbs at 37°C for 30 minutes prior to incubating with streptavidin-FITC at 4°C for 20 minutes. Thereafter, the intermediary incubation step was removed and PBMCs were incubated with PE-conjugated CD4+ and CD8+ MAbs. Both CD4+ and CD8+ T cell apoptosis appeared enhanced in the indirect method. The significant differences were abolished after subtraction of data from simultaneously studied time-matched controls. CD4+ and CD8+ T cell apoptosis were significantly higher in HIV-infected study subjects than in simultaneously studied seronegative controls. PBMCs were assayed immediately ex vivo and after overnight culture after stimulation by an anti-TCR MAb as well as spontaneously. There was a direct correlation between CD4+ and CD8+ T cell apoptosis and CD4+ T cell depletion. A significant correlation was also shown between apoptosis immediately ex vivo and after overnight culture. I then studied apoptosis in a South African population comprising 18 symptomatic children and 4 seroreverters. CD4+ and CD8+ T cell apoptosis were significantly higher in symptomatic HIV-1-infected children than in seroreverters and seronegative controls. CD4+ T cell apoptosis correlated with depletion of CD4+ T cell percentage in symptomatic HIV-1-infected children. I also noted elevated CD4+ T cell apoptosis in patients recovering from intercurrent disease in comparison to those who were either acutely ill or relatively asymptomatic outpatient attendees. Lastly, I compared CD4+ and CD8+ T cell apoptosis in cohorts from Denver, CO and Tygerberg Children’s Hospital, South Africa. I selected only patients with moderate or severe HIV infection from both centers. South African patients were significantly younger, more malnourished, had higher gamma globulin levels and were less likely to receive ART. CD8+ T cell apoptosis was higher in North American patients suggesting a possible impairment in CD8+ activity in the South African study subjects. / AFRIKAANSE OPSOMMING: ‘n Eenvoudige en vinnige vloei sitometriese toets is ontwikkel om apoptose aan te toon vanuit ‘n gemengde populasie selle. Dit moes geskik wees vir kinders van wie net klein volumes bloed getrek kan word. Die teenwoordigheid van apoptotiese perifere bloed mononuklere selle (PBMS) was vasgestel deur morfologiese beoordeling in kliniese monsters ex vivo en na oornag kultuur. Die ondersoek is gebasseer op die verstrooings patroon van bestraalde PBMS wat apoptose induseer. PBMS is gemerk met a CD4 MAb. CD4+ selle is gesorteer in apoptotiese en lewensvatbare populasies deur verstrooings karakteristieke. Morfologie is beoordeel deur fluoreserende mikroskopie. Die meerderheid van selle met apoptotiese verstrooings karakteristieke (verminderde voorwaartse en verhoogde sywaartse verstrooings patroon) het apoptotiese karakteristieke gehad (80.6%). Twee-en-negentig persent van selle van die lewensvatbare area het normale morfologie gehad. Verstrooings patroon is ook gekorreleer met die TdT meting vir DNA fragmentasie in kliniese monsters van MIV-geinfekteerde kinders. Daarna is Annexin V gekorreleer met verstrooings patroon, apoptotiese morfologie en trypan blou opname in selle wat blootgestel is na verskillende konsentrasies van beauvericin. Apoptose van CD4+ en CD8+ T-selle is bewys in PBMS van MIV-geinfekteerde volwassenes na oornag kultuur. Omdat sel dood ‘n artefak van in vitro kultuur kan wees, en omdat daar min inligting is oor apoptose in paediatriese MIV siekte, het ek onderneem om ‘n deursnee analiese te doen in PBMS wat onmiddelik ex vivo geanaliseer is vanaf MlV-geinfekteerde kinders en volwassenes. Die pasiente is bestudeer in Denver, Colorado, VS A. PBMS van 22 kinders, 4 adolessente en 9 volwassenes en seronegatiewe ouderdoms-gepasde kontroles is gekleur vir CD4+ en CD8+ oppervlaksmerkers. Apoptotiese selle is vloeisitometries aangedui deur verandering in verstrooings patroon. Vir die doeleindes van die verstrooings assay is die PBMS aanvanklik deur ‘n indirekte metode gemerk, wat ‘n intermediere inkubasie in die teenwoordigheid van biogetinileerde MAbs by 37°C vir 30 minute voor dit geinkubeer is met streptavidin- FITC by 4°C vir 20 minute behels. Daarna is die intermediere inkubasie stap verwyder en PBMC is geinkubeer met PE - gekonjugeerde CD4+ and CD8+ MAbs. Beide die CD4+ en CD8+ T-sel apoptose het verhoog voorgekom met die indirekte metode. Die betekenisvolle verskille het verdwyn na data van gelyktydige tyd - gepaarde kontroles afgetrek is. CD4+ en CD8+ T-sel apoptose was betekenisvol hoër in MIV-geinfekteerde studie gevalle as in gelyktydig bestudeerde seronegatiewe kontroles. PBMS assays is gedoen onmiddelik ex vivo en na oornag inkubasie na stimulasie deur ‘n anti-TCR MAb, sowel as spontaan. Daar was ‘n direkte korrelasie tussen CD4+ en CD8+ T sel apoptosis en CD4+ T sel vermindering. ‘n Beduidende korrelasie is ook getoon tussen apoptose onmiddelik ex vivo en na oornag kultuur. Daaropvolgend het ek apoptose in ‘n Suid Afrikaanse populasie van 18 simptomatiese kinders en 4 serologies terukerende gevalle bestudeer. CD4+ en CD8+ T sel apoptose was aansienlik hoër in siptomatiese MIV - 1-geinfekteerde kinders as in die serologies terukerende gevalle en seronegatiewe kontroles. CD4+ T sel apoptose het gekorrelleer met vermindering van CD4+ T sel persentasie. Ek het ook opgemerk dat daar ‘n tendens bestaan het tot verhoogde CD4+ T sel apoptose in pasiente wat besig was om te herstel van bykomende siektes. Ek het CD4+ en CD8+ T sel apoptose in kohorte van Denver, Colorado en Tygerberg, Suid Afrika vergelyk. Suid Afrikaanse pasiente was jonger en meer wangevoed as hul Noord Amerikaanse ewekniee. Suid Afrikaanse kinders het ook meer gevorderde siekte gehad. Wanneer pasiente gepas is vir die graad van ernstigheid van siekte en slegs die minder ernstige (B) en ernstige siekte (C) vergelyk is, was CD8+ T sel apoptose beduidend hoër in Noord Amerikaanse pasiente. Hierdie waarneming ondersteun die hipotese dat CD 8+ T sel aktiwiteit moontlik onderdruk mag wees in simptomatiese Suid Afrikaanse MIV-1-geinfekteerde kinders.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The following dissertation discusses the role of Aβ42 dependent hyperactivation of AMPK mediating synaptic loss through coordinated Mff-dependent mitochondrial fission and Ulk2-dpendent mitophagy in dendrites of PNs. In Chapter 1, I provide a brief background on Alzheimer’s disease and the cellular and molecular mechanisms that have been relevant to the pathogenesis of the disease including disruption on mitochondrial homeostasis and autophagy. In Chapter 2, I discuss the findings of my main project describing the role of Aβ42 induced mitochondrial remodeling leading to synapse loss in vitro and in vivo in part by hyperactivation of CAMKKII-AMPK. Chapter 3 covers a review article that I participated in in examining the role of mitochondria in various ND. In Chapter 4, I discuss about a project I was involved in in examining the mechanism behind maintaining mitochondrial morphology in axon versus dendrite and its functional consequence. In Chapter 5, I end the dissertation by highlighting key findings, potential future studies, and concluding remarks.

No description available.

Submitted in fulfilment of the requirements of Master's Degree in Chemistry, Durban University of Technology, 2017. / DNA damage plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD) therefore, an innovative ss-DNA/dopamine/TiO2/FTO electrode strategy was developed to detect the genotoxicity upon photocatalytic reactions. This study involves a computational and electrochemical investigation towards the direct measurement of DNA damage. Computational chemistry was useful to resolve the intricate chemistry problems behind electrode constructions. The computational protocols were simultaneously carried out comprising of density functional theory (DFT) calculations, Metropolis Monte Carlo (MC) adsorption studies, and molecular dynamics (MD) simulations. The DFT calculations elucidated the structural, electronics, and vibrational properties of the electrode components resulting in a good agreement with the experimental parameters. The MC simulations carried out using simulated annealing predicted the adsorption process within layer-by-layer electrode as well generating reliable inputs prior to MD simulations. A 100 ns MD simulations were performed using a canonical ensemble provided information on the thermodynamics parameters such as total energy, temperature, and potential energy profiles, including radius of gyrations and atomic density profiles. Binding energies calculated from the MD trajectories revealed increasing interaction energies for the layer-by-layer electrode, in agreement with the electrochemical characterization studies (i.e. gradual decrease of cyclic voltammogram (CV) as well as increasing diameter of electrochemical impedance spectroscopy (EIS) semicircle upon electrode modification). The higher binding energies may lead to smaller changes in the electrochemical polarizability which directly affect to the decreasing of redox peak current and charge transfer resistance enhancement. Instead, HOMO-LUMO DFT levels are also taken into account to explain electron transfer phenomena within layer construction leading to the alteration of CV behaviours. Experimentally, the ss-DNA was electronically linked to TiO2/FTO surface through dopamine as a molecular anchor. Electrochemical measurements using cyclic voltammetry and EIS were employed to characterize the electrode modifications. The square wave voltammetry was subsequently used to measure the DNA damage and the potency of antioxidant treatment using ascorbic acid (AA) due to its ability in protecting the DNA from the damages. The presence of AA significantly protected the DNA from the damage, therefore was able to be used as a potential treatment in AD. Theoretically, guanine residues predicted by DFT as the most reactive sites of the ss-DNA involved in the genotoxic reactions. Overall, the theoretical studies successfully validated the experimental study as well as providing the molecular basis of interaction phenomena towards electrode constructions. Our results highlight the potential application of this methodology to screen the genotoxicity in Alzheimer’s, suggesting the important role of theoretical studies to predict the molecular interaction and validation of the DNA-based sensors and bioelectronics. / M

The aim of this dissertation was 1) to explore early stage aggregation kinetic behavior of Amyloid-β 1-40 (Aβ1-40) by incorporation of unnatural amino acid pcyanophenylalanine as a site-specific fluorescence reporter, 2) to explore the effect of macromolecules on the aggregation of Aβ1-40. Chapter One provides an introduction of Alzheimer’s disease as an amyloidogenic disease, amyloidogenic peptide and amyloid formation. Details were shown about the research progress of Aβ1-40 aggregation and Aβ1-40’s interaction with polyelectrolytes, and how treatments studies were designed. In Chapter two, using Aβ1-23 as a model molecule, the distinct site-specific dynamics was identified, during amyloid formation, and the structural characteristics of amyloid fibrils were defined by using an unnatural amino acid, p-cyanophenylalanine, as a sensitive fluorescent and Raman probe. The results reveal distinct local environmental changes of specific residues during the aggregation of Aβ1-23. The results also suggest that an edge-to-face aromatic interaction between the F4 and F19 residues from the adjacent in-register β-strands plays a key role in the conformational conversion to form and stabilize β-sheet structure. In Chapter Three, p-cyanophenylalanine was incorporated in the full sequence of Aβ1-40. Site-specific information from p-cyanophenylalanine fluorescence was studied and summarized. In Chapter Four, the inhibiting effect of an anionic polyelectrolyte poly(4- styrenesulfonate) (PSS) on the aggregation of Aβ1-40 peptide was reported. The results demonstrate the strong inhibition potential of PSS on the aggregation of Aβ1-40. Additional studies indicate that the presence of both aliphatic backbone as well as aromatic side chain group in PSS is essential for its inhibition activity. In Chapter Five, it was investigated the effect of two polyelectrolytes, chitosan (CHT) and N-trimethyl chitosan chloride (TMC), on the aggregation of Aβ1-40. Results show that both CHT and TMC exhibit a concentration-dependent decrease of amyloid aggregation suggesting their application as amyloid assembly inhibitors. Their binding mechanism was investigated by computational modeling which shows that Aβ1-40 monomer was primarily stabilized by electrostatic interactions with charged amine and quaternary amines of CHT and TMC respectively. Chapter Six, describes all experimental procedures and instrument setup in detail. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

"December, 2002" Includes bibliographical references (leaves 211-238)