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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
51

Comparison of the neuroprotective potential of theanine and minocycline

Mpofu, Tariro Ann-Maureen 20 September 2010 (has links)
Stroke is one of the most common causes of disability and death worldwide. The most commonly experienced stroke in the clinical setting is focal ischaemia in which the middle cerebral artery (MCA) is occluded and leads to a complex series of various pathophysiological pathways that ultimately lead to neuronal cell death. Several studies have been conducted on various therapeutic agents in the search for a neuroprotective drug and various animal models have been used to carry out this research. While theanine, a component of green tea and minocycline, a tetracycline antibiotic, have been shown to possess some neuroprotective properties, the mechanisms by which these two agents carry out these effects still remains unclear. The objectives of this study were to investigate the mechanisms by which these drugs carry out these neuroprotective effects and their neuroprotective ability in a MCA occlusion model of focal ischaemia. Ischaemia leads to oxidative stress due to the imbalance of free radicals and the endogenous antioxidant defence system. An antioxidant assay using the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH●) radical was used to assess the antiradical properties of each drug. It was found that minocycline showed superior antioxidant activity in vitro when compared to theanine. Further studies on the drugs‟ ability to attenuate the Fenton reaction (in which iron catalyses the formation of reactive species) were elucidated using electrochemical analysis, UV/VIS studies, ferrozine and ferritin assays. It was found that minocycline, in contrast to theanine, was able to bind to iron ions and thus potentially prevent the participation of iron in metal catalysed radical reaction. The antioxidant activity of both drugs was further investigated by assessing their effect on cyanide-induced superoxide generation and quinolinic acid (QA)-induced lipid peroxidation (LP). Experimental evidence shows that both drugs had no significant effect on the generation of superoxide in vitro and that there was a significant decrease in LP for minocycline in vitro and theanine in vivo. The metal binding and antioxidant properties were postulated to be a possible mechanism through which these agents reduced lipid peroxidation. A study was conducted to determine the effects of the drugs on the biosynthesis of the neurotoxin, QA and it was found that minocycline increases the levels of holoenzyme activity of tryptophan-2, 3-dioxygenase (TDO) in vitro and that theanine reduces the levels of the same enzyme in vivo after treatment for 10 days. TDO is the enzyme that converts tryptophan to other products that enable enzymatic activity to change it to QA. Minocycline was thought to bring about this effect as it has been shown from preceding experimental studies that it is an effective reducing agent. Theanine on the other hand is hypothesised to bring about a reduction in holoenzyme activity by changing the binding of tryptophan to the enzyme or affecting the radicals that participate in the enzymatic degradation of tryptophan. A focal ischaemic model of stroke was induced by occluding the MCA. Histological examination of the hippocampus post -ischaemia shows a reduction in the size of the infarct after pre-treatment with minocycline only. A further study into the effects of the drugs on the generation of superoxide and on the levels of the endogenous glutathione after a stroke was carried out. Pre-treatment of the animals with either theanine or minocycline showed no significant effects on the generation of the radical species or of the endogenous antioxidant which ruled out these as a mechanism of neuroprotection of both drugs, post-ischaemia.The findings of this study provide novel information on the possible mechanisms by which both theanine and minocycline act to bring about neuroprotection. In particular in this study, pre-treatment with minocycline has shown promise in the focal ischaemic model of stroke.
52

Metal interactions with neural substrates and their role in neurodegeneration

Lack, Barbara Anne January 2003 (has links)
"Life" may be characterized as a controlled stationary flow equilibrium, maintained by energy consuming chemical reactions. The physiological functioning of these life systems include at least 28 of the elements isolated on the periodic table thus far, most of which are metals. However, as with Paracelsus Principle: "The dose makes the poison", there exists a definite link between metal levels, essential and toxic, and the onset of neurodegenerative diseases. The economic costs of brain dysfunction are enormous, but this pales in comparison to the staggering emotional toll on the victims themselves and their families. In an attempt to improve the understanding of the causes of neurodegeneration, this study focuses on one potential aspect: the possible link between metals and neurotransmitter homeostasis utilising a variety of electronanalytical techniques. Adsorptive cathodic stripping voltammetry was employed to investigate the binding affinities and complex formation of melatonin and its precursor serotonin with calcium, potassium, sodium, lithium and aluminium. The results showed that all the metals studied formed complexes with both pineal indoleamines. However, the stability and affmity of the ligands toward the various metals varied greatly. The study suggests a further role for melatonin, that of metalloregulator and possible metal detoxifier in the brain, the in vivo studies which followed will further substantiate this notion. This research additionally focused on the cholinergic system, in particular acetylcholine complex formation studies with mercury, lead, cadmium, copper and zinc using the adsorptive cathodic stripping voltammetry method. The formation and characterisation of a solid mercury-acetylcholine complex lent further strength to the in situ electrochemical complex formation observed. The results showed the preference of acetylcholine for environmentally toxic heavy metals (such as Cd²⁺) over those divalent cations that occur naturally in the body. The possible metalloregulatory role melatonin played in the three brain regIOns: cerebellum, cortex and corpus striatum of male Wistar rats was studied as an in vivo extension of the earlier in vitro studies. Anodic stripping voltammetry was employed to detect metal levels present. The results showed that daily injections of melatonin was responsible for significantly decreasing copper(I), cadmium(II) and lead(II) levels in various regions of the rat brain of those animals that had undergone a pinealectomy in comparison to the saline injected group having undergone the same treatment. Histological and electrochemical stripping techniques were applied to investigate the implications of high A1³⁺ levels in the brain regions, particularly the hippocampus. Melatonin showed signs of promise in indirect symptom alleviation and by significantly decreasing A1³⁺ levels in rats that had been dosed with melatonin prior to A1³⁺ treatments in comparison with the control groups. Finally a preliminary study outlining a method for the production of a calcium selective microelectrode was undertaken. Further work is still needed to optimise the microelectrode production as well as its possible applications. However, whilst the overall conclusions of this entire multidisciplinary study may indeed only be in effect one piece of a very large puzzle on neurodegenerative diseases, this piece will no doubt serve as a building block for further ideas and work in this field.
53

An investigation into the neuroprotective and neurotoxic properties of levodopa, dopamine and selegiline

Scheepers, Mark Wesley January 2008 (has links)
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a profound loss of dopaminergic neurons from the substantia nigra (SN). Among the many pathogenic mechanisms thought to be responsible for the demise of these cells, dopamine (DA)-dependent oxidative stress and oxidative damage has taken center stage due to extensive experimental evidence showing that DA-derived reactive oxygen species (ROS) and oxidized DA metabolites are toxic to SN neurons. Despite its being the most efficacious drug for symptom reversal in PD, there is concern that levodopa (LD) may contribute to the neuronal degeneration and progression of PD by enhancing DA concentrations and turnover in surviving dopaminergic neurons. The present study investigates the potential neurotoxic and neuroprotective effects of DA in vitro. These effects are compared to the toxicity and neuroprotective effects observed in the rat striatum after the administration of LD and selegiline (SEL), both of which increase striatal DA levels. The effects of exogenous LD and/or SEL administration on both the oxidative stress caused by increased striatal iron (II) levels and its consequences have also been investigated. 6-Hydroxydopamine (6-OHDA) is a potent neurotoxin used to mimic dopaminergic degeneration in animal models of PD. The formation of 6-OHDA in vivo could destroy central dopaminergic nerve terminals and enhance the progression of PD. Inorganic studies using high performance liquid chromatography with electrochemical detection (HPLC-ECD) show that hydroxyl radicals can react with DA to form 6-OHDA in vitro. SEL results in a significant decrease in the formation of 6-OHDA in vitro, probably as a result of its antioxidant properties. However, the exogenous administration of LD, with or without SEL, either does not lead to the formation of striatal 6-OHDA in vivo or produces concentrations below the detection limit of the assay. This is despite the fact that striatal DA levels in these rats are significantly elevated (two-fold) compared to the control group. The auto-oxidation and monoamine oxidase (MAO)-mediated metabolism of DA causes an increase in the production of superoxide anions in whole rat brain homogenate in vitro. In addition to this, DA is able to enhance the production of hydroxyl radicals by Fenton chemistry (Fe(III)-EDTA/H2O2) in a cell free environment. Treatment with systemic LD elevates the production of striatal superoxide anions, but does not lead to a detectable increase in striatal hydroxyl radical production in vivo. The co-adminstration of SEL with LD is able to prevent the LD induced rise in striatal superoxide levels. It has been found that the presence of DA or 6-OHDA is able to reduce lipid peroxidation in whole rat brain homogenate induced by Fe(II)-EDTA/H2O2 and ascorbate (Fenton system). However, DA and 6-OHDA increase protein oxidation in rat brain homogenate, which is further increased in the presence of the Fenton system. In addition to this, the incubation of rat brain homogenate with DA or 6-OHDA is also accompanied by a significant reduction in the total GSH content of the homogenate. The exogenous administration of LD and/or SEL was found to have no detrimental effects on striatal lipids, proteins or total GSH levels. Systemic LD administration actually had a neuroprotective effect in the striatum by inhibiting iron (II) induced lipid peroxidation. Inorganic studies, including electrochemistry and the ferrozine assay show that DA and 6-OHDA are able to release iron from ferritin, as iron (II), and that DA can bind iron (III), a fact that may easily impede the availability of this metal ion for participation in the Fenton reaction. The binding of iron (III) by DA appears to discard the involvement of the Fenton reaction in the increased production of hydroxyl radicals induced by the addition of DA to mixtures containing Fe(II)-EDTA and hydrogen peroxide. 6-OHDA did not form a metal-ligand complex with iron (II) or iron (III). In addition to the antioxidant activity and MAO-B inhibitory activity of SEL, the iron binding studies show that SEL has weak iron (II) chelating activity and that it can also form complexes with iron (III). This may therefore be another mechanism involved in the neuroprotective action of SEL. The results of the pineal indole metabolism study show that the systemic administration of SEL increases the production of N-acetylserotonin (NAS) by the pineal gland. NAS has been demonstrated to be a potent antioxidant in the brain and protects against 6-OHDA induced toxicity. The results of this study show that DA displays antioxidant properties in relation to lipid eroxidation and exhibits pro-oxidant properties by causing an increase in the production of hydroxyl radicals and superoxide anions, as well as protein oxidation and a loss of total GSH content. Despite the toxic effects of DA in vitro, the treatment of rats with exogenous LD does not cause oxidative stress or oxidative damage. The results also show that LD and SEL have some neuroprotective properties which make these agents useful in the treatment of PD.
54

An investigation into the neuroprotective properties of acyclovir

Müller, Adrienne Carmel January 2006 (has links)
Accumulating evidence suggests that quinolinic acid has a role to play in disorders involving impairment of learning and memory. In the present study, the effect of the guanosine analogue antiherpetic, acyclovir, on quinolinic acid-induced spatial memory deficits was investigated, as well as some of the mechanisms which underlie this effect. Behavioural studies using a Morris water maze show that post-treatment of rats with acyclovir significantly improves spatial memory deficits induced by intrahippocampal injections of quinolinic acid. Histological analysis of the hippocampi show that the effect of acyclovir is related to its ability to alleviate quinolinic acid-induced necrotic cell death, through interference with some of the mechanisms of neurodegeneration. However, acyclovir is unable to alter a quinolinic acid-induced increase in glutamate release in the rat hippocampus, even though it alleviates quinolinic acid induced oxidative stress by scavenging the superoxide anion in vitro and in vivo in whole rat brain and hippocampus respectively. Due to the inverse relationship which exists between superoxide anion and glutathione levels, acyclovir also curtails the quinolinic acid-induced decrease in hippocampal glutathione levels. Acyclovir suppresses quinolinic acid-induced lipid peroxidation in vitro and in vivo, in whole rat brain and hippocampus respectively, through its alleviation of oxidative stress and possibly through the binding of iron (II) and / or iron (III), preventing the participation and redox recycling of iron (II) in the Fenton reaction, which quinolinic acid is thought to enhance by weak binding of ferrous ions. This argument is further strengthened by the ability of the drug to suppress iron (II)-induced lipid peroxidation in vitro directly. Inorganic studies including ultraviolet and visible spectroscopy, electrochemistry and the ferrozine assay show that acyclovir binds to iron (II) and iron (III) and that quinolinic acid forms an easily oxidisable association with iron (II). Acyclovir inhibits the endogenous biosynthesis of quinolinic acid by inhibiting the activity of liver tryptophan-2,3-dioxygenase, intestinal indoleamine-2,3-dioxygenase and rat liver 3-hydroxyanthranillic acid oxygenase in vitro and in vivo, possibly through competitive inhibition of haeme, scavenging of superoxide anion and binding of iron (II) respectively. An inverse relationship exists between tryptophan-2,3-dioxygenase activity and brain serotonin levels. Acyclovir administration in rats induces a rise in forebrain serotonin and 5-hydroxyindole acetic acid and reduces the turnover of forebrain serotonin to 5-hydroxyindole acetic acid. Furthermore, it shows that acyclovir does not alter forebrain norepinephrine levels. The results of the pineal indole metabolism study show that acyclovir increases 5-hydroxytryptophol, N-acetylserotonin and the neurohormone melatonin, but decreases 5-hydroxyindole acetic acid. The results of this study show that acyclovir has some neuroprotective properties which may make it useful in the alleviation of the anomalous neurobiology in neurodegenerative disorders.
55

Specific aspects of neurodegenerative disease

Biro, Andrew J. January 1989 (has links)
This thesis is broken into four chapters. The first two chapters summarize two separate lines of investigation into the role of a putative neurotoxin in the pathogenesis of Huntington's Disease (HD). The third chapter outlines an investigation of the putative role of beta-N-methylamino-L-alanine (BMAA) in the pathogenesis of amyotrophic lateral sclerosis (ALS), while the final chapter details a post-mortem investigation of the contents of biogenic amines and amino acids in the brain of a man who died of a familial form of parkinsonism. Chapter I is a description of a chromatographic technique developed to isolate quinolinic acid (QA), an endogenous compound implicated in the pathogenesis of HD, from deproteinized human sera. A cation exchange column was used to selectively isolate QA, which was eluted with 10 mM HCl. The eluted fractions were analyzed by UV spectrometry to isolate and quantify QA. Once the fractions corresponding the elution of authentic QA were isolated, concentrated and the excess HCl removed, the fractions were added to growing fetal rat striatal explant cultures as an assay of neurotoxicity. Since HD involves the selective degeneration of GABAergic neurons in the striatum, the activity of glutamic acid decarboxylase, the final enzyme in the synthesis of GABA, was used to determine the viability of the cultures. Unfortunately, the method was confounded by the contamination of all effluents by compounds originating from the cation exchange resin, which were discovered to be neurotoxic to the striatal cultures, and as a result the investigation had to be abandoned. Chapter II describes an investigation designed to further characterize the nature of neurotoxicity observed in the sera obtained from patients with HD (Perry et al. 1987). Compounds with the capacity to selectively stimulate neurons at the N-methyl-D-aspartate (NMDA) receptor have been implicated in a variety of neurodegenerative disorders, including HD. Selective antagonists at the NMDA receptor have been shown to protect neurons from the degenerative effects of such "excitotoxins". The investigation described used MK-801, a potent noncompetitive NMDA antagonist, in an attempt to protect fetal rat striatal cultures from the neurodegenerative effects of the sera obtained from HD patients. The results obtained were equivocal. No evidence was obtained to support a role of the NMDA receptor in the mediation of the neurotoxicity, and in addition the neurodegenerative effects of HD sera were not reproduced in the present investigation. A variety of possible explanations for the apparent discrepancy are suggested. Chapter III describes an experiment intended to produce an animal model of ALS based on the observations by Spencer et al. 1987 that chronic oral administration of BMAA in monkeys produced the histological and behavioural characteristics of this disease. In the present investigation synthetic D,L-BMAA was given by gavage to mice over an eleven week period. Since BMAA is known to act at the NMDA receptor, a subset of the mice were also given MK-801 in an effort to protect them from any deleterious effects based on the action of BMAA at this receptor. The animals were sacrificed at the end of the experiment, and biochemical analyses were performed on the striata and cortices of the animals. In addition, neuropathological studies were performed on the spinal cords, basal ganglia and related structures. The results indicated no biochemical or neuropathological abnormality as a result of BMAA administration. Chapter IV describes a post-mortem investigation of a man who was a member of a well described pedigree which carries an autosomal dominant form of parkinsonism. The object of the investigation was to determine post-mortem levels of dopamine, noradrenaline, serotonin and their metabolites, in addition to amino acids in various regions of brain. Although conflicting evidence was obtained during life, neuropathological findings and the present neurochemical analyses confirm the degeneration of the nigrostriatal dopaminergic tract, characteristic of parkinsonism, in this man. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate
56

Neuronal Survival After Dendrite Amputation: Investigation of Injury Current Blockage

Shi, Ri Yi 12 1900 (has links)
After dendrite transection, two primary injury current pathways may acount for cell death: (1) the lesion current at the site of injury and (2) the voltage sensitive calcium channels along the dendrite. Lesions were made with a laser microbeam in mouse spinal monolayer cell cultures. Polylysine was tried as a positively charged "molecular bandage" to block the lesion current. The calcium channel blockers, verapamil and nifedipine, were used to reduce the calcium channel current. Control toxicity curves were obtained for all three compounds. The results show that neither verapamil, nifedipine, nor polylysine (MW: 3,300) protect nerve cells after dendrite amputation 100 ptm from the soma. The data also indicate that these compounds do not slow the process of cell death after such physical trauma.
57

Inhibitory-excitatory imbalance in hippocampal subfield cornu ammonis 2 circuitry in a mouse model of temporal lobe epilepsy

Whitebirch, Alexander Craig January 2021 (has links)
Temporal lobe epilepsy (TLE) is among the most common forms of epilepsy in adults. A significant proportion of patients experience drug-resistant seizures associated with hippocampal sclerosis (HS), in which there is extensive cell loss in the hippocampal cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) subfields. The dentate gyrus (DG) and cornu ammonis 2 (CA2) subfield are more resilient to neurodegeneration, and a prior report found that CA2 neurons in tissue from TLE patients show interictal-like firing and receive aberrant perisomatic excitatory synapses from DG granule cell (GC) mossy fibers (Wittner et al. Brain. 2009;132:3032–3046). Furthermore, findings from a collaborative study in the laboratory of Dr. Helen Scharfman demonstrated that chronic chemogenetic inhibition of CA2 pyramidal neurons (PNs) in vivo significantly reduced the frequency of spontaneous recurring convulsive seizures in epileptic mice. I therefore explored the hypothesis that pathophysiological changes to CA2 PN excitability or synaptic connectivity may be associated with chronic epilepsy by examining CA2 properties in a mouse model of TLE.Pilocarpine-induced status epilepticus in mice leads to a pattern of hippocampal sclerosis-like neurodegeneration and recurring spontaneous seizures, and thus recapitulates key features of TLE. I performed whole-cell electrophysiological recordings from PNs in acute hippocampal slices from pilocarpine (PILO)-treated mice in the chronic phase of epilepsy as well as age-matched controls. In some experiments I used Cre-expressing mouse lines to selectively express a light-activated excitatory channel in CA2 PNs or DG GCs. I also performed immunohistochemistry to examine CA2 interneuron (IN) populations following PILO-induced status epilepticus. I found that in healthy tissue CA2 PNs, like those in CA3, both directly excited other CA2 PNs via a recurrent CA2-CA2 PN circuit and indirectly inhibited other CA2 PNs by recruiting local INs. The CA2 and CA3 subfields also form reciprocal excitatory and feedforward inhibitory circuits. These recurrent and reciprocal circuits constitute an auto-associative network in which INs crucially control CA2/CA3 population excitability. DG GC mossy fibers made direct but relatively weak excitatory synapses onto CA2 PNs. Following PILO-induced status epilepticus, feedforward inhibition is diminished in the DG GC mossy fiber circuit to CA2, in the CA2/CA3 recurrent network, and in the forward-projecting circuit from CA2 PNs to CA1. I found a modest decrease in the density of parvalbumin-immunopositive INs and a profound decrease of cholecystokinin-immunopositive IN density, combined with degradation of the pyramidal neuron-associated perisomatic perineuronal net, which together may contribute to this inhibitory disruption. DG GC mossy fiber excitatory input to CA2 PNs is strengthened, along with CA2 PN excitatory input to CA1 PNs. Finally, in hippocampal slices from PILO-treated mice I found an increase in CA2 PN input resistance and thus elevated intrinsic excitability, leading to a higher firing rate upon direct current injection. The combined effect of these changes may drive the emergence of epileptiform synchronization in the CA2 network and facilitate the propagation of seizure activity from the DG and entorhinal cortex directly to CA1 via the CA2-centered disynaptic (EC LII --> CA2 --> CA1) and alternate trisynaptic circuits (EC LII --> DG --> CA2 --> CA1).
58

The contribution of 14-3-3 proteins to protein aggregate homeostasis

Herod, Sarah Grace January 2022 (has links)
Amyloids are fibrous protein aggregates associated with age-related diseases, such as Alzheimer’s disease and Parkinson’s disease. The role of amyloids in the etiology of neurodegeneration is debatable, but genetic and molecular evidence supports a causative relationship between amyloidogenesis and disease. Amyloidogenic proteins are constitutively expressed throughout the lifespan of an organism, and yet only become pathogenic in certain situations. This led to a hunt to understand how amyloidogenic proteins could be modified in order to become aggregation-prone. One possibility that has garnered attention is phosphorylation, primarily because several amyloid aggregates such as tau and α-synuclein are often highly phosphorylated in disease. However, the contribution of phosphorylation to disease progression remains unclear.While amyloid aggregates are typically described as irreversible and pathogenic, some cells utilize reversible amyloid-like structures that serve important functions. One example is the RNA-binding protein Rim4 which forms amyloid-like assemblies that are essential for translational control during S. cerevisiae meiosis. If Rim4 is unable to translationally repress its mRNA targets, cells mis-segregate chromosomes during meiosis resulting in aneuploid gametes. Importantly, Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner at meiosis II onset which allows previously repressed transcripts to become translated. In Chapter 1, I describe the significance and complexity of protein phosphorylation as it relates to disease-associated amyloids and why Rim4 is an ideal model for studying this phenomenon. The objective of this thesis is to examine the mechanisms underlying clearance of Rim4 amyloid-like assemblies. The work described in Chapter 2 focuses on identifying co-factors that mediate clearance of amyloid-like assemblies in a physiological setting. I demonstrate that yeast 14-3-3 proteins, Bmh1 and Bmh2, bind to Rim4 assemblies and facilitate their subsequent phosphorylation and timely clearance. Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. In Chapter 3, I explore the mechanism underlying 14-3-3 contribution to Rim4 amyloid-like disassembly. I find that 14-3-3 proteins are critical for the interaction between Rim4 and its primary kinase Ime2, thus facilitating downstream multi-site phosphorylation of Rim4. In Chapter 4, I explore additional roles for 14-3-3 proteins in general protein aggregate homeostasis. I find that 14-3-3 mutants exhibit greater protein aggregate burdens. Additionally, 14-3-3 mutants accumulate ubiquitinated proteins and are sensitized to proteasome mutations, suggesting a role for 14-3-3 proteins in proteasome function. Collectively, the studies described in this thesis support a protective role for 14-3-3 proteins in protein aggregation that may have implications for amyloid biology in human disease.
59

Multiplexed high-throughput screening identifies broadly active rescuers of proteotoxicity

Resnick, Samuel Jackson January 2022 (has links)
The accumulation of misfolded proteins within intracellular aggregates is a distinctive feature observed within multiple neurodegenerative diseases (NDDs). However, the genes and pathways that regulate protein misfolding, aggregation, and subsequent cellular toxicity remain poorly understood. Here I describe a high-throughput discovery platform that enables the simultaneous screening of dozens of neurodegenerative disease models to rapidly uncover genetic modifiers that alter the solubility and toxicity of a wide variety of aggregation-prone proteins. From these studies, I identify the human HSP40 chaperone, DNAJB6 as a potent rescuer of the misfolding and proteotoxicity of multiple RNA-binding proteins implicated in Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) including FUS, TDP-43, and hnRNPA1. I, with collaborator help, further demonstrate that DNAJB6 has an intrinsic ability to phase separate under physiologic conditions and can alter the properties of FUS containing condensates by maintaining them in a gel-like state over long periods, preventing FUS aggregation. By conducting domain mapping studies and a deep mutational scan on DNAJB6, I am able to gain detailed insight into its mechanism of action while also uncovering a series of novel variants with enhanced activity. During the development of this multiplexed screening approach for neurodegenerative disease models, research was interrupted by a global pandemic caused by SARS-CoV-2. I realized that the themes of studying proteotoxicity of multiple related, yet distinct models could be applied towards drug development to identify inhibitors of the essential 3CL proteases encoded by multiple coronaviruses, which cause proteotoxicity when expressed in cells. As such, I develop and describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This essay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, I identify compounds with broad 3CLpro inhibitory activity. I also adapt the assay for use in compound screening and in doing so uncover additional SARS-CoV-2 3CLpro inhibitors. I observe strong concordance between data emerging from this assay and those obtained from live virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. I identify two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested including 3CLpro enzymes from understudied zoonotic coronaviruses.
60

Improving Accessible and Personalized Airway Protective Rehabilitation in Neurodegenerative Disease

Sevitz, Jordanna Sarah January 2023 (has links)
Utilization of airway protective rehabilitation among individuals with neurodegenerative disease is astoundingly low. Yet, due to progressive decline in airway protective function and resulting health consequences such as aspiration pneumonia, the need for rehabilitation is clear. Moreover, a growing literature supports the benefit of airway protective rehabilitation in neurodegenerative populations. Therefore, it is a healthcare priority to increase treatment utilization in order to improve health and quality of life for individuals with neurodegenerative disease. Improving treatment accessibility and relevance are two approaches that have the potential to improve utilization. Despite the need to increase treatment accessibility and the growing evidence base to support the use of telehealth to increase access, a significant gap remains in our understanding of the feasibility and acceptability of telehealth to manage dysphagia in neurodegenerative disease. Moreover, little is known about patient perspectives which are critical to refine person-centered models of care that are relevant to patient’s needs. To address this important clinical research gap, this dissertation includes a series of three research studies aimed at improving accessible and relevant rehabilitation for airway protective dysfunction in neurodegenerative disease. Chapter 1 will provide an overview of the current literature as it relates to airway protective dysfunction in neurodegenerative disease, existing rehabilitation approaches, telehealth to manage dysphagia, and the need for personalized care. Chapter 2 will examine the feasibility of rehabilitating airway protection via telehealth in individuals with neurodegenerative movement disorders. Chapter 3 will then explore speech language pathologists’ (SLPs) perspectives and experiences using telehealth to manage dysphagia. Chapter 4 will characterize patient perspectives on airway protective dysfunction and treatment experience following cough skill training (CST). I will conclude (Chapter 5) by synthesizing the findings from chapters 2-4 and suggesting directions for future research.

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