One of the most foundational and personal philosophical questions one can ask is what makes you, you? In large part, you are made up of your relationships, experiences, and memories. The hippocampus, a brain region which is critical for the formation of memories, has been the focus of neuroscience research for decades due partially to this function, which is foundational to our individuality. In Alzheimer’s disease (AD), one of the most common and well-researched neurodegenerative diseases in the world, the hippocampus is one of the earliest targets. Despite extensive work on AD, we still lack a coherent understanding of what is causing the disease, the mechanisms by which it is causing neuronal dysfunction and death within the hippocampus and other brain regions, and how it ultimately causes deficits in cognition and behavior, leading to an erosion of our selves. In this thesis, I explore three independent but related questions: 1) what molecular mechanisms are causing early synaptic loss in AD, specifically within the hippocampus, 2) what molecular effectors are responsible for establishing and maintaining intracellular architecture in hippocampal neurons, which are exploited in early AD, and 3) how and when does the hippocampal circuit dysfunction in AD progression? Using a variety of experimental techniques, ranging from in utero and ex utero electroporation, primary murine and human neuronal cell culture, longitudinal confocal microscopy, immunohistochemistry, biochemistry, cell and molecular biology, in vivo two-photon calcium imaging, and behavioral assays, I have found that, within CA1 of the hippocampus, synapse loss requires degradation of the dendritic mitochondrial network, activity and input specificity are driving mitochondrial compartmentalization within CA1 neurons through the same pathway that is aberrantly overactivated in AD, and the hippocampal circuit is overly rigid in encoding the environment as the disease progresses.

<p>Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease that results in a myriad of physical and mental symptoms. Current disease-modifying therapies do not prevent long-term disability accumulation and are particularly ineffective for patients with a progressive disease onset. Exercise may represent an alternative strategy for managing symptoms and disability accumulation, particularly in progressive MS.</p> <p>Whereas the benefits of exercise have been established primarily in ambulatory MS patients with a relapsing disease course, few studies have investigated the benefits of exercise for patients with progressive MS with greater impairment. Therefore, the purpose of this dissertation was to determine the short-term, long-term, and maintenance effects of adapted exercise interventions for patients with progressive MS of high disability which was addressed by conducting two adapted exercise interventions.</p> <p>The first intervention examined the effects of 24 weeks of body weight supported treadmill training (BWSTT) on outcomes of physical and mental functioning, fatigue, quality of life, and brain health. Outcomes were evaluated at baseline, 12, and 24 weeks following the intervention, and again 12 weeks post-intervention. The second intervention evaluated and compared the effects of 12 weeks of total-body recumbent stepper training (TBRST) to BWSTT on outcomes of safety, physical and mental functioning, fatigue, quality of life, and equipment preference.</p> <p>Safety of BWSTT and TBRST was established. Significant improvements in fatigue and QoL were observed with both training modalities; however, neither significantly improved physical function. There was some evidence to suggest long-term BWSTT may improve cognitive performance and brain health, and that TBRST was the preferred exercise modality. Furthermore, most beneficial effects of long-term BWSTT tended not to be maintained when exercise was discontinued.</p> <p>This dissertation established evidence for the potential benefits of BWSTT and TBRST in patients with progressive MS with high disability. BWSTT and TBRST may represent viable alternative strategies for disease management.<strong></strong></p> / Doctor of Philosophy (PhD)

<p>Huntington’s disease (HD) is a progressive, neurodegenerative disorder that leads to the selective loss of neurons in the striatum and the cerebral cortex. HD is caused by a CAG trinucleotide repeat expansion beyond the normal length in the <em>IT15 </em>(<em>Htt</em>) gene. The CAG stretch codes for an elongated polyglutamine tract within the amino‐terminus of the huntingtin protein. Polyglutamine tracts with lengths exceeding 37 repeats cause HD whereas repeat lengths below do not. This phenomenon has plagued the HD community since the discovery of the gene in 1993. In this thesis, we sought to elucidate the molecular mechanism by which huntingtin becomes toxic at polyglutamine lengths above 37. Using Förster resonance energy transfer (FRET) techniques, we describe an intramolecular proximity between the first 17 residues (N17) and the proline-rich regions, which flank the polyglutamine tract of huntingtin. We report that we can precisely measure differences between the conformations adopted by the huntingtin protein with polyglutamine tracts below and above the pathogenic repeat threshold of 37 repeats. Our data supports the hypothesis that polyglutamine tracts below the pathogenic threshold can act as a flexible hinge allowing the N17 domain to freely fold back upon huntingtin and come into close 3D proximity with the polyproline region. This flexibility is lost in polyglutamine tracts with >37 repeats resulting in a diminished spatial proximity between N17 and the polyproline domain.</p> / Doctor of Philosophy (PhD)

Stroke is a leading cause of long-term disability, affecting thousands of individuals annually and significantly impairing their mobility, independence, and quality of life. Traditional methods for assessing motor impairments are often costly and invasive, creating substantial barriers to effective rehabilitation. This thesis explores the use of DeepLabCut (DLC), a deep-learning-based pose estimation tool, to extract clinically meaningful kinematic features from video data of stroke survivors with upper-extremity (UE) impairments. To conduct this investigation, a specialized protocol was developed to tailor DLC for analyzing movements characteristic of UE impairments in stroke survivors. This protocol was validated through comparative analysis using peak acceleration (PA), mean squared jerk (MSJ), and area under the curve (AUC) as kinematic features. These features were extracted from the DLC output and compared to those derived from the assumed ground-truth data from IMU sensors worn by the participants. The accuracy of this analysis was quantified using percent mean squared error (PMSE) between each IMU sensor and DLC. PMSE analysis indicates that DLC-based kinematic features capture aspects of both accelerometer and gyroscope for the control participant. PA (8.78%) and AUC (3.28%) align more closely with the gyroscope, while MSJ (5.20%) demonstrates greater agreement with the accelerometer. On the other hand, for the stroke participant, DLC estimations for all kinematic features predominantly reflect data from the accelerometer. Across all datasets, AUC has the smallest PMSE values, suggesting that, based on our data, motor effort and energy expenditure in the tasks are best represented by DLC. Additionally, PMSE values for the stroke dataset are higher than those for the control, highlighting DLC's limitations in accurately detecting finer details of motion data in individuals with UE impairments. The results indicate that DLC reasonably estimates kinematic data for both participants, although further refinement of the methods is necessary to enhance the analysis of stroke data.

The dysregulation of immune system function has been centrally implicated in numerous age-related and neurodegenerative disorders, including Alzheimer’s disease (AD). Genetic susceptibility studies have positioned microglia, brain-resident immune cells, as critical actors in the development and the progression of the disease. Microglia are highly plastic cells with diverse functions across many modalities, and the appropriate regulation of their activities are a prerequisite for central nervous system homeostasis and cognitive health. Aging and pathogenic contexts are posited to modify microglial behavior, inhibiting their neuroprotective function and promoting a dysfunctional state that drives disease. However, the mechanisms underlying these pathogenic alterations in microglial state and function are complex and poorly understood. This thesis identifies three elements that are altered in the AD brain and investigates how these mechanisms may serve as triggers producing microglial dysregulation in AD. Chapter 3 examines the role of expression of the transposable element LINE-1 in AD-related microglial dysfunction. Chapter 4 explores the regulation of PLCG2, which encodes a critical AD-associated signaling enzyme. Chapter 5 investigates the role of the AD-linked sorting receptor SORL1 in microglia. Together, these data expand our understanding of mechanisms driving altered microglial pathophysiology in AD and illuminate pathways of interest with potential therapeutic applications meriting deeper exploration.

Parkinson's Disease (PD) is a neurodegenerative disorder characterized by motor and nonmotor symptoms, including gastrointestinal (GI) dysfunction. Symptoms such as constipation, bloating, and nausea often show early signs before the onset of motor symptoms. Recent research has suggested an essential role of the human gut microbiome and gut dysbiosis, an imbalance in gut microbiota composition, in the pathogenesis of PD. This thesis aims to assess the potential relationship between gut dysbiosis and PD by conducting a meta-analysis, focusing on differences in gut microbiota between PD patients and healthy controls and evaluating their implications for GI dysfunction and PD progression. The primary objectives of this meta-analysis are to (1) compare gut microbiota composition between PD patients and healthy controls via quantitative statistical metrics; (2) identify bacterial taxa differences and their functional roles; (3) assess whether these changes support the hypothesis that gut dysbiosis contributes to α-synuclein (α-SYN) aggregation and PD progression; and (4) evaluate the impact of study methodology and heterogeneity on the reported outcomes. The results revealed no statistically significant differences in alpha diversity between PD patients and controls, likely influenced by substantial heterogeneity across studies. However, specific bacterial taxa were consistently altered, with increased levels of pro-inflammatory bacteria and decreased levels of SCFA-producing bacteria in PD patients. This meta-analysis provides insights into the potential role of gut dysbiosis in PD, suggesting that changes in specific bacterial taxa may contribute to gut inflammation, increased gut permeability, and α-SYN aggregation. The findings highlight the complexity of the gut-brain axis in PD and the need for further longitudinal studies to understand the underlying mechanisms and explore targeted therapeutic intervention.

Proteases play an indispensable role in medicine, serving both as drug targets and as therapeuticagents. Protease inhibitors are a key component of our antiviral arsenal, and are widely used to combat human immunodeficiency virus (HIV), hepatitis C virus (HCV), and, most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nirmatrelvir, an inhibitor of the 3-chymotrypsin like (3CL) protease essential for SARS-CoV-2 replication, was granted emergency use authorization in late 2021, formulated with ritonavir and sold under the brand name Paxlovid. Since then, Paxlovid use has become widespread, raising the possibility that nirmatrelvir resistance could emerge in circulating SARS-CoV-2. It is therefore important to understand how the 3CL protease might mutate to lose nirmatrelvir sensitivity so that circulating variants can be monitored for these mutations and future generations of inhibitors can be designed to prevent cross-resistance. The most widely used therapeutic proteases are the botulinum neurotoxins (BoNTs),which are effective in the treatment of a wide array of movement, pain, and autonomic disorders. These toxins exert their therapeutic effect by cleaving members of the SNARE protein family inside neuronal cytosol, preventing neurotransmitter release. Much work has been dedicated to engineering members of the BoNT family to extend their therapeutic utility, including altering receptor tropism, extending half-life, and modifying protease specificity. While significant progress has been made in each of these areas, the extent to which these proteases can be reprogrammed to target the proteins that cause human disease remains unexplored. In this dissertation, we employ diverse methods to study and direct protease evolutionand to facilitate protein engineering more broadly. To investigate the emergence of nirmatrelvir resistance, we passage SARS-CoV-2 in increasing concentrations of the drug and sequence the 3CL protease gene over time in resistant lineages. We then validate the observed 3CL protease mutations by incorporating them into recombinant SARS-CoV-2 and testing the nirmatrelvir sensitivity of the resulting viruses. We find that the development of nirmatrelvir resistance typically begins with the acquisition of a precursor mutation such as T21I, P252L, or T304I, which confers a low level of resistance and enables strong resistance conferring mutations, such as E166V, to emerge without imposing a significant fitness cost. To explore the programmability of the BoNT proteases, we engineer type E (BoNT/E) to cleave proteins involved in neurodegenerative disease. To accomplish this, we employ targeted mutation based on a structural model as well as the continuous evolution platform known as OrthoRep, selecting for variants that cleave the desired substrates with a circuit that links protease activity to the growth of a Saccharomyces cerevisiae (yeast) cellular chassis. We first use this approach to target ATXN3, the protein whose aggregation causes type 3 spinocerebellar ataxia (SCA3). We then profile the substrate specificities of the BoNT/E variants that emerged during our ATXN3 engineering, identifying patterns that can inform the selection of new targets. Based on one of these patterns, we generate a new BoNT/E variant capable of cleaving TDP-43, a protein implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Finally, to facilitate future protein engineering campaigns, we develop a machine learning pipeline that uses deep mutational scanning (DMS) data to model a protein’s fitness landscape and predict optimized variants with user-defined constraints. We name this pipeline OptiProt, and we demonstrate its utility by applying it to the human chaperone DNAJB6, which rescues cellular toxicity in a yeast model of ALS. In this context, we probe OptiProt’s engineering capabilities with a range of challenges. We first predict hyper-functional variants with up to 50 mutations. We then restore activity to variants harboring one of two known loss-offunction mutations. Finally, to demonstrate the OptiProt’s amenability to complex engineering constraints, we restore activity to a variant harboring a deleterious mutation while simultaneously mutating a set of highly conserved residues. Altogether, the work presented in this dissertation highlights the value of laboratory protease evolution in two clinically relevant applications and provides a tool that can facilitate future work in this field.

Thesis (DPhil)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: This qualitative study comprised an in-depth investigation into the subjective - the explicitly human - experience of those suffering from Myalgic Encephalomyelitis (ME). I was, firstly, concerned with the nature and meaning of the social side of illness, that is, the sufferer's encounters with doctor, family member, friend and acquaintance alike. I was, secondly, set to develop greater insight into the essentially personal experience of being ill. I was able to conclude that, even amidst the inhumane presence of utter ignorance that permeate the very experience of ME, no ME sufferer is inevitably doomed a victim. They can always make a deliberate decision to have a say in their situation, in their own experience of their circumstances. They have the power of personal choice. Recommendations are, therefore, directed at expanding the potential extent and magnitude of this dynamic power. The substantive focus of my study was enriched by a deliberate concern with the methodological implications of my own intimate involvement within the research process. I was here primarily concerned with my personal contribution to the research process as well as the influence thereof on the research relationships developed and the research strategies chosen and applied. I could not but conclude that the understanding I explicate is, as all social science theory, essentially a human construction, developed by me, in my distinctly human capacity. Recommendations are, therefore, geared to sensitise all social researchers to their own contribution to the construction of that which is eventually presented (and taken) as truthful knowl~dge. In conclusion, I am thoroughly convinced that the experience of both illness and research is fundamentally human. This "humanness" cannot and should not be denied. Instead, I advocate a more deliberate focus on the human dimension of illness and research. Without such a focus, a more comprehensive understanding of either realm will continue to linger as but an elusive ideal. / AFRIKAANSE OPSOMMING: Hierdie kwalitatiewe studie is gebaseer op 'n in-diepte ondersoek gerig op die subjektiewe - die onteenseglik menslike - ervaring van diegene wat ly aan Myaligië Enkefalomiëlitis (ME). Ek was, eerstens, geïnteresseerd in die aard en betekenis van die sosiale dimensie van siekte, dit wil sê, die lyer se ervaring van sosiale kontak met dokters, gesinslede, vriende en kennisse. Ek was, tweedens, gerig op die ontwikkeling van 'n grondige insig in die uiters persoonlike ervaring van siek-wees. Ek het tot die gevolgtrekking gekom dat, selfs te midde van die onmenslike teenwoordigheid van blatante onkunde wat die ganse ervaring van ME kenmerk, geen ME lyer noodwendig tot 'n slagoffer-status gedoem is nie. Hulle kan altyd 'n doelbewuste besluit neem om 'n sê te hê in hul eie situasie, in hul eie ervaring van hul omstandighede. Hulle het die mag van persoonlike keuse. Aanbevelings is dus daarop gerig om die potentiële trefwydte en impak van hierdie dinamiese mag uit te brei. Die substantiewe dimensie van my studie is verryk deur 'n doelbewuste fokus op die metodologiese implikasies van my eie intieme betrokkenheid in die navorsingsproses. Ek was hoofsaaklik gemoeid met my persoonlike bydrae tot die navorsingsproses en die invloed daarvan op die ontwikkel van navorsingsverhoudings en die toepassing van gekose navorsingstrategieë. Hierdie fokus het gelei tot die besef dat die beskrywing wat ek aanbied, soos inderdaad alle sosiale teorie, essensieël 'n menslike konstruksie is, soos ontwikkel deur my, in my uitdruklik menslike kapasiteit. Aanbevelings is dus daarop gerig om alle sosiale navorsers te sensitiseer ten opsigte van hul eie bydrae tot die konstruksie van dit wat uiteindelik voorgestel (en geag) word as die waarheidsgetroue kennis. In slotsom, is ek oortuig dat die ervaring van beide siekte en navorsing fundamenteel menslik is. Hierdie "mensheid" kan en behoort nie ontken te word nie. Inteendeel, ek bepleit 'n doelbewuste fokus op die menslike dimensie van siekte en navorsing. Sonder só 'n fokus sal 'n meer diepgaande begrip van iedere area bloot 'n onbereikbare ideaal bly.

Blast-induced traumatic brain injury (bTBI) has been described as the defining injury of Operations Enduring Freedom and Iraqi Freedom (OEF/OIF). Previously, most blast injury research has focused on the effects of blast on internal, gas filled organs due to their increased susceptibility. However, due to a change in enemy tactics combined with better armor and front-line medical care, bTBI has become one of the most common injuries due to blast. Though there has been a significant amount of research characterizing the brain injury produced by blast, a sound understanding of the contribution of each component of the shockwave to the injury is needed. Large animal models of bTBI utilize chemical explosives as their shockwave source while small animal models predominantly utilize compressed air-driven membrane rupture as their shockwave source. We designed and built a multi-mode shock tube capable of utilizing compressed gas (air or helium)-driven membrane rupture or chemical explosives (oxyhydrogen – a 2:1 mixture of hydrogen and oxygen gasses, or RDX – high order explosive) to produce a shockwave. Analysis of the shockwaves produced by each mode of the McMillan Blast Device (MBD) revealed that compressed air-driven shockwaves exhibited longer duration positive phases than compressed helium-, oxyhydrogen-, or RDX-driven shockwaves of similar peak overpressure. The longer duration of compressed air-driven shockwaves results in greater energy being imparted on a test subject than would be imparted by shockwaves of identical peak overpressures from the other sources. Animals exposed to compressed air-driven shockwaves exhibited more extensive brain surface hematoma, more blood-brain barrier compromise, more extensive reactive astrocytosis, and greater numbers of activated microglia in their brains than did animals exposed to oxyhydrogen-driven shockwaves of even greater peak overpressure. Taken together, these data suggest that compressed air-driven shockwaves contain more energy than their chemical explosive-derived counterparts of equal peak overpressure and can result in greater injury in an experimental animal model. Additionally, these data suggest that exposure to longer duration shockwaves, which is common in certain realworld scenarios, can result in more severe bTBI. The results of this study can be used to aid design of blast wave mitigation technology and future clinical intervention.

Noninvasive assessment of diabetic cardiovascular autonomic neuropathy (AN): Cardiac and vascular dysfunctions resulting from AN are complications of diabetes, often undiagnosed. Our objectives were to: 1) determine sympathetic and parasympathetic components of compromised blood pressure regulation in patients with polyneuropathy, and 2) rank noninvasive indexes for their sensitivity in diagnosing AN. Continuous 12-lead electrocardiography (ECG), blood pressure (BP), respiration, regional blood flow and bio-impedance were recorded from 12 able-bodied subjects (AB), 7 diabetics without (D0), 7 with possible (D1) and 8 with definite polyneuropathy (D2), during 10 minutes supine control, 30 minutes 70-degree head-up tilt and 5 minutes supine recovery. During the first 3 minutes of tilt, systolic BP decreased in D2 while increased in AB. Parasympathetic control of heart rate, baroreflex sensitivity, and baroreflex effectiveness and sympathetic control of heart rate and vasomotion were reduced in D2, compared with AB. Baroreflex effectiveness index was identified as the most sensitive index to discriminate diabetic AN. Four-dimensional multiscale modeling of ECG indexes of diabetic autonomic neuropathy: QT interval prolongation which predicts long-term mortality in diabetics with AN, is well known. The mechanism of QT interval prolongation is still unknown, but correlation of regional sympathetic denervation of the heart (revealed by cardiac imaging) with QT interval in 12-lead ECG has been proposed. The goal of this study is to 1) reproduce QT interval prolongation seen in diabetics, and 2) develop a computer model to link QT interval prolongation to regional cardiac sympathetic denervation at the cellular level. From the 12-lead ECG acquired in the study above, heart rate-corrected QT interval (QTc) was computed and a reduced ionic whole heart mathematical model was constructed. Twelve-lead ECG was produced as a forward solution from an equivalent cardiac source. Different patterns of regional denervation in cardiac images of diabetic patients guided the simulation of pathological changes. Minimum QTc interval of lateral leads tended to be longer in D2 than in AB. Prolonging action potential duration in the basal septal region in the model produced ECG and QT interval similar to that of D2 subjects, suggesting sympathetic denervation in this region in patients with definite neuropathy.