Background Up to 59% of tuberculosis (TB)/human immunodeficiency virus (HIV) co-treated patients develop paradoxical TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) after addition of antiretroviral (ARV) therapy to anti-tuberculous therapy (ATT). The course can be prolonged and the average mortality rate is 2% (75% for TB-IRIS involving the central nervous system (CNS)). Immune elements – including neutrophils - involved in the anti-Mycobacterium tuberculosis (Mtb) response are implicated in pathogenesis, which remains incompletely understood. Diagnosis is one of exclusion, no reliable laboratory markers exist, corticosteroid-mediated prophylaxis and therapy are only partially effective, and no treatment targets tissue damage. Disentangling cause and effect in complex disorders such as TB-IRIS requires techniques capable of interrogating complex biological systems. Neutrophils are the major circulating leukocyte population, the earliest innate system responders, and exhibit various unusual immunometabolic functional specialisations. Proteins represent the most functionally-proximal and commonly pharmacologically-targeted cellular biomolecules. Label-free high-performance liquid chromatography-coupled tandem mass spectrometry (HPLCMS/MS) is well-suited to differentially profiling the ex vivo neutrophil proteome in an unbiased manner, in order to investigate TB-IRIS predisposition and pathogenesis. Methods Applying first principles to existing human literature, the most parsimonious holistic hypothetical model regarding paradoxical TB-IRIS predisposition and pathogenesis was inferred. A clinical cohort of control (CTRL) and matched TB-IRIS case (IRIS) study participants was assembled. Demographic, clinical, and biochemical characteristics were analysed for statistically-significant differences and to identify potential risk/protective factors (relative risk (RR) with 95% confidence interval (CI)). A small group (n = 9) of TB-HIV- healthy volunteers (HVs) was also assembled. Phlebotomy occurred at two timepoints: just prior to ARV initiation (week 0) and at the typical time of IRIS manifestation (week 2). Neutrophils were isolated and lysed, proteins underwent on-filter protein trypsinisation, peptide salts and detergents were removed, and neutrophil-optimised HPLC-MS/MS was conducted. Spectra were submitted to MaxQuant for parent protein identification and quantitation. Comparisons of (a) CTRL0 and IRIS0 to HV (and resultant differences) identified class-differential impacts of partial ATT-treated coinfection (IRIS predisposition) and of (b) CTRL2 to CTRL0 and IRIS2 to IRIS0 (and resultant differences) identified class-differential impacts of ARV therapy (IRIS pathogenesis). Class-discriminating proteomic differences were visualised using principal components analysis (PCA), protein differential expression analysis was performed (including for detectable/undetectable and significantly differentiallyexpressed (SDE) proteins), and results informed differential functional profiling via gene ontology overrepresentation analysis (GO-ORA) and pathway activation state prediction. To address shortcomings of current knowledgebases and automated tools, a novel deep manual analysis approach focused on key inference-friendly proteins, convergent findings, and neutrophil-specific functional modules. Integrated findings extended the literature-derived TB-IRIS model, generating testable novel hypotheses, one of which was partially validated using live-cell fluorescence microscopy. Proteomic data were additionally analysed to detect Mtb proteins, preliminarily analyse variable post-translational modifications (PTMs) of interest, and identify candidate prognostic and diagnostic biomarkers. Finally, mechanistic hypotheses facilitated identification of novel potential prophylactic and therapeutic targets. Results (1) Literature suggests advanced TB/HIV-coinfection (including a higher Mtb/antigen load) as the major TB-IRIS risk factor. Attendant significant immunometabolic state perturbations include myeloid overactivation, metabolic stress (possibly including adaptogen depletion), a lack of regulatory receptors, impaired pro-inflammatory signal transduction, and impaired antigen clearance. These likely predispose to lytic cell death - including release of host- and pathogen-derived inflammatory/cytotoxic molecules and proteolytic enzymes - and less restrained/more abnormal inflammation as well as tissue damage, on restoration of HIV-suppressed inflammatory signalling pathways by ARV therapy. (2) Regarding the clinical cohort, a sample size of > 42 participants per characteristic-matched comparison class provides > 95% power to detect a two-fold change with 99% confidence. Cases exhibited known TB-IRIS risk factors, and largely expected white cell count (WCC), body mass index (BMI), and C-reactive protein (CRP) level changes in response to ARV therapy (e.g. WCC increase and BMI decrease). None of the few participants on alternate (efavirenz (EFV)- or tenofovir (TDF)-lacking) regimens developed TB-IRIS. Pre-ARV prednisone (or incidental antihistamine/anti-fungal) use was associated with a non-significantly decreased TB-IRIS risk. Interestingly, smoking is associated with a significant decrease in TB-IRIS risk by 60%. (3) Regarding sample processing and analysis, the average sample collection to neutrophil isolation interval was within recommended limits. Isolation yield exceeded 15 x 106 and 25 x 106 per sample in the CTRL and IRIS groups, respectively. Isolated neutrophil purity exceeded 80% in both groups; the few low-purity samples were excluded from subsequent proteomic analysis. Lysates from 5 x 106 neutrophils routinely yielded over 100μg total protein, tryptic digestion was efficient (on average < 96% missed cleavages), and equivalent peptide injection volumes yielded comparable total ion chromatogram (TIC) profiles and intensities. An average 23% spectral identification rate resulted in a total of 2532 protein group identifications, the deepest neutrophil proteome coverage achieved to date without pre-fractionation, representing ~12% of human protein coding genes, and ~25% of the detectable human proteome. Samples were analysed in two (randomised) batches, producing independent datasets A (N = 37) and B (N =74). Withindataset technical replicates exhibit excellent agreement in protein identities and quantities; betweendataset protein identities and functional inferences also exhibit excellent agreement. We identify a number of proteins apparently not known to be expressed by human neutrophils, as well as one predicted human protein never before observed empirically. Overall, parent pathways of level-altered proteins suggest perturbation of nine major neutrophil function modules at both time-points: (a) signal transduction, (b) pattern recognition receptor (PRR) and cytokine signalling, (c) the eicosanoid cascade, (d) neutrophil antimicrobial functions, (e) carbon-energy metabolism, (f) protein homeostasis, (g) integrated nitrogen-sulfur-B-vitamin and redox/xenobiotic/glyoxal metabolism, (h) gene expression, and (i) cytoskeletal dynamics. (4) Regarding impacts of partially ATT-treated co-infection (week 0), neutrophil proteomic profiles successfully distinguish between HV and IRIS0 or CTRL0. Many differences from HV are shared between IRIS0 and CTRL0 (i.e. driven by partially ATT-treated co-infection), but some are class-unique (i.e. driven by factors predisposing to or protecting from TB-IRIS). Findings are supported by a head-to-head comparison of the CTRL0 and IRIS0 proteomes, including changes suggesting: more prevalent type I IFN, TGFβ, and Th2-type cytokine signalling; poorer capacity for restraint of alternate complement activation; mitochondrial and oxidative stress (including proneness to necrosis); impaired function (e.g. microbicidality, TLR/IL-1R-MyD88-NFκB signalling, and caspase 1- mediated IL-1β and IL-18 maturation) of activated neutrophils; and enhanced lipid and upstream (but inhibited downstream) isoprenoid synthesis (including decreased steroidogenesis). Candidate biomarkers distinguish CTRL- and IRIS-class partially ATT-treated neutrophils from HV neutrophils and from each other. (5) Regarding impacts of ARV therapy (week 2), both IRIS and CTRL neutrophil proteomes exhibit significant changes in response to ARV therapy. Many changes are shared between IRIS and CTRL (i.e. driven by ARV therapy and declining viral load (VL)), but some are class-unique (i.e. driven by factors preventing/contributing to TB-IRIS pathogenesis). Findings are supported by a head-to-head comparison of the CTRL2 and IRIS2 proteomes, including changes suggesting: a slower decline in type I IFN signalling; increased inflammatory cytokine (e.g. IL-6, TNFα, and IFNγ) signalling and protease (e.g. MMP-8) activity; decreased sensitivity to immunoregulatory glucocorticoids and vitamin A; and increased mitochondrial, endoplasmic reticulum (ER), and oxidative stress. Candidate biomarkers distinguish CTRL- and IRIS-group ARV-exposed neutrophils from baseline and from each other. (6) Livecell fluorescence microscopy of HV neutrophils suggests that in vivo-equivalent levels of EFV rapidly alter mitochondrial, lysosomal, and aggresomal architecture in a manner consistent with organelle and protein folding stress, and suggesting cell death commitment. (7) Integrated neutrophil immunometabolic changes suggested by proteomic findings support and extend the biologically compelling literature-derived model. Model highlights include more advanced baseline TB/HIV (including higher type I IFN, TGFβ, and possibly Th2-type cytokine levels), with consequent impaired myeloid-mediated Mtb antigen clearance and depletion of cellular adaptogens. The resultant abnormal immunometabolic state produces myeloid cells less able to counteract metabolic stress and primed for less-restrained inflammation. Introduction of mitotoxic ARV drugs and rapid lifting of HIVmediated immune embargoes escalates myeloid metabolic (including oxidative) stress and overactivation (including via NLRC4, CASP4/5, TLR/IL-1R-MyD88-NFκB, and MAPK-AP1 signalling), producing - instead of Mtb clearance - inflammatory cell death with release of immune-activating and tissue-damaging host- and Mtb-derived molecules. Reactivation of Mtb lymphocyte memory responses likely only produces clinically-apparent inflammation (TB-IRIS) when multiple simultaneous but incompatible immune programmes (e.g. overzealous myeloid activity, Th1, Th2, Th17, and Treg) coexist. Based on this model, existing compounds with the potential for rational, safe, effective TB-IRIS prophylaxis/therapy are identified (e.g. glutathione, vitamins B-complex and A/D/E, rapamycin, and metformin) which may assist in restoring system homeostasis.

Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Master of Science in the School of Informatics, Indiana University May 2003 / Alzheimer's disease is a progressive, age-related, degenerative brain disorder, which is one of the most serious diseases in old people. The patients' memory is lost and their personality and behavior are changed gradually; furthermore, this process is irreversible until the patients die [1]. Alzheimer's disease first attacks the entorhinal cortex; then to the hippocampus, which help to control short-term memory; then to other regions, especially the cerebral cortex, which is very important in using language and reasoning [1 , 2]. After its attack, the neurons degenerate and lose synapses and eventually die [1 , 2]. According to the age of having this disease, Alzheimer's disease can be divided to early-onset (usually at age 30 to 60) and late-onset (at age of 65 or older) [1]. About 5% to 10% of Alzheimer's disease cases are early onset [1 ]. Another way to describe Alzheimer's disease is according to the inheritance pattern. In this way, Alzheimer's disease also can be divided to: sporadic Alzheimer's disease, which has no certain inheritance pattern; and familial Alzheimer's disease (FAD), which has certain inheritance pattern [1]. All FAD are early onset [1 ]. Alzheimer's disease is a progressive disease and the progression of symptoms can be divided into mild, moderate and severe phases [2, 3]. The symptoms of mild Alzheimer's disease include loss of memory, disorientation, and difficulty of performing routine tasks. [2]. Patients in this phase can live independently [3]. The moderate symptoms include having great difficulty in daily living, wandering, personality changes, agitation and anxiety [2]. Patients in this phase should be cared by other people. People in severe phase lose all communication functions, almost cannot think, and need total care [2].

Chronic respiratory disease (CRD), comprised mainly of asthma and chronic obstructive lung disease (COPD), is responsible for significant morbidity and mortality worldwide. Although asthma and COPD cannot be cured, they can be controlled using appropriate medications. Poorly controlled CRD is associated with significantly poorer quality of life and mortality for patients, an increased burden on the healthcare system, and a negative economic impact due to loss of productivity. CRD is underdiagnosed, undertreated and poorly controlled, especially in low- and middle-income countries. Improving control of CRD would result in improved quality of life for patients and a reduced burden on the healthcare system and economy. Despite the increase in burden of CRD globally, limited data are available on the burden of CRD in South Africa. Such data are essential if appropriate measures are to be put in place to address these needs. In this mini-dissertation, I aimed to describe the symptomatic burden of disease and levels of treatment in adults with CRD attending primary healthcare facilities in the Western Cape. Additionally, I aimed to identify predictors of both the quality of life and receipt of treatment in this population. This study was a secondary analysis of the baseline data collected during the Primary Care 101 (PC101) trial, a large pragmatic cluster randomised controlled trial conducted in 38 primary healthcare clinics in the Eden and Overberg districts of the Western Cape between 2011 and 2012. The study population for the current study was limited to the 1 157 participants enrolled in the CRD cohort of the PC101 trial. Part A of this mini-dissertation comprises the research protocol which was submitted to, and approved by, the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee. Part B comprises the literature review, which outlines the prevalence and increasing burden of CRD both globally and in South Africa. As demonstrated in many studies from a wide variety of countries, the literature supports that CRD is underdiagnosed, undertreated, and has a significant impact both on affected individuals as well as healthcare systems and economies. Part C includes the journal-ready manuscript. Findings confirm a high burden of symptoms and activity limitation, indicating a poor quality of life amongst this population. Findings also suggest undertreatment, with 40% of patients not receiving treatment for CRD despite being symptomatic. More respiratory symptoms were associated with male sex, a positive screen for depression, previous tuberculosis, previous smoking, more activity limitation and current receipt of treatment for CRD. Greater activity limitation was associated with unemployment, diabetes, a positive screen for depression, more respiratory symptoms, recent hospital admission and receiving treatment for CRD. Participants were more likely to be on treatment if they were older, more symptomatic or had greater activity limitation due to their respiratory condition. Treatment was less likely in participants who screened positive for depression, were current smokers, had increased recent clinic visits or a recent hospital admission. In summary, we found a high burden of symptoms and activity limitation, and of undertreatment, possibly contributed to by under-recognition of respiratory disease among patients attending primary care clinics in the Western Cape. Depression, a history of previous tuberculosis and unemployment are common features in such patients. Potential interventions are to introduce a systematic approach to CRD diagnosis in primary care clinics that includes screening for depression, improving availability of essential drugs for the management of CRD, and preventive strategies such as more effective tuberculosis control, and support and pharmacotherapy to assist smokers to quit.

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Peripheral neuropathies, affect approximately 20 million people in the United States and are often a complication of conditions such as diabetes that can result in amputation of affected areas such as the feet and toes. In vitro methodologies to facilitate the understanding and treatment of these disorders often lack the cellular and functional complexity required to accurately model peripheral neuropathies. In particular, they are often 2-D and functional readouts, such as electrical activity, are limited to cell bodies thereby limiting the understanding of axonopathy which often characterizes these disorders. We have developed a functional 3-D model of peripheral nerves using a capillary alginate gel (Capgel™), as a scaffold. We hypothesize that: 1) The unique microcapillary structure of Capgel™ allows for the modeling of the 3-D microstructure of the peripheral nerve, and 2) That axon bundling in the capillary allows for the detection of axonal electrical activity. In our initial studies, we demonstrate that culturing embryonic dorsal root ganglia (DRG) within the Capgel™ environment allows for the separation of cell bodies from axons and recreates many of the features of an in vivo peripheral nerve fascicle including myelinated axons and the formation of a rudimentary perineurium. To develop functionality for this model we have integrated the DRG Capgel™ culture with a microelectrode array to examine spontaneous activity in axon bundles, which we find demonstrates superiority to other widely used 2-D models of the same tissue. Furthermore, by analyzing the activity on individual electrodes, we were able to record action potentials from multiple axons within the same bundle indicating a functional complexity comparable to that observed in fascicles in vivo. This 3D model of the peripheral nerve can be used to study the functional complexities of peripheral neuropathies and nerve regeneration as well as being utilized in the development of novel therapeutics.

The gastrointestinal tract forms the body’s largest interface with the external environment and is exposed to a vast amount of foreign material, including pathogenic and commensal bacteria, as well as food antigens. The gastrointestinal tract has multiple functions that are performed through complex interactions by its different components. It must be able to degrade food, absorb nutrients and eliminate waste, while at the same time maintain a balance between immune tolerance and protection against pathogenic and antigenic material. This concept of mucosally-induced tolerance is a key feature of the gut immune system, whereby a state of local and systemic unresponsiveness to food protein or systemic ignorance of commensal bacteria is maintained under homeostatic conditions through interactions between the host, dietary factors, and the intestinal microbiota. Dysfunctional interactions can lead to a breakdown in tolerance to otherwise innocuous antigens. One of the best characterized food sensitivities is celiac disease (CD). CD is a chronic immune-mediated disease triggered by the ingestion of gluten, the water insoluble protein fraction in wheat, rye and barley, in patients who are HLA/DQ2 or DQ8 positive. Celiac patients can experience a loss of oral tolerance to gluten any time throughout life. The clinical presentation of CD is variable and is often associated with extra-intestinal autoimmune diseases, such as type 1 diabetes (T1D). The increasing incidence of CD and the observation that only a small proportion of genetically susceptible individuals go on to develop active inflammation suggest a role for additional environmental factors in disease pathogenesis. The current treatment for CD is a strict, life-long adherence to a gluten-free diet (GFD), which is very demanding. Frequent gluten contamination can lead to persistent mucosal damage and symptoms, which have a negative effect on quality of life. Understanding the environmental and host factors that contribute to gluten tolerance is critical for the development of adjuvant therapies to the GFD. Therefore, the overall aim of my thesis is to characterize a humanized mouse model of gluten sensitivity in order to study factors that influence host-responses to gluten and to investigate potential therapeutic strategies. In chapter 3 of this thesis I characterized host responses to gluten using transgenic non-obese diabetic (NOD)/DQ8 mice. I found that gluten sensitization in NOD/DQ8 mice induced barrier dysfunction with a moderate degree of enteropathy and the development of anti-gliadin and anti-tissue-transglutaminase antibodies. I also explored the potential role of gluten in the development of T1D and found that gluten-induced barrier dysfunction was not sufficient to induce insulitis; a partial depletion of regulatory T cells (Tregs) plus gluten sensitization was required. In chapter 4, I utilized this model to demonstrate that the microbiota can modulate host responses to gluten. I found that both the presence and absence of a microbiota, as well as the composition of the microbiota influenced host responses to gluten in NOD/DQ8 mice. Finally, Chapters 5 and 6 of this thesis utilized transgenic DQ8 mice to explore two different adjuvant therapies for CD. In chapter 5, I showed that administration of a gluten binding polymer, P(HEMA-co-SS, to gluten-sensitive HLA/DQ8 mice reduced short-term and long-term gluten-induced barrier dysfunction and inflammation. In chapter 6, I discovered that elafin, a human anti-protease, was decreased in patients with active CD and in vitro, it inhibited the deamidation of gliadin peptides, a key step in the pathogenesis of CD. I showed that administration of elafin prevented gluten-induced barrier dysfunction and intraepithelial lymphocytosis. Together these results, (a) provide an in depth characterization of a humanized animal model for studying gluten-induced intestinal and extra-intestinal immune responses, (b) demonstrate the role of the microbiota as an environmental modulator of gluten-induced immune responses, (c) support the preclinical potential of two novel adjuvant therapies to the GFD. These findings emphasize the translational value of using relevant animal models to study the complex interactions between environmental and host factors that contribute to intestinal health and disease. / Thesis / Doctor of Philosophy (Medical Science)

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