published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Bibliography: leaves 107-138.

Alzheimer’s Disease (AD) is a complex brain disorder that affects at least one in every ten persons aged 65 and above worldwide. The pathogenesis of this disorder remains elusive. In this work, we utilized a rich set of publicly available gene expression data to elucidate the genes and molecular processes that may underlie its pathogenesis. We developed a new ranking score to prioritize molecular pathways enriched in differentially expressed genes during AD. After applying our new ranking score, GO categories such as cotranslational protein targeting to membrane, SRP-dependent cotranslational protein targeting to membrane, and spliceosomal snRNP assembly were found to be significantly associated with AD. We also confirm the protein-protein interaction between APP, NPAS4 and ARNT2 and explain that this interaction could be implicated in AD. This interaction could serve as a theoretical framework for further analyses into the role of NPAS4 and other immediate-early genes in AD pathogenesis. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Non-alcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease worldwide, with 25% of these patients developing nonalcoholic steatohepatitis (NASH). NASH is characterized by steatosis, inflammation, cell death, and liver fibrosis and significantly increases the risk of cirrhosis and decompensated liver failure. There are currently no approved drugs on the market for treating NASH leaving a major unmet medical need for drug discovery research. The aim of this dissertation is to better understand the pathophysiology of NASH and elucidate disease driving molecular mechanisms that can be used for drug targeting. Analysis of human liver samples using state of the art mass spectrometry proteomics identified dysregulation of one-carbon metabolism in NASH. This dissertation details the molecular mechanisms for how these proteomic changes can drive NASH pathogenesis and be targeted for therapeutic purposes. Chapter 1 provides an extensive background on NASH prevalence and pathophysiology and the association of one-carbon metabolism and NASH. Chapter 2 presents the identification of reduced glycine N-methyltransferase, a key regulator of one-carbon metabolism, in human NASH patients. Using a diet-induced animal model of NASH and targeted proteomics and metabolomics it was found glycine N-methyltransferase reduction in NASH leads to an accumulation of S-adenosylmethionine, activation of polyamine metabolism, and production of oxidative stress. Oxidative stress is a key component to NASH pathogenesis and this work identifies a novel mechanism for how oxidative stress is produced during NASH. Chapter 3 covers the discovery of increased folate receptor gamma (FOLR3) specifically in the liver of NASH patients. Initially, FOLR3 was predicted to impact one-carbon metabolism through folate metabolism, but molecular characterization found FOLR3 drives liver fibrogenesis independent of one-carbon metabolism. Chapter 3 details the molecular mechanism for how FOLR3 drives liver fibrosis by enhancing transforming growth factor beta activation of hepatic stellate cells through its interaction with serine protease HTRA1. The effect of FOLR3 was then validated in an in vivo model of NASH showing FOLR3 treatment can induce severe liver fibrosis in mice comparable to human liver fibrosis. These findings provide a translational animal model that can be used for NASH drug development and introduce a novel drug target in FOLR3. Chapter 4 discusses the innovation and translational impact of these findings and Chapter 5 summarizes the main results of this dissertation. / Pharmaceutical Sciences

Beta-Amyloid (1-40) (Aβ40) is an aggregation prone protein, which undergoes a nucleation-dependent aggregation process causing the pathological neurodegeneration by amyloid plaque formation implicated in Alzheimer’s disease. In this thesis, we investigated the effects of small molecule modulators extracted from the marine invertebrate Pseudopterogorgia elisabethae on the Aβ40 amyloidogenic process using in- vitro ThT fluorescence assay and atomic force microscopy. We also investigated the effects of neutral and anionic phospholipid liposomes on Aβ40 aggregation. Our results show that a marine natural product Pseudopterosin-A and its derivatives can suppress and modulate the Aβ40 aggregation process. Furthermore, our results demonstrate that a neutral phospholipid liposome inhibits Aβ40 fibril formation, whereas the anionic liposomes promote it. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Despite the tremendous advances of the last century, the cause of Alzheimer disease (AD) remains unclear. Genetic analysis of families with Alzheimer disease has revealed a disease-associated variant of the APOE gene, which encodes apolipoprotein E, a transporter of lipids in the blood and central nervous system. The effect of the AD-associated isotype, termed ApoE-E4, on disease risk has been validated, though it is unclear by what mechanism apoE-E4 confers AD risk. Mitochondria have long been implicated in AD pathogenesis, as the canonical histopathological findings of amyloid plaques and tau tangles occur in the setting of mitochondrial dysfunction. The disrupted processes include calcium homeostasis, cholesterol metabolism, phospholipid synthesis, and mitochondrial dynamics, and are all regulated by a subcompartment of the ER that is in physical contact with mitochondria. This compartment, called the mitochondria-associated ER membrane, or MAM, has been found to be overactive in AD patient cell lines and cell models of AD. Given that MAM is dysfunctional in AD and that ApoE-ε4 is the most important risk factor for AD, this dissertation examines if ApoE4 contributes to the MAM dysfunction seen in AD. The MAM dysfunction seen in AD patients and in cell models of AD has been best characterized in the context of familial AD, and it is the purpose of this study to extend those findings to the more common, sporadic, form of the disease. Familial AD is the result of autosomal dominant mutations in one of three genes, amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2). APP is the protein from which amyloid-beta, the component of amyloid plaques, is cleaved. The presenilins constitute the enzymatic core of the γ-secretase complex, which cleaves amyloid-beta from a precursor APP molecule. Both PSEN1 (PS1) and PSEN2 (PS2) localize at the MAM, and their action is speculated to influence MAM activity. Fibroblasts from familial AD patients, which contained mutations in APP, PSEN1 or PSEN2, showed a marked increase in MAM activity when compared to that of age-matched controls. In mouse embryonic fibroblasts, one can recapitulate this increased MAM activity by knocking out presenilins 1 and 2. In these Psen1/2 double knockout (DKO) cells, the typical measures of MAM function, i.e. increased cholesteryl ester and phosphatidylethanolamine synthesis, calcium transport from ER to mitochondria, and co-localization of ER and mitochondria by confocal and electron microscopy, mimicked the same phenotype found in fibroblasts obtained from familial AD patients, which suggests that the presenilins are negative regulators of ER-mitochondrial apposition. The precise mechanism by which they regulate the ER-mitochondria interface, whether directly as part of a tethering complex, or indirectly though the metabolism of APP-derived substrates, is unclear. While the effect of familial AD mutations on MAM has been characterized, the mechanism of mitochondrial dysfunction seen in the more common sporadic form of the disease remains obscure. Sporadic AD patients harbor no mutations in APP, PSEN1, or PSEN2, but rather inherit mutations in other genes which do not guarantee the development of the disease, but are instead considered risk factors. The most important of these risk factors, in terms of both amount of AD risk conferred and prevalence in the population, is ApoE. Embedded in the phospholipid monolayer of lipoproteins, ApoE is involved in the transport of phospholipids, cholesterol, and cholesteryl esters in plasma and the central nervous system (CNS). In the CNS, it is the most abundant apolipoprotein, and is secreted primarily by astrocytes and taken up by neurons. Once endocytosed, ApoE can follow three different pathways: degradation by the lysosome, intracellular retention in early endosomes, or re-lipidation and re-secretion out of the cell. Our approach takes advantage of the physiological role of ApoE as part of a high densitylike lipoprotein particle (HDL). Using astrocytes from ApoE targeted gene replacement mice in which murine APOE has been replaced by either human APOE-E3 or human APOE-E4, cultured media containing ApoE3 and ApoE4-lipoproteins can be produced and applied to target cells that do not express ApoE, such as neurons or fibroblasts. These target cells can then be analyzed for MAM activity. To examine the contribution of ApoE towards MAM dysfunction, target cells, either neurons or fibroblasts, were grown in the presence of astrocyte conditioned media (ACM) from ApoE targeted gene replacement mice. Several measures of phospholipid and cholesteryl ester synthesis were performed to analyzed MAM function. To confirm that the alterations in phospholipid synthesis were the result of altered MAM activity, the same assay was performed in cells in which a protein tethers that bind mitochondria and ER were genetically ablated. Finally, to confirm that the effects seen were the result of the HDL particles and not the result of other components of the ACM, lipoproteins were extracted from ACM by density ultracentrifugation and applied to fibroblasts. In all of the assays performed, ApoE4 conditioned media or ApoE4 isolated lipoproteins were able to induce a significant increase in MAM activity, whereas ApoE4 from recombinant sources did not. These data suggest a contribution of ApoE4 towards MAM dysfunction seen in AD. The mechanism of these ApoE4 induced MAM alterations remains to be deduced. One may speculate that given the role of ApoE in cholesterol transport outside of the cell, its intracellular retention may impact the distribution of cholesterol within the cell. MAM is a cholesterol rich subdomain with lipid raft-like properties, and any change in the cholesterol content or lipid nature of this membrane may alter its activity. To test this hypothesis, MAM was biochemically extracted from ApoE3 and ApoE4 treated cells and analyzed for cholesterol and lipidomic content. The results described in this thesis demonstrate an AD-like effect in wildtype cells when treated with ApoE-E4, and that the mechanism for these alterations may be due to disturbances in cholesterol distribution in the MAM.

La maladie d’Alzheimer (MA) est caractérisée par deux lésions neuropathologiques: les plaques séniles (composées essentiellement du peptide amyloïde Ab) et les dégénérescences neurofibrillaires (DNF, composées de formes hyperphosphorylées de protéines tau). Les mécanismes de formation des DNF sont encore mal compris et notre travail expérimental a eu pour objectifs d’étudier certaines hypothèses de formation des DNF.<p>1° Une hypothèse étiopathogénique de la MA est la “cascade amyloïde”, selon laquelle le peptide amyloïde Ab exercerait un effet toxique entraînant la phosphorylation de tau et la formation de DNF. Certaines formes familiales de MA sont dues à des mutations du gène du précurseur du peptide amyloïde (APP) ou des présénilines et nous avons voulu déterminer si la surexpression de ces protéines pouvait entraîner la formation de DNF. Nous avons d’abord étudié une lignée murine double transgénique surexprimant l’isoforme 0N3R de protéine tau humaine “sauvage” et une forme mutée de préseniline 1 (M146L). Nous y avons démontré une co-expression neuronale des deux protéines et une augmentation de la phosphorylation de tau mais nous n’y avons pas observé de formation de DNF, chez des animaux examinés jusqu’à 17 mois. Nous avons ensuite étudié une lignée murine triple transgénique surexprimant l’isoforme 0N3R de protéine tau “sauvage”, une forme mutée de préseniline 1 (M146L) et une forme mutée de l’APP 751 (mutations Swedish K670N, M671L et London V717I). Ces animaux ont développé précocement (2.5 mois) des dépôts extracellulaires de peptide Ab. Nous y avons observé une augmentation de la phosphorylation de tau dans les prolongements neuronaux en contact avec les dépôts amyloïdes et des anomalies de l’organisation du cytosquelette, mais pas de DNF, chez des animaux examinés jusqu’à 18 mois.<p>2° Certaines mutations du gène de tau sont responsables de formes familiales de démence frontotemporales dans lesquelles se développent des DNF. Ces mutations favoriseraient l’agrégation de tau où entraîneraient un déséquilibre de l’expression relative des isoformes de tau. Un tel déséquilibre pourrait également être induit dans les formes sporadiques de MA, en l’absence de mutations de tau. Afin d’investiguer cette hypothèse, nous avons étudié le profil d’expression des ARNm de tau et des isoformes de protéines tau dans plusieurs régions cérébrales de sujets contrôles ou atteints de MA. Un même profil d’expression a été observé dans les deux groupes. Une augmentation relative de l’expression de l’isoforme 0N3R de tau dans le cortex temporal pourrait être liée à la sensibilité de cette région au développement de DNF. Nous avons également étudié des lignées stables de cellules CHO exprimant des formes mutées (P301L, R406W) et non-mutées de protéines tau. Nous n’avons cependant pas observé d’augmentation de l’agrégation de tau dans les lignées exprimant les formes mutées de tau.<p>Nos résultats indiquent que la simple surexpression de formes mutées de l’APP et des présénilines, même en présence d’une protéine tau humaine, ne suffit pas à entraîner la formation de DNF. En outre, l’absence de différence dans le profil d’expression cérébrale des isoformes de tau entre sujets contrôles et atteints de MA suggère que les modifications post-traductionnelles de cette protéine jouent un rôle plus important dans la genèse des DNF.<p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Background: Air pollution is one of the leading environmental issues in the world today. In 2015, pollution-related diseases accounted for 16% of all deaths worldwide — that is an estimated 9 million premature deaths were linked to air pollution. In addition to the substantial effects on human health, air pollution-related diseases result in productivity losses that reduce countries’ gross domestic product. Although air pollution disproportionately affects middle- and low-income countries, it is still a major issue in high-income countries, such as the United States, where 25% of Americans breath air with pollutant levels above the national regulatory standards. Fine particle matter (particles with diameter ≤ 2.5 μm, PM₂.₅ ) is the most extensively studied air pollutant and it has been causally linked with a wide range of adverse health outcomes, including cardiovascular and pulmonary disease, myocardial infarction, hypertension, congestive heart failure, arrhythmias, chronic obstructive pulmonary disease, and lung cancer. Moreover, recent scientific evidence suggests that PM₂.₅ affects the nervous system and possibly contributes to the development and exacerbation of neurodegenerative diseases. This is increasingly relevant as populations are aging and the number of adults living with neurodegenerative diseases increases, negatively affecting families, communities, and health-care systems around the world. Although millions of people suffer from neurodegenerative diseases, there is currently no treatment that slows the progression of these conditions and no known cure or cause. Thus, determining whether a link exists between air pollution and neurodegenerative diseases is a goal of increasing importance. Objective: The research presented in this dissertation has two main objectives: (1) to characterize the relationship between long-term exposure to PM₂.₅ and disease aggravation in two of the most prevalent neurodegenerative diseases worldwide: Alzheimer’s (AD) and Parkinson’s disease (PD), as well as in the rare and devastating neurodegenerative motor disorder amyotrophic lateral sclerosis (ALS); (2) to identify the specific PM₂.₅ chemical components that are associated with disease aggravation in PD. Methods: We used data from the New York Department of Health Statewide Planning and Research Cooperative System from 2000–2014 to identify patients’ first hospitalization with a primary or secondary diagnosis of AD, PD, or ALS. With these data, we constructed annual AD, PD, and ALS first hospitalization county counts (total and sex- and age-stratified) for all of New York State (NYS). A patient’s first hospital admission was used as a surrogate for disease aggravation, indicating the crossing point into a more severe stage of the disease. We used prediction estimates from well-validated models that incorporate satellite information and ground-based monitoring data to estimate annual PM₂.₅ and PM₂.₅ chemical component (nitrate, sulfate, organic matter, sea salt, black carbon, and soil) concentrations across NYS at a high spatial resolution. In Chapter 2, we used outcome-specific (AD, PD, or ALS) mixed quasi-Poisson models with county-specific random intercepts to assess the relationship between long-term exposure to PM₂.₅ and disease aggravation. In Chapter 3, we used a multi-pollutant mixed quasi-Poisson model with county-specific random intercepts to identify specific PM₂.₅ components associated with disease aggravation in PD. In all analyses, we evaluated potential nonlinear exposure–outcome relationships using penalized splines and accounted for potential confounders. Results: We observed a total of 264,075 AD, 114,514 PD, and 5,569 ALS first admissions between 2000 and 2014. The hospitalization annual average counts per county were 284, 131, and 6 for AD, PD, and ALS, respectively. In Chapter 2, we found a nonlinear association between total PM₂.₅ exposure and PD hospitalizations, which plateaued at higher concentrations of PM₂.₅ (> 13 μg/m³, RR=1.08, 95% CI: 1.04–1.13 for a PM₂.₅ increase from 8 to 10 μg/m³, Figure 2.3). We also found that patients with a first PD hospitalization at age 70 or younger are at slightly higher risk for disease aggravation at lower PM₂.₅ concentrations relative to those age >70. In the case of AD, we observed evidence of a potential association between annual increases in PM₂.₅ exposure and disease aggravation, but only in a sensitivity analysis aiming to decrease outcome misclassification. We found no association for ALS in the main analysis, but we observed an unexpected negative association in those <70 years in the stratified analysis. We found no evidence of effect modification by sex for any of the outcomes. In Chapter 3, we observed a linear association between disease aggravation in PD and long-term exposure to the PM₂.₅ components nitrate (RR = 1.05, 95%CI: 1.02–1.09 per one standard deviation (SD) increase) and organic matter (RR = 1.05, 95%CI: 1.02– 1.07 per one SD increase), and a nonlinear association for black carbon with a negative association above the 96th percentile of the BC concentration distribution (Figure 3.4). We found no evidence of an association with sulfate, sea salt or soil. Conclusion: Overall, our studies provide an analysis of the potential association between long-term exposure to PM₂.₅ , both as an overall pollution mixture and by chem- ical composition, and disease aggravation in AD, PD, and ALS. Our findings suggest that annual increases in county-level PM₂.₅ concentrations are associated with disease aggravation in PD and possibly AD. We found that the PM₂.₅ components organic matter and nitrate are particularly harmful in the association between PM₂.₅ and dis- ease aggravation in PD. Additionally, our results indicate that current national PM₂.₅ standards may not be strict enough to safeguard the population’s neurological health. Specifically, in Chapter 2, we observed that the PM₂.₅ –PD association has a steeper slope at lower concentrations that are well below the current annual National Ambient Air Quality Standards for PM₂.₅ . Thus, our findings warrant further investigation into the potential link between long-term PM₂.₅ exposure and disease aggravation, particularly in the context of PD. Our results also indicate that the chemical composition of PM2.5 affects its neurotoxicity. Further research into how PM₂.₅ composition influences the overall PM₂.₅ adverse effects is needed to fully understand the mechanisms that underlie the association between exposure to PM₂.₅ and aggravation of neurodegenerative diseases.

Inhibition of nitric oxide (NO) signalling may contribute to the pathogenesis of severe malaria. This thesis examines the impact of Plasmodium infection on three key determinants of nitric oxide synthase (NOS) biochemistry: substrate availability, substrate/inhibitor homeostasis and cofactor availability. Arginine, the NOS substrate, is depleted in human patients with severe Plasmodium falciparum malaria and mice infected with P. berghei ANKA. Using heavy isotope tracer infusions to quantify arginine metabolism in infected mice, we found no evidence of increased catabolism by the enzyme arginase, widely assumed to be responsible for arginine depletion. Genetic knock-out of parasite arginase had no effect on arginine depletion in mice. Instead, our findings link arginine depletion to decreased rates of arginine and citrulline appearance in the plasma of infected mice. Asymmetric dimethylarginine (ADMA) competes with arginine for binding to the NOS catalytic site. We observed elevation of the ADMA/arginine ratio in Gambian children with severe malaria, favouring NOS inhibition. In mice infected with P. berghei ANKA, we found evidence of degradation of dimethylarginine dimethylaminohydrolase 1 (DDAH1), the enzyme primarily responsible for ADMA metabolism. We also observed reduced DDAH activity and accumulation of intracellular ADMA in hepatic tissue of infected mice, suggesting that DDAH dysfunction could contribute to disruption of ADMA/arginine homeostasis. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. In P. berghei ANKA-infected mice, BH4 concentrations were decreased in plasma, erythrocytes and brain tissue, which could inhibit NO synthesis and promote NOS-dependent superoxide production. To reverse deficiencies of NOS substrate and cofactor availability, we infused P. berghei ANKA-infected mice with citrulline, an arginine precursor, and sepiapterin, a BH4 precursor. Restoration of systemic arginine and BH4 availability in infected mice improved whole blood nitrite concentrations, a biomarker of NO synthesis, but did not prevent onset of disease symptoms. These studies have identified biochemical disturbances that may contribute to severe malaria pathogenesis by inhibiting NO synthesis.