Theses/Dissertations - Biomedical Studies

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    An in vitro, architecturally relevant, alveolar model to assess potential for respiratory sensitization.
    (May 2023) Gibb, Matthew James, 1983-; Sayes, Christie M.
    With greater numbers of the global population entering urban areas and subsequently noting increases in respiratory illness and disease, it is of utmost importance to understand the mechanisms and potential for inhaled air (containing both chemical and particulate) to lead to adverse human health effects. Novel substances, specifically chemicals and particulates, are well-documented for their ability to cause respiratory allergies and sensitizing reactions. Many of these materials are produced to advance industry, consumer, and medicinal products. The standard for toxicological testing involves the use of a single cell type relevant to the potentially exposed organ, e.g., epithelial cells for dermal testing. However, in any biological system multiple cell-types exist where inter and intracellular communication, cell turn-over, clearance mechanisms, and a variety of movements are involved. The goal of this dissertation was to develop and utilize an architecturally relevant respiratory model to assess and study the potential biological impact of both known respiratory sensitizers and unknown materials to gain important insights into the early steps involved in forming a sensitizing reaction in the lungs. While it is possible for sensitization to occur within any compartment within the lungs, a simplistic, reliable, reproducible model where endpoint measurements can be modified has yet to be established. In this study, an in vitro alveolar model designed to mimic in vivo architecture was utilized to characterize cell types and analyze responses at the cellular, biochemical, and gene expression levels after exposure to known respiratory sensitizers, known non-respiratory sensitizers, and unknown materials. First, a chemical respiratory sensitizer was compared to a general cell activator to determine differences between overall cell activation and specifics of sensitization. Next, a respiratory irritating particulate and a suspected respiratory sensitizing particulate were used to understand differences between irritation and sensitization. Finally, an in-depth gene expression analysis was performed on two known respiratory sensitizers and compared to two known non-respiratory sensitizers to gain insight of the total gene expression analyses that occurs after chemical respiratory sensitizers are inhaled. Ultimately, the aim of this work is to advance the knowledge base of respiratory immunotoxicology by improving the understanding of what cellular perturbations may occur after inhalation to potential sensitizing substances; aid in the preventing risky materials from entering production and the environment; aid in risk assessment and regulations; and understand how adverse health outcomes can be prevented or treated.
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    Metabolomic analysis in Parkinson’s and Alzheimer’s disease.
    (May 2023) Kalecký, Karel, 1991-; Bottiglieri, Teodoro, 1958-
    Parkinson’s disease (PD) and Alzheimer’s disease (AD) are two most prevalent forms of neurodegeneration with progressively disabling symptoms and no cure, leading to a high burden to caregivers and society in general. The underlying disease mechanisms remain unclear and more research is required to develop novel therapies. Metabolomics is a scientific area, which explores biochemical processes happening inside an organism by measuring their intermediates. Thus, metabolomics studies the high-level functioning as also influenced by external factors including nutrition, environment, and microorganisms, directly reflecting the final phenotype. Recent technological advancements have enabled performing accurate metabolomic measurements across many pathways of metabolism simultaneously. Here, we present a series of metabolomic experiments that allows new insights into the disease mechanisms in PD and AD. Specifically, we searched for disease-related metabolic differences via case-control association studies. We focused on human brain frontal cortex and putamen, the direct location of the pathologies, combining broad investigation across many metabolic pathways with detailed analysis focused on one-carbon metabolism. Other experiments were conducted with human plasma to search for potential diagnostic biomarkers in AD and PD. Additionally, we included a mouse model of PD to compare its metabolic changes with real disease in human. The majority of experiments were targeted using liquid chromatography and flow injection analysis coupled to tandem mass spectrometry. Untargeted analysis was based on comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. Furthermore, we emphasize the importance of data processing and analysis, for which we have developed several new methods. Our results are summarized in individual chapters. The most fascinating finding implicates the anti-parkinsonian medication levodopa as a direct contributor to dementia in susceptible individuals through the accumulation of homocysteine. The susceptibility is largely explained by low amounts of betaine (trimethylglycine) and B vitamins that act as enzymatic cofactors in one-carbon metabolism, combined with specific genetic polymorphisms. Similar changes were observed in AD. The risk of developing dementia, especially in PD, could thus be potentially reduced by low-cost nutritional intervention. Collectively, our work represents a systematic effort in metabolomics in PD and AD, and constitutes a major scientific contribution in biomedical research of these diseases.
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    Induction of protective humoral immune responses at steady-state.
    (December 2022) Bouteau, Aurélie, 1989-; Igyártó, Botond Z.
    The specific roles of dendritic cell (DC) subsets in regulating antibody responses are essentially unknown. Here, we used a steady-state targeting system to determine the contribution of two skin dendritic cell (DC) subsets in the induction and regulation of humoral immunity to a foreign antigen. We found that Langerhans cells (LCs) targeting, but not cDC1s, through the same receptor (Langerin) led to a robust and protective humoral immune response. LCs, unlike cDC1s, supported the formation of germinal center T follicular helper cells (GC-Tfh), and then, likely licensed by these T cells, some of the LCs migrated to the B cell area to initiate B cell responses. Furthermore, we found that the cDC1s prevented the LCs from inducing GC-Tfh cells and humoral immune responses. cDC1s likely achieved this through their superior T cell activation capacity. Overall, these data suggest that an adjuvant-free vaccine could be a viable option to promote protective immune responses if targeted to a specific DC subset. Our findings also revealed that contrary to the accepted danger model, specific DC subsets could support adaptive immune responses without inflammation. Here, we showed that type I interferon and IL-6 signaling pathways, significant contributors to Tfh cell differentiation in inflammatory settings, played no role in LC-induced adaptive immune responses at steady-state. However, the membrane-bound co-stimulatory molecule ICOS/ICOSL regulates germinal center formation. Therefore, these data suggest that adaptive immune responses against foreign antigens in the absence of inflammation are generated through a mechanism that likely does not involve inflammatory cytokines. Identifying DC factors promoting humoral immune responses in a non-inflammatory environment could help understand how antibody-based autoimmune diseases develop and devise more efficient immuno-therapeutics.
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    Regulation of islet cell differentiation and function by calcineurin/NFATc2 signaling and applications for islet cell transplantation.
    (August 2022) Darden, Carly M. 1992-; Lawrence, Michael C.
    Diabetes is a leading cause of death globally and is a result of high blood glucose concentrations contributing to metabolic impairments and high associated risk of cardiovascular disease, nephropathy, neuropathy, stroke, blindness, and loss of limbs by amputation. Islet cell transplantation represents a promising minimally invasive procedure to reverse diabetes and prevent hypoglycemia by replacing all types of islet endocrine cells that regulate blood glucose. Challenges that limit widespread use of islet replacement therapy include immune-mediated loss of islet graft function and lack of available donor pancreas tissue for transplantation. Pancreatic β cells succumb to metabolic and inflammatory stress by loss of β-cell identity genes, induction of β-cell disallowed genes, and loss of function. In the first aim, I elucidated the role of CN/NFATc2 in islet stress response. This work identified CN/NFAT as a key therapeutic target for pro-differentiation and maintenance of β-cell function during stress. Specifically, I show that small molecule differentiation inducer isoxazole-9 (ISX9) could preserve CN/NFATc2 signaling by preventing overstimulation and exhaustion of intracellular calcium stores and subsequent β-cell dedifferentiation. ISX9 also preserved glucose-responsive insulin secreting capacity in islets and improved islet graft function in a mouse islet transplant model. In the second aim, I developed methods to expand and pro-differentiate islet cell precursors in culture and in vivo by HDAC inhibition and induction of CN/NFATc2. Specifically, I identified a subpopulation of RGS16+ islet cell precursors that could differentiate into functional islet organoids which produced both β- and α-like cells within 14 d exposure to ISX9. This was attributed in part to NFATc2-mediated expression of the early islet endocrine progenitor specification gene RFX6 which induced a cascade of downstream transcription factors required for islet cell differentiation. Lastly, I present data and explore the potential of Hedgehog-GLI (HH-GLI) signaling targets for driving islet cell precursors toward β- and α-like cell type specification. My preliminary data indicate that manipulation of HH signaling in islet cell precursors can influence β- and α-cells fate in islet organoids. These findings provide proof of principle that adult islet cell precursors can be expanded and engineered for potential use in islet cell replacement therapies.
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    Peptidoglycome of gram-positive bacteria.
    (2021-10-08) Rimal, Binayak, 1988-; Sim, Cheolho.; Kim, Sung Joon.
    Our modern healthcare system faces imminent threats from multidrug-resistant pathogens due to their unprecedented increase in the frequency of infections in clinical environment compounded by the lack of novel antibiotic development. One of the leading targets for the development of novel antimicrobial agents is the bacterial cell wall. The cell wall is a unique feature found in all eubacteria with peptidoglycan (PG) as its primary component. PG plays a crucial role in protecting the bacteria from fluctuating osmotic pressures and the external environment. Thus, chemotherapeutic agents that interfere with PG biosynthesis or target its assembly can potentially lead to the development of novel antibiotics. To facilitate the understanding of how bacteria become resistant to antibiotics and the development of new-generation cell-wall targeting antimicrobial agents, our research focuses on the comprehensive in-depth study of PG composition analysis of clinically important Gram-positive pathogens (Enterococcus faecalis and Mycobacterium species) through liquid chromatography/mass spectrometry (LC/MS). This dissertation provides an innovative approach to PG composition analysis with i) a protocol that enables efficient PG isolation and purification without harsh chemical treatment to preserve PG modifications, and ii) a method for liquid chromatography/mass spectrometry (LC/MS) analysis using in silico muropeptide library for identification and quantification. Our PG data shows that bacterial PG composition is dynamic, remarkably able to modify their PG composition in response to the challenging external environment including growth conditions and antibiotic exposures in order to better adapt to survive. Our approach provides unprecedented molecular details into variations in PG modifications by pathogens and offers fundamental insight into bacterial physiology and mechanisms of microbial pathogenesis.
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    Plasmacytoid dendritic cell TLR7/9 response modulation through EGA and α-CD138 antibody.
    (2021-05-28) Wiest, Matthew, 1990-; Oh, SangKon.
    Plasmacytoid dendritic cells (pDC) have the unique ability to produce an inordinate amount of type 1 interferon (IFN) and other cytokines in response to stimulation with pathogenic and self-nucleic acids mainly through the toll-like receptors, TLR7 and TLR9. In vitro cultures of pDCs exhibit a bifurcated response to distinct TLR7 and TLR9 agonists: a response dominated by type 1 IFN production or pro-inflammatory cytokine production and maturation. The bifurcation of response is hypothesized to occur in distinct signaling endosomes within pDCs. We report that EGA alters the TLR7 and TLR9 response in pDCs by targeting the transition from the early to late endosome. EGA enhances imidazoquinoline-induced pro-inflammatory cytokine while diminishing type 1 IFN production. EGA abrogated the IFN and pro-­inflammatory cytokine response of pDCs to both influenza and ODN stimulation. Intriguingly, EGA enhanced the IFN and cytokine response of a "pDC-like" cell line, Gen2.2, to TLR9 stimulation. Mechanistically, EGA pre-incubation caused the retention of ODNs and imidazoquinolines within endosomes characterized by an early/recycling phenotype. These results suggest the presence of unique signaling localizations for TLR7 and TLR9 agonists within pDCs which were not reproducible within a "pDC-like" cell line, Gen2.2. EGA also shows therapeutic potential in SLE patients in diminishing type 1 IFN responsiveness to TLR7 stimulation which is known to be play a role in SLE pathogenesis. It has been suggested that heparan sulfate proteoglycans (HSPG) play a role in the uptake of pathogenic nucleic acid-antimicrobial peptide complexes. We have found that pDCs in the blood of pediatric SLE patients express syndecan-1 (CD138). We also found that α-CD138 antibody treatment resulted in decreased IFNα expression by pDCs stimulated with CpG ODNs and genomic DNAs without affecting the TLR7 response in pDCs. This study demonstrates that pDC response to nucleic acids and their analogues can be controlled by both EGA and α-CD 138. While the mechanisms of action of both EGA and α-CD138 need to be further studied, our findings will help us understand molecular mechanisms for pDC expression of type 1 IFN in response to TLR7/9 ligands.
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    MISTR : a conserved MItochondrial STress Response network revealed by signatures of evolutionary conflict.
    (2021-04-01) Sorouri, Mahsa, 1987-; Hancks, Dustin C.
    As pathogens manipulate host-encoded master regulators, we hypothesized that molecular scars of host-pathogen conflicts-such as signatures of rapid evolution and viral mimics-could lead to the discovery of novel cellular functions. Indeed, our evolution-guided screens have identified MItochondrial STress Response (MISTR), a cellular circuit conserved in vertebrates. MISTR proteins are associated with electron transport chain (ETC) factors and activated by stress signals such as interferon-gamma and hypoxia. Importantly, MISTR homologs are present in yeast, Plasmodium, plants, and diverse viruses. Upon stress, ultraconserved miRNAs downregulate MISTR1 followed by replacement with paralogs MISTRAV and/or MISTRH. While cells lacking MISTR1 are more sensitive to chemical and viral apoptotic triggers, cells lacking MISTRAV or expressing the squirrelpox virus-encoded vMISTRAV exhibit resistance to the same insults. MISTR1, which acts as the central axis of the MISTR circuit, is a constitutively expressed protein associated with Complex IV (CIV) of the ETC. In agreement with a role in oxidative phosphorylation (OXPHOS), MISTR1 KO cells display reduced proliferation and cellular ATP levels compared to WT cells when switched from glucose-containing media; yet this defect is grossly attenuated when the cells are grown in galactose-containing media for several days prior to assay indicating the presence of a compensatory mechanism. A recently published structure of CIV illustrates that MISTR1 lies at the dimeric interface of CIV homodimers, which may prevent CIV dimerization. Given CIV exhibits higher activity in its monomeric form, we hypothesize that the observed defects in cellular proliferation and ATP production in the absence of MISTR1 are due to dimerization of CIV. Furthermore, our data support a model where stress-induced miRNAs downregulate MISTR1 to decrease ETC activity and reduce reactive oxygen species generation. Overall, the findings presented in this dissertation suggest that MISTR1 is a master regulator of OXPHOS and cell death in a stress response circuit regulated by related miRNAs and targeted by viruses. Furthermore, the discovery of MISTR circuitry highlights the use of evolution-guided studies to reveal fundamental biological processes.
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    Impact of neuroinflammatory and mTOR signaling inhibition following flurothyl seizures on the acute development of autistic-like behavior in C57BL/6 mice.
    (2020-03-13) Hodges, Samantha L., 1992-; Lugo, Joaquin N.
    Epilepsy is a common neurological disorder, with individuals having an increased susceptibility of seizures in the first few years of life. Children with epilepsy are at risk of developing a multitude of cognitive and behavioral comorbidities throughout development. In addition, approximately one third of individuals with epilepsy are resistant to anti-seizure drugs, emphasizing the need for therapeutics that extend beyond manipulation of neuronal transmission and target alternative mechanisms. The present study examined the role of PI3K/Akt/mTOR pathway activity and neuroinflammatory signaling in the development of autistic-like behavior following seizures in the neonatal period. Male and female C57BL/6 mice were administered 3 flurothyl seizures on postnatal (PD) 10, followed by administration of minocycline, the mTOR inhibitor rapamycin, or a combined treatment of both therapeutics. On PD12, isolation-induced ultrasonic vocalizations (USVs) of mice were examined to determine the impact of seizures and treatment on communicative behaviors, a component of the autistic-like phenotype. Hippocampal tissue was collected on PD12 to examine proinflammatory cytokine expression with qRT-PCR, along with western blotting to examine mTOR protein expression and astrocyte and microglial reactivity. Seizures on PD10 increased the quantity of USVs in female mice and reduced the amount of complex call types emitted in males compared to controls. Inhibition of mTOR with rapamycin significantly reduced the quantity and duration of USVs in both sexes. Changes in USVs were associated with increases in mTOR activity and astrocyte reactivity in male mice, however, three PD10 seizures did not result in enhanced proinflammatory cytokine expression in either sex. Rapamycin treatment significantly reduced % total pS6(235,236) and % total pS6(240,244) expression on PD12, however, minocycline did not impact any of the examined proteins. These findings emphasize the importance of differences that may exist across preclinical seizure models, as three flurothyl seizures did not induce as drastic of changes in mTOR activity or inflammation as observed in other models. Early-life seizures can have a profound impact on the developing brain, and thus it is critical to continue investigating potential therapeutics that target the underlying pathology of seizures and could prevent the development of cognitive and behavioral comorbidities.
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    Innovating recombinant production of short peptide targeted antimicrobial peptides utilizing clean purification in plants and E. coli bioreactors.
    (2019-11-05) Ghidey, Meron Fessehaie, 1991-; Kearney, Christopher Michel, 1958-
    Antibiotic-resistant pathogens continue to become a pressing issue globally and current broad spectrum therapies fail to meet this challenge. Therapeutics equipped with targeting moieties would alleviate this issue without facilitating the rise of opportunistic pathogens, and employing small antimicrobial peptides (AMPs) would avoid resistant strains. Fusion of the two could be produced recombinantly at commercial levels in bioreactors such as plants with a purification tag, but issues with phytotoxicity and unexplained low recombinant AMP yield from plant tissue are major limiting factors. This dissertation addresses the issues of the plant expression platform for AMPs as well as properly assessing the selectivity of a previously discovered 12mer targeting domain (A12C) specific to Staphylococci. To understand why AMPs are poorly expressed in plant expressions systems, meta-analysis of peptide databases was performed and revealed plant-derived AMPs are less cationic in net charge compared to AMPs from organisms like animals and fungi. Using the elastin-like polypeptide as a tag for increased recombinant production and clean temperature shift purification, a survey of AMPs falling under the cysteine-stabilized motif ranging in net charge were produced in Nicotiana benthamiana. It was observed that only the anionic fusions were expressed, and at record levels as high as 563 µg/gram fresh leaf weight with retained antimicrobial activity only pre-protease cleavage. This phenomenon of activity maintained while still fused was studied in E. coli, and it was found the size of the ELP fusion partner dictates antimicrobial potential. For quicker turnover to facilitate targeting studies, cationic AMPs plectasin and eurocin were expressed in E. coli with the SUMO solubility tag. The targeting domain was assessed for antimicrobial selectivity against a panel of gram positive bacterium. Unexpectedly, there was no enhancement of activity against target bacterium, but a significant decrease in antimicrobial activity against non-target genus. These studies elucidate that the peptide net charge dictates recombinant AMP expression in plants, and a genus-specific targeting domain can be derived from as small peptide sparing commensal non-target bacteria. Together, these discoveries provide the foundation for the inexpensive production of targeted AMPs in plant bioreactors or in transgenic seed for poultry or livestock.
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    Statistical methods for high-throughput data integration : methodologies in disease research and drug discovery.
    (2019-07-09) Chan, Jinyan, 1990-; Gu, Jinghua.
    The wide application of high-throughput technologies in biomedical research calls for integrative approaches for data mining and knowledge discovery. Consequently, methodologies that deliver robust and systems integrations are in unprecedented demand. Two important sub-disciplines in biomedical research, namely disease research and drug discovery, have become ever-evolving frontiers for integration of “big data”. In disease research, p-value combination has been broadly employed to integrate statistical evidences from multiple studies. Common assumptions of conventional p-value combination methods include independence and homogeneity of the combined tests, which are constantly challenged by the complex nature of high-throughput biomedical datasets. In this dissertation, we propose a novel and robust p-value combination algorithm based on the Pareto Dominance principle from multi-objective optimization, which accounts for dependency and heterogeneity in data. Compared to existing methods, the Pareto method attains adaptive rejection regions from “learning” the multivariate null distribution estimated by permutations, therefore achieves superior performance when combining heterogeneous effects from multiple datasets, meanwhile remains appropriate error control for correlated tests. The Pareto meta-gene-set-analysis tool, PEACH, was developed and tested on a 16-cancer pan-cancer dataset from The Cancer Genome Atlas (TCGA). Significantly improved statistical power of the PEACH algorithm and its ability to detect important pathways related to sub-groups of cancers were demonstrated. On the other hand, computational drug repurposing based on gene expression data has gained increasing popularity in the field of drug discovery. The Connectivity Map (CMap) is a major database to repurpose new drugs from gene expression data. However, key limitations of the current signature-based drug-repurposing paradigm have prohibited accurate and unbiased repurposing. In the second part of this dissertation, we developed a frame-breaking statistical approach, namely Dr. Insight, to remove the requirement of subjective selection of a gene signature to query CMap database. We performed comprehensive studies using simulation data and disease datasets and validated the superior performance of Dr. Insight compared to previous methods. A TCGA breast cancer case study was also performed to showcase the application of Dr. Insight to breast cancer drug repurposing, from drug redirection to systematic construction of disease-specific drug-target networks.
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    Towards less hazardous chemicals : identifying chemical bioactivity through fish behavioral profiles.
    (2019-04-02) Steele, William Baylor, 1983-; Brooks, Bryan W.
    Because most chemicals in commerce lack empirical toxicology information, innovative approaches are needed to identify substances presenting elevated hazards and risks to public health and the environment. When larval fish models are employed with automated tracking technologies, behavioral studies can be used to perform rapid, diagnostic toxicity screens for large volumes of chemicals. Beyond utility in chemical safety screening applications, behaviors are integrated processes that are critical for organism survival and reproduction. This dissertation developed novel approaches to diagnostically examine chemicals for toxicity using larval fish models, and further extended these efforts beyond lab raised models to examine effects of neuroactive substances on wild fish populations. In Chapter two, methods used in the biomedical sciences to study therapeutic attributes of novel molecules were adapted for environmental screening applications. Using automated tracking software, a protocol was developed to quantify locomotor and photomotor responses (PMRs) of two common larval fish models, the zebrafish and fathead minnow. These developed methods were applied to study the behavioral effects of a common aquatic contaminant and neuro-stimulant, caffeine, which exerted photomotor and locomotor responses at environmentally relevant levels. In Chapter three, these methods were broadly applied to develop larval fish behavioral response profiles for a variety of different chemicals from diverse mode of action (MOA) categories. Both fish models demonstrated unique behavioral responses upon exposure to each chemical indicating that behavioral response may be informative of compound specific MOAs. Chapter four demonstrated that in the two most common larval fish models, refractory PMR and locomotor patterns appear informative of electrophilic properties associated with oxidative stress for SN2 chemicals. Property-based quantum mechanical modeling of electrophile reaction energies were predictive of experimental in vivo acute and sublethal toxicity, which provide important implications for identifying and designing less hazardous industrial chemicals. Because lab fish models cannot be expected to be representative of wild fish populations, Chapter five examined behavioral effects of two commonly prescribed psychiatric medications, oxazepam and sertraline, on perch collected from natural waters. Results from this study identified time related effects on fish boldness and neuroactive pharmaceutical related effects on fish activity levels.
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    Inhibition of TLR4 minimizes islet damage due to sterile inflammation and improves islet transplant outcomes.
    (2018-06-01) Chang, Charles A., 1991-; Kane, Robert R.; Naziruddin, Bashoo.
    Islet transplantation has emerged as an important treatment option for brittle type 1 diabetes and as an adjunct procedure after total pancreatectomy to prevent brittle diabetes. The efficacy and long-term function of islet transplantation have significantly improved over the last two decades. However, transplant outcomes are still largely compromised due to inflammation mediated prior to and after transplantation which results in the loss of as much as 50% of the islet graft. Toll-like receptor 4 (TLR4) has been identified as a major pro-inflammatory mediator of sterile inflammation by sensing damage-associated molecular patterns (DAMPs) and compromising graft function, making it a putative therapeutic target. Here, we study the effects of TLR4 blockade during the peri-transplant period on islet transplant outcomes using TAK-242, a small molecule inhibitor of TLR4, and a combination of basic biological assays as well as in vivo transplant models in mice. The results of early TLR4 blockade during islet isolation demonstrate a markedly reduced inflammatory profile in islets post-isolation which translated to reduced islet damage post-transplant and overall improved transplant outcomes with a cure rate of 75% for treated islets and 29% for untreated islets. Next, we developed a TLR4-antagonist prodrug and a chemical conjugation method to link the prodrug to the surface of islets which is slowly released, creating drug-eluting islets. Transplantation of a marginal dose of 100 modified islets into the kidney subcapsular space resulted in a cure rate of 100% compared to 0% for unmodified islets. In conclusion, we demonstrate that TLR4 is a major mediator of islet graft loss during the peri-transplant period. Therapies directed to inhibit this receptor, before and after transplant, are a promising avenue for improving islet transplant outcomes. The addition of TAK-242 to media during the isolation process is a rapidly translatable approach to clinical use, while the surface modification technique opens a broad range of possible transplant applications.
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    Quantitative cellular and molecular imaging of the intact tumor microenvironment.
    (2018-07-23) Martinek, Jan, 1986-; Palucka, Karolina.
    The causes underlying the extent and character of tumor-associated immune responses in cancer are not well defined and are likely multifactorial including cancer cell heterogeneity, host genotype, and the immune status of individual patients. Tumors are complex and organized tissues that include multiple cell types, which together compose the tumor microenvironment (TME). The myeloid cells play a major role in TME and can be composed of heterogeneous cells with functions that can be grossly summarized as: (1) Antigen capture for presentation (dendritic cells, DCs) or for degradation (macrophages); (2) Tissue repair (macrophages) and (3) effector function (mast cells, monocytes, granulocytes). However, the functional status of myeloid cells in human tumors and variation between tumors and patients is not completely understood. This is an important gap in knowledge that needs to be addressed because myeloid antigen presenting cells (mAPCs) control cancer antigen presentation to T cells thereby launching and regulating anti-cancer immunity. Our studies focused therefore on two key approaches necessary to improve our understanding of myeloid cells in TME: 1. Analysis of myeloid cells in situ in tumors. To this end, we developed and applied a microscopy-based approach for quantitative and qualitative mapping of non-dissociated tumors. Indeed, current methods are based on tissue dissociation into single cell suspension, which is associated with cell loss and activation, possibly impacting observed phenotypes. This was combined with laser capture microdissection to lift the cells and transcriptional profiling of APCs based on their tissue location and antigen content; and 2. Humanized mouse models recapitulating human TME. Indeed, while syngeneic and genetically modified mouse models enable in vivo studies of the TME, substantial differences exist between human and mouse immune systems, possibly impacting translation of pre-clinical studies to the clinic. Therefore, we studied human myeloid cells in novel humanized mouse models that support the tumor progression and metastatic spread of human melanoma. Our results show that location of myeloid cells within the tissue, as well as antigen cargo, have a significant impact on cell’s transcriptomic profile and potentially function. Thus, our approach developed in the course of studies discussed herein might bring a new resolution to unraveling the biology of APCs within the TME. This in turn could have an impact beyond melanoma. Furthermore, the new humanized mouse models that we have studied bring the in vivo proof that human myeloid cells and macrophages contribute to tumor development and metastatic colonization.
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    Electrostatic control of Cu, Zn superoxide dismutase aggregation in Amyotrophic Lateral Sclerosis : from lysine modification to interaction with lipid membranes.
    (2018-07-20) Rasouli, Sanaz, 1982-; Shaw, Bryan Francis, 1976-
    Cu, Zn superoxide dismutase (SOD1) is a ubiquitous metalloprotein, which is responsible for protecting living cells from oxidative stress via disproportionation of superoxide ions. Misfolding and subsequent aggregation of SOD1 is casually linked to familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Symptoms of ALS included weakness in arms and legs and respiratory failure, which leads to death within a few years. The deposition of SOD1 amyloid fibrils (i.e., gain of toxic function) in axons leads to selective death of motor neurons and neurodegeneration. In this dissertation, I use various bioanalytical and biophysical techniques to study aggregation of wild-type (WT) and ALS-variant apo-SOD1 in both in vitro and in vivo conditions, in the context of ALS. In Chapter Two of this dissertation, I first show how acylation of lysine residues in WT apo-SOD1 affects the kinetics and morphology of amyloid formation during microplate-based (in vitro) assays. Moreover, the ability of WT SOD1 fibrils to seed the aggregation of ALS-variant SOD1 in organotypic spinal cord derived from transgenic YFP-G85R-SOD1 mice is studied. I show, for the first time, that minimal acylation of apo-SOD1 with small moieties carrying a high negative charge increases the probability of formation of non-toxic SOD1 oligomers. The results from this study are crucial for designing small molecules that can selectively bind to and inhibit the propagation of toxic SOD1 fibrils. In Chapter Three of this dissertation, I determine the rate, mechanism of formation, and morphology of aggregated forms of WT apo-SOD1 in the presence of small unilamellar vesicles (SUVs) composed of identical hydrophobic chains, but headgroups of variable charge i.e., anionic, zwitterionic, and cationic. The aggregation of eight different ALS variants of apo-SOD1 was also examined in the presence of SUVs. I found that SOD1 aggregation can be triggered by negatively-charged liposomes, which is very significant considering that cell-to-cell propagation of SOD1 is mediated by its interaction with lipid membranes.
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    Mechanism of C-type lectin receptor Dectin-1 and DC-ASGPR-mediated immune modulation.
    (2018-03-23) Gu, Chao, 1987-; Oh, SangKon.
    Dendritic cells (DCs) are major antigen-presenting cells (APCs) and play a critical role in directing host immune responses towards either immunity or tolerance. C-type lectin receptors (CLRs) expressed on DCs not only facilitate antigen capture and uptake for presentation, but also deliver diverse intracellular signals that modulate DC functions and result in altered immune responses. Among numerous CLRs, Dectin-1 is critical to induce both Th1 and Th17 cell responses that are essential to host immune defense against fungi and mycobacteria whereas DC-ASGPR modulates immune responses by promoting antigen-specific regulatory T cells. Herein, we report that activation of CD11c+ myeloid DCs via Dectin-1 significantly downregulates TSLP-induced inflammatory Th2 cell responses by (1) subverting the Th2-permissive microenvironment via IL-10, (2) suppressing OX40L expression by downregulating the transcriptional activity of p50-RelB heterodimer and (3) decreasing Th2 cell-attracting chemokine CCL17 secretion. In addition, we dissected DC-ASGPR-mediated signaling pathway and found that DC-ASGPR ligation by specific monoclonal antibody (mAb) induces Syk activation. Similar to Dectin-1, engagement of PLCγ2 and PKCδ is conserved downstream of Syk activation upon DC-ASGPR triggering. Unexpectedly, however, DC-ASGPR ligation by mAb does not induce NF-κB activation. Instead, it selectively activates MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 leads to activation of p90RSK and CREB, promoting IL-10 expression in DCs. Moreover, DC-ASGPR ligation activates PI3K-Akt pathway, which differentially regulates the activities of GSK-3α/β and β-Catenin for cytokine expression. Our results provide a molecular explanation for the ability of DC-ASGPR-interacting ligands to preferentially evoke immune modulation. Data from this study support that both Dectin-1 and DC-ASGPR represent promising targets that may allow us to control host immune responses, particularly inflammatory responses.
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    Machine learning-assisted prediction of structure and function of cystine-stabilized peptides and optimization of expression in an E. coli system.
    (2018-03-22) Islam, S. M. Ashiqul, 1988-; Kearney, Christopher Michel, 1958-
    Cystine-stabilized peptides are promising prospects for the pharmaceutical industry as biologics. These peptides carry out a variety of useful functions which could be exploited to treat diseases and kill unwanted organisms. As well, an array of disulfide bonds makes the peptides highly stable against temperature, enzymatic degradation, pH and other adverse physiological conditions. There is a vast number of cystine-stabilized peptides serving as antimicrobial peptides, immunological modulators, ion channel blockers and other functions across a wide array of taxa, from fungi and bacteria to plants and humans. Practical access to these promising bioactive molecules could be greatly accelerated if it were possible to efficiently mine cystine-stabilized peptide sequences from genomic databases, determine the function and structure of each candidate from only the primary sequence, and then express the top candidates in E. coli for biological analysis. In this way, only the natural, presumably functional, variants of a particular family of cystine-stabilized peptides could be collected in large quantities. Going further, it would be desirable to convert the nonspecific activity of antimicrobial peptides to a specific activity, targeting a specific pathogen and leaving the rest of the microbiome intact; in essence, developing a targeted antibiotic. To contribute to developing this pipeline, I developed the machine learningassisted algorithms PredSTP and CSPred to predict structural and functional characteristics, respectively, of cystine-stabilized peptides from primary sequence data. In addition, I developed an E. coli-based expression system for high yield production of recombinant antimicrobial peptides specifically targeted to Staphylococcus aureus. These techniques are now available to collect large libraries of cysteine-stabilized peptide sequences, to express top candidates in E. coli, and to target the peptides to specific pathogens.
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    The chromatin accessibility signature of aging in human blood leukocytes stem from CD8+ T cells.
    (2017-07-26) Chung, Cheng-Han, 1982-; Banchereau, Jacques.
    Human aging is linked to changes in immune function that contribute to decreased responses to pathogens and increased systemic inflammation. Human aging is also associated with profound epigenetic changes across cell types and tissues. How these changes affect the aging –associated decline of the immune system is unknown. The Assay for Transposase Accessible Chromatin with sequencing technology (ATAC-seq) allowed us to study, at a system biology level, the open chromatin landscapes of human peripheral blood mononuclear cells (PBMCs), monocytes, purified B and T cell subsets from healthy young and healthy elderly individuals. We captured aging-associated epigenomic remodeling in PBMCs consisting of (1) systematic chromatin closing at promoters and enhancers targeting the T cell signaling and development and (2) chromatin opening, mostly at quiescent and repressed sites associated with cytotoxicity. Transcriptome profiling of the same individuals revealed gene expression changes concordant with epigenomic changes. Analysis of naïve and memory CD4+ and CD8+ T cell subsets demonstrated that the epigenomic signature of aging in PBMCs arises mostly from memory CD8+ T cells, indicating that aging differentially affects T cell epigenomes in a subset-specific manner. This study provides the first systems-level description of chromatin accessibility changes associated with immune aging in human PBMCs and T cell subsets. It revealed in PBMCs significant chromatin closing at promoters and enhancers, including at the IL7R locus and the IL-7 signaling pathway. Our study revealed individual-level variability in aging-associated chromatin remodeling and provided a systematic and modular tool for assessing deviations from chronological age. The open chromatin profiling of sorted T cell subsets, concluded that the chromatin “aging signature” captured in PBMCs, mostly stems from memory CD8+ T cells. The combined ATAC-seq/RNA-seq analyses uncovered epigenetic changes poised for expression changes and active noncoding elements (e.g., enhancers), both of which will be essential for understanding the regulatory mechanisms underlying immunosenescence. Nevertheless, ATAC-seq based open chromatin profiling is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. In our lab, ATAC-seq is utilized to understand the epigenetics differences in different immune cells and diseases.
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    Evolution to pollution in Gulf killifish (Fundulus grandis) from Galveston Bay, TX, USA.
    (2017-07-27) Oziolor, Elias M., 1990-; Matson, Cole W.
    Anthropogenic contamination associated with industrial activity is a widespread and active threat to the stability of organisms. The Houston Ship Channel (HSC) is one example of a heavily impacted environment, where industrial activity has contributed to extreme levels of pollution with various classes of contaminants, such as polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). This dissertation studies the impacts of chronic multi-generational exposure to industrial contamination on the population structure, resistance and demography in a keystone coastal species – Gulf killifish (Fundulus grandis). We have characterized their sensitivity to contaminants in populations from 12 locations across Galveston Bay, as well as the contamination levels at those sites. We found a gradient of resistance that was positively correlated with contaminant concentrations. This resistance was also correlated to a suppression of the aryl hydrocarbon receptor pathway (AHR), as estimated via the activity of a down-stream regulated enzyme – cytochrome P450 1A (CYP1A). To better understand the impacts of this adaptation, we evaluated the cross-resistance and fitness cost of populations to mechanistically and environmentally relevant stressors, but we were unable to confirm any fitness costs. We showed that the heritability of this resistance is biparental and multi-generational, which suggest that this is a genetic trait. Finally, we performed a full genome resequencing of seven populations along this gradient of resistance and discovered that genomic regions under selection in adapted populations included the AHR pathway. To determine if regulatory benchmarks on the compounds driving this adaptation would protect from such events, we performed a meta-analysis of all evolutionary events to contamination with PCBs and PAHs and found several locations, in which populations have adapted at values below regulation. Here we show that Gulf killifish populations have undergone a rapid evolutionary adaptation to a gradient of anthropogenic contaminants in Galveston Bay. In addition, we suggest that evolutionary toxicology studies, as described here, can be informative for regulatory purposes for compounds that may drive population-wide change.
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    Preclinical characterization of small molecule compounds as therapeutics for cancer and Chagas’ disease : tumor vascular disrupting agents and cysteine protease inhibitors.
    (2017-03-31) Odutola, Samuel O., 1986-; Trawick, Mary Lynn.
    The purpose of this study was the pre-clinical exploration of specific small molecule compounds as vascular disrupting agents and cysteine protease inhibitors, targeted towards the development of therapeutic agents for the treatment of cancer and Chaga's disease. Disruption of tumor vasculature is a selective approach to cancer therapy that results in tremendous tumor necrosis while leaving normal blood vessels relatively unaffected. The first part of this study describes an examination of the vascular disrupting ability and the mechanism of action of the indole-based tubulin-binding compound, OXi8006, and its water-soluble phosphate prodrug OXi8007. Treatment of rapidly proliferating human umbilical vein endothelial cells, used as a model for the tumor vasculature, with OXi8006 caused a dramatic disruption of the microtubule network and subsequent increase in cell contractility as observed from increased actin stress fiber and focal adhesion formation. The induced signaling cascade included increased phosphorylation of myosin light chain and focal adhesion kinase, and activation of the LIM kinase-cofilin pathway. It was demonstrated that these events were mediated by the intracellular G protein switch RhoA and its downstream target RhoA kinase, via the microtubule-binding guanine nucleotide exchange factor GEF-H1. A separate research project focused on the evaluation of synthetic thiosemicarbazone inhibitors of cysteine proteases as anticancer agents. Cathepsins L, K, and B are cysteine proteases that are implicated in tumor growth and metastasis. In addition, the cysteine protease cruzain, an essential enzyme in the life cycle of parasite Trypanosoma cruzi, is a validated therapeutic target for Chagas' disease. In this project, preliminary in vitro analysis of a group of synthetic thiosemicarbazones were carried out in order to characterize their potency and mode of inhibition against these enzymes. A number of compounds were found to be potent inhibitors of each enzyme. Results demonstrated that the most potent inhibitors in this library have slow binding, slowly reversible, competitive mechanisms of inhibition. Furthermore, results suggested that the best thiosemicarbazone inhibitors form a reversible covalent bond with each enzyme. Cathepsin L inhibitors were able to delay cell invasion of the MDA-MB-231 breast cancer cell line in a concentration dependent manner.
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    Immune profiles of allergic asthma patients treated with anti-IgE.
    (2016-12-01) Upchurch, Katherine C., 1987-; Oh, SangKon.
    Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyper-reactivity, mucus overproduction and airway remodeling and narrowing. Of the various phenotypes of asthma, by far the most common is allergic asthma, in which the airway inflammation is triggered by allergen exposure in sensitized individuals. The diagnosis of allergic asthma is usually done through an allergen prick test; high allergen reactivity is correlated with allergy and these patients should also contain allergen-specific IgE antibodies (abs). One of the more effective medications for moderate-to-severe, uncontrollable allergic asthma is omalizumab, an anti-IgE ab that targets free IgE, thus preventing the continuation of IgE-dependent allergic responses. However, the mechanisms behind how anti-IgE treatment influences the pathophysiologic responses remain to be fully revealed. Additionally, only a 21-64% response rate is seen after 16 weeks, despite the underlying allergy pathogenesis. Thus, in order to better understand the role of IgE in the pathogenesis of human allergic asthma and to identify potential biomarkers for response to anti-IgE therapy, we studied the mechanisms of action of anti-IgE abs in allergic asthma patients through the comparison of immune cell composition and activation status and whole blood transcriptional profiling. By acquiring patient samples before the start of anti-IgE treatment, we were also able to compare healthy donors with allergic asthma patients to gleam additional insights into allergic asthma. This study offers a unique global examination on the impacts of anti-IgE on the immune response, as observed in peripheral blood and also gains a further step towards the development of a biomarker for response to anti-IgE treatment.