Theses/Dissertations - Biomedical Studies

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Advancing chemical and biologically-based “Read across” through in silico, in vitro and in vivo techniques.
(2014-09-05) Connors, Kristin A.; Brooks, Bryan W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Read across approaches include data leveraging across chemicals or among biological scales of organization or species, and may focus limited resources to support chemical hazard and risk assessment. Sustainable molecular design of less toxic industrial chemicals promises to preempt the production of organic contaminants with elevated toxicity. In chapter 2, probabilistic hazard assessment approaches were used to model potential reduction of aquatic toxicity hazards by using the ‘rule of two’ in the chemical design stage. Implementing this 'rule of two' was predicted to appreciably decrease chemicals designated of 'high' and 'very high' concern for standardized acute and chronic toxicity in common aquatic species. However, select modes of action and chemical classes will not have a reduced toxicity profile after following the 'rule of two.' Other read across techniques were examined to support chemical prioritization for safety assessments or to generate hypotheses for advanced research. Unlike most industrial chemicals, robust data exists for pharmaceutical physicochemical and biological properties, including human metabolism. Thus, it may be possible to employ existing mammalian pharmacological safety data to support screening-level bioaccumulation assessments. In chapter 3, a comparative pharmacology approach was used to determine whether rainbow trout biotransform pharmaceuticals known to be substrates for specific human CYPs. Only two general CYP substrates demonstrated measureable intrinsic clearance in vitro. No significant biotranformation was observed for substrates of human CYP2D6, CYP2C9, or CYP3A4. In chapter 4, enantiomer specific biotransformation profiles of chiral pharmaceuticals also deviated from human clearance rates. These observations demonstrate that relative clearance rates in trout are not predictable from human pharmacology data. Biological read across extrapolations were further explored in chapter 5 to characterize hazards of next generation therapeutics in zebrafish. Mechanistic and behavioral responses identified potential adverse outcomes from emerging antidepressants.
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.
Analysis of inflammatory changes in human pancreatic islet cells.
(2009-02-27) Jackson, Andrew M.; Naziruddin, Bashoo.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Pancreatic islet cell transplantation is a promising investigational research treatment for labile type 1 diabetes mellitus. However, several obstacles still exist to the implementation of clinical islet cell transplantation as a standard therapy. These obstacles include a shortage of donor pancreata, an imperfect islet isolation procedure, significant loss of islets during the peri-transplant period, and islet toxicity of immunosuppressive drugs. The present study is focused on addressing two of these major hurdles; namely, identification of factors affecting the islet isolation, and understanding the mechanism involved in the peri-transplant loss of islets. Initial analysis was centered on the effect of cold ischemia time (CIT) on the islet isolation outcome. Comparison of varying CIT and several factors that determine the quantity and quality of islets obtained from 52 isolations was performed. This analysis showed that CIT of less than four hours significantly improved islet isolation results. This finding could help improve the current strategy used in clinical islet transplantation. We hypothesized that following transplantation, the inflammatory environment will specifically alter gene expression in transplanted islets, and also will induce surface expression of HLA-class II molecules. This, in turn, could cause anti-donor response resulting in islet destruction. To test the above hypotheses, two studies were performed. Islet cells were treated with control or type 1 diabetic serum. Gene expression was then analyzed using micro array and confirmed by real-time PCR. Islets treated with diabetic serum demonstrated specific induction of multiple genes reported to have secondary roles in angiogenesis while inhibiting transcription of genes with protective attributes against environmental stresses. Islet cells were treated with IFNγ and TNFα and analyzed for HLA class II induction by real-time PCR analysis, flow cytometry, and immunofluorescent imaging.Cytokine treated islets demonstrated significantly upregulated HLA class II gene transcription and surface expression. Importantly, islet transplant recipient serum showed increased binding and cytotoxicity specifically directed against cytokine treated islets. Together these data suggest that in the context of hepatic portal vein transplantation, islet cells contribute to the innate and adaptive immune response during the peri-transplant period resulting in islet destruction.
Anti-CD47 targeting therapy for the treatment of colon and pancreas cancers.
(2016-03-10) Cha, Adriel C., 1985-; Weissman, Irving L.
CD47 is a “don’t eat me” signal over expressed on cancer cells inhibiting their engulfment by phagocytes. A novel monoclonal antibody, Hu5F9-G4, blocking CD47’s interaction with its ligand SIRPα allowed for phagocytosis of colorectal and pancreas tumors by macrophages. In some models, treatment of xenotransplanted tumors in immunodeficient mice with anti-CD47 antibodies strongly reduced tumor growth. In other models, the combination of anti-CD47 and anti-EGFR antibodies regressed tumor burden, where neither single agent therapy was as effective. Even in KRAS mutated tumors, where use of anti-EGFR antibodies is not indicated, the combination of antibodies lead to major tumor reduction. Taken collectively, Hu5F9-G4 is an effective treatment in colorectal and pancreas cancers, and in the combination of Hu5F9-G4 with an anti-EGFR antibody is therapeutically effective in KRAS mutated tumors in mouse xenografts. Cancer immunotherapy, including anti-CD47 blockade, tries to utilize the host’s own immune system to mount an immune response to cancer. To date, there have been a multitude of new therapies from cytokine treatments, to cell based vaccines, to antibody therapies which all recruit immune cells that have been somehow silenced by the tumor microenvironment. The Weissman lab’s own studies into the adaptive immune system suggest that upon phagocytosis, mediated by anti-CD47 antibodies, the subsequent T cell presentation preferentially recruits cytotoxic T cells. Here, we propose two additional model systems to further investigate the role CD47 plays in antigen presentation and downstream T cell recruitment to target cancer cells. Recent advances with immunotherapy agents for the treatment of cancer has provided remarkable, and in some cases, curative results. Blockade of the CD47:SIRPα axis between tumor cells and innate immune cells increases tumor cell phagocytosis in both solid tumors and hematological malignancies. These phagocytic innate cells are also professional antigen presenting cells, providing a link from innate to adaptive anti-tumor immunity. Preliminary studies have demonstrated that APCs present antigens from phagocytosed tumor cells, causing T cell activation. Therefore, agents that block CD47:SIRP-α engagement are attractive therapeutic targets as a monotherapy or in combination with additional immune modulating agents for activating anti-tumor T cells in vivo.
Anti-tumor activity of an oncolytic adenoviral construct expressing a small interfering RNA transgene.
(Philadelphia, PA : American Association for Cancer Research., 2006-10-01) Samuel, Shirley Kulangara.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Cancer is a leading cause of mortality in the world today. Mutation in the K-ras oncogene is common in most human cancers. K-ras oncogene expression was specifically downregulated by 58.7% by K-ras silencing siRNA, and this was accompanied by 66% growth inhibition of NCI-H441 lung cancer cells. To improve siRNA delivery and cause its stable expression in cancer cells, we used ONYX-411, an oncolytic adenovirus as a backbone to clone the K-ras silencing siRNA. This new adenoviral construct called Internavec, significantly downregulated mutant K-ras expression by 48.9% (p<0.05, n=3) as compared with 11.9% downregulation by parental ONYX-411 in HTB-79 pancreatic cancer cells. The anti-tumor activity of Internavec was examined in various cancer cell lines with and without the relevant K-ras mutation to observe the specificity of the siRNA transgene against the glycine to valine mutation on codon 12. Internavec showed enhanced anti-tumor activity in cell lines with the relevant mutation, compared with ONYX-411. Internavec (5 @ 1x108 pfu) significantly reduced the growth of subcutaneous HTB-79 pancreatic tumor xenografts in vivo by 85.5%, including complete growth suppression in 3 of 5 mice. Parental ONYX-411 or ONYX-411-siRNAGFP was markedly less effective (47.8% and 44.1% growth reduction, p<0.05, respectively). To characterize interferon-inducing activity of Internavec, landmark gene expression of the interferon pathway (OAS1, MX1) was examined following Internavec treatment, using HEK-293 cells as positive control. HEK-293 cells displayed an upregulation of OAS1 and MX1 following Poly (I:C) treatment. However, Internavec did not upregulate these interferon-pathway genes in HEK-293 or H79 lines, suggesting a lack of interferon activation by Internavec. To delineate underlying molecular events contributing to the enhanced growth inhibition, microarray experiments were performed on cells treated with Internavec. Internavec, but not ONYX-411, downregulated the expression of multiple Ras signaling-related genes (MAP4K5, PLCε1, IKBKB, FOXO3A and RAB28). These findings indicate that the knockdown of mutant K-ras serves to enhance oncolytic virus anti-tumor activity through the perturbation of additional cellular signaling events.
Application of genomics technology in the study of human disease.
(2015-07-16) Anguiano, Esperanza, 1969-; Pascual, Virginia.
Genomic technologies are helping advance our understanding of human diseases at a very fast pace, especially in the fields of cancer, auto inflammatory diseases as well as immunodeficiencies. High throughput technology permits to survey the entire genome and assess individuals genetic variation. This application has proven successful in identifying causal genes in Mendelian diseases and has also provided insight into the genetic etiology in complex diseases. In the past, we have applied applied blood transcriptional profiling to identify biomarkers and therapeutic targets such as IL-1B for patients with systemic onset juvenile idiopathic arthritis (sJIA), one of the major causes of chronic inflammatory arthritis in children. Indeed, clinical trials to evaluate the effect of blocking IL-1B in this disease have proven successful. This discovery supports that sJIA belongs to the recently described category of autoinflammatory diseases, which share similar clinical phenotypes and respond to IL1 blockade. Most autoinflammatory diseases have been described as Mendelian diseases affecting different genes in the IL-1B pathway. In the work presented here, we investigated a potential genetic basis for sJIA, which is a sporadic disease. We applied high throughput next generation sequencing technology to survey the known coding regions of the human genome, the exome, in 6 sJIA trios (individual patients and parents) and in an additional group of 11 patients. In the analysis of trios we identified several potentially pathogenic variants in genes involved in biological functions associated with IL1; we also found mutations in genes shared by two or more sJIA patients. In addition, a rare variant was found in the S100A12 gene, which encodes a biomarker and potential pathogenic factor in the disease, in one family. Overall, our study revealed potential genetic models of compound heterozygous and/or multigenic inheritance in this complex and heterogeneous disease.
Automated sequence homology : using empirical correlations to create graph-based networks for the elucidation of protein relationships.
(2008-10-02T18:47:42Z) Bush, Stephen J.; Baker, Erich J.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Identification of sequence homology has presented a formidable obstacle despite significant increases in both technological capability and detailed knowledge of genomes and proteomes. While PSI-BLAST remains the popular tool for the job, it often returns inaccurate results with unacceptable levels of false positives. In order to increase the sensitivity and accuracy of homology finding, we have developed a software application called Automated Sequence Homology that bypasses these shortcomings and provides reliable and precise results. The system presented here is based upon the creation of a graph-based network highlighting the relational connections between proteins using empirical correlations. It takes a step back from PSI-BLAST to the acclaimed BLAST algorithm to create a sampling of the protein relational network.
Comparative toxicity evaluations for polybrominated diphenyl ethers (PBDEs) : investigation of developmental toxicity, uptake and mechanisms of action in embryonic zebrafish (Danio rerio)
(2012-11-29) Usenko, Crystal Y.; Bruce, Erica Dawn.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Recently, polybrominated diphenyl ethers (PBDEs) were banned due to extensive environmental contamination and potential health effects. There is little understanding of the comparative toxicity of individual congeners. Seven congeners were selected over a wide-range of physical-chemical properties for investigation into relative toxicity, uptake, and mechanisms/modes of action. Based on the occurrences in environmental and human samples, BDE 28, 47, 99, 100, 153, 183, and 209 were chosen for evaluations, using embryonic zebrafish as the model organism. Exposure to PBDEs disrupted behavior during different windows of development, as well as induced curved body malformations and mortality over the exposure period from 6-168 hours post fertilization (hpf). There was a direct correlation between log Kow and the toxicity of the compounds as well as their subsequent uptake in embryos. In addition to uptake, metabolites and potential metabolic pathways were investigated. Several genes were assessed to elucidate the primary pathway of metabolism for different congeners. Several genes were significantly upregulated following exposure to most congeners (i.e. deiodinase 1 and 2, CYP1A1, CYP1B1, and CYP3A1), with the exception of BDE 153. Interestingly, BDE 153 also had the greatest amount of debromination. The analysis of phase II metabolism pathways demonstrated that UGT5g was significantly upregulated following exposure to all congeners. Two pathways were further investigated for BDE 28, 47, 99, and 100: oxidative stress and thyroid disruption. There was not a significant oxidative stress-related response for any of the treatments until 120 hpf, where we observed upregulation. We concluded that oxidative stress was not a primary pathway of toxicity for these congeners. Further analysis demonstrated that the adverse effects observed in zebrafish are most likely due to disruption of thyroid hormone homeostasis. In addition to metabolism, the toxicity of three hydroxylated metabolites was assessed, with very different responses to exposure than the parent congeners. Individual congeners did not appear to act through different mechanisms/modes of action; however the actual dose was a primary determinant in the toxicity of PBDEs.
Design, optimization, and evaluation of conditionally active gene therapy vectors.
(2008-06-09T11:51:54Z) Wood, David Rowe.; Ding, Jiahuan.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
It is the objective of this project to produce gene therapy vectors that are active and/or significantly up-regulated due to specific physiological conditions. The significance of such constructs is that it imparts a greater degree of control in the implementation of gene therapy. In general, it is desirable for a gene therapy vector to be active only when and where it is needed. The majority of gene therapy research to date has focused primarily on obtaining expression levels high enough to elicit a therapeutic response, as well as, distributing the vector to enough tissues to provide a corrective effect to the disorder being addressed. However, simply having a gene adequately delivered to enough cells to treat disease and having the gene product be produced in sufficient amounts to have a therapeutic effect cannot be the end of the story. Not unlike genes found naturally in the body, artificially delivered genes also need to be regulated. The construction of such vectors could prove useful for the treatment of disorders; such as Coronary Artery Disease (CAD) or even Cardiomyopathy, that occur in a specific tissue type or that are associated with an abnormal physiological state, such as hypoxia. Our vector constructs are a small step towards this ultimate goal. In this study, we present data on DNA vectors that were designed, constructed and evaluated in vitro and in vivo; both qualitatively and quantitatively. We report success in the creation of vectors/plasmids that are primarily cardiac tissue specific (pMHCI, pMHCII), vectors that are regulated by cellular oxygen levels (pHAL, pHAM), and even some success in combining the two (pHMHC).
Development of an aptamer-based detection assay against Bacillus anthracis lethal factor and Escherichia coli.
(2013-09-24) Lahousse, Mieke.; Kim, Sung-Kun, 1968-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
The impressive capacity of bacteria to adapt to their environment has led to the development of mechanisms that can convey resistance to currently FDA approved antibiotics. In an attempt to find a solution to the possibility that soon there will be no antibiotic treatment available for bacterial infections, new approaches are being explored. One such approach relies on the effective detection and/or diagnosis of pathogens or unique bacterial proteins to aid in prevention or better treatment of infectious diseases. Within this work we describe the development of ssDNA aptamers by SELEX that can bind to their target with high affinity and can be used to develop colorimetric assays that can positively detect the presence of the target. Our targets for aptamer development were whole E. coli cells, which are currently used in water quality assessment, and a unique protein toxin (lethal factor) produced by B. anthracis, the etiological agent of anthrax. As a result of the SELEX experiments we recovered one aptamer that binds to LF protein with high affinity and we demonstrated that its use in the development of an aptamer-based colorimetric assay for LF was successful. We also demonstrate that the aptamer found for LF protein can act as an inhibitor of the catalytic activity of LF, suggests its potential use as a therapeutic agent. The SELEX experiment using whole E. coli cells generated a diverse number of aptamers with binding affinities in the low µM range. Additionally, studies of aptamer specificity showed that three aptamers had no binding affinity for other coliform strains, making them suitable aptamers for further characterization. This work also describes the characterization of a glutaredoxin protein expressed by Synechocystis sp. involved in arsenate reduction. Elucidation of the mechanism by which cyanobacteria can survive in highly arsenic contaminated environments could enlighten possible remediation approaches. In conclusion, we successfully developed ssDNA aptamers that can bind with high affinity and specificity to its targets with demonstrated potential as detecting agents and contributed to the enrichment of the knowledge available for glutaredoxin GrxA involved in arsenate oxidoreduction by Synechocystis sp.
Development of dendritic cell-based vaccines for the treatment of HIV-infected patients.
(2011-09-14) Cobb, Amanda J., 1979-; Palucka, Karolina.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Dendritic cells (DCs) are professional antigen-presenting cells of the immune system with the ability to induce and control T and B cells responses. Monocytes have been used as precursors to generate DC vaccines ex vivo. Monocyte-derived DCs have been used as vaccines in clinical trials to treat cancer and infectious disease by eliciting potent T cell responses. Infection with Human Immunodeficiency Virus (HIV) almost invariably leads to a chronic disease. Eventually the destruction of the immune system culminates into Acquired Immune Deficiency Syndrome (AIDS). Since the advent of Highly Active Antiretroviral Therapy (HAART), HIV patients have been able to control viral levels. However, with prolonged use of treatment, these patients experience serious adverse effects such as liver and mitochondrial toxicity that can potentially become fatal. Long-Term Non-Progressors (LTNP) are a unique subpopulation of HIV patients that are able to control their viral load without HAART. Studies revealed HIV-specific polyfunctional CD8⁺ T cells to be vital for viral control. Thus, we have focused our efforts on devising therapeutic HIV DC vaccines to increase the quality of CD8⁺ T cell responses in chronic HIV infected patients. First, we conducted a series of preclinical development assays of GM-CSF/IFN-α and GM-CSF/IL-15 HIV DC vaccines loaded with a mixture of HIV-1-antigen lipopeptides (ANRS HIV-LIPO-5 vaccine), which is comprised of five HIV-1-antigen peptides (Gag₁₇₋₃₅, Gag₂₅₃₋₂₈₄, Nef₆₆₋₉₇, Nef₁₁₆₋₁₄₅, and Pol₃₂₅₋₃₅₅) each covalently linked to a palmitoyl-lysylamide moiety, and activated with LPS. Functional assays demonstrated that the DC vaccines expressed DC differentiation markers and displayed typical morphology which included the presence of dendrites important for cellular interaction. Potency assays proved the DC vaccines were capable of eliciting HIV-1-antigen-specific CD4⁺ and CD8⁺ T cell responses. Our studies have demonstrated the use of DC vaccines in eliciting HIV-specific CD8⁺ T cells responses in vitro. Furthermore, the pre-clinical development of the GM-CSF/IFN-α DC vaccine activated with LPS led to the evaluation of safety and immunogenicity in a Phase I/II clinical trial in chronically HIV-1-infected patients on HAART (clinicaltrials.gov identifier: NCT00796770).
The effect of dietary folate deficiency and age on methylation metabolites, neurotransmitters, and behavior in mice.
(2011-01-05T19:46:26Z) Wasek, Brandi Lynn.; Bottiglieri, Teodoro, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Severe folate deficiency caused by inborn errors of metabolism has profound deleterious effects in the CNS such as mental retardation, psychiatric disorders, seizures and myelopathy. Mild folate deficiency, due to dietary insufficiency, drugs, or a common mutation in the gene encoding methylenetetrahydrofolate reductase (MTHFR C677T), are associated with an increased risk for depression and dementia, especially in the aging population. The metabolism of folate is intimately linked with the synthesis of S-adenosylmethionine (SAM), the sole source of methyl groups required in methyltransferase reactions. S-adenosylhomocysteine (SAH), a product of methylation reactions, accumulates in folate deficiency due to increased conversion from homocysteine. Recent studies have linked hyperhomocysteinemia and hypomethylation to gene activity, as well as methylation dependent post-translational modification of proteins and neurotransmitter metabolism in depression and dementia. To better define the role of folate deficiency in CNS function, we fed young and old C57BL/6J mice folate deficient diets for 3 months and old heterozygous tg-MTHFR mice a low folate diet for 6 months. Mice were tested for grip strength, coordination, open field activity, and spatial memory. After treatment mice were sacrificed by CO2 asphyxiation or microwave radiation. Blood, peripheral and regional brain tissues were processed for the analysis of methylation and neurotransmitter metabolites. Age did not influence brain methylation metabolites in C57BL/6J mice. Low folate and folate deficient diets decreased the SAM/SAH ratio, an indicator of methylation status in most brain regions from C57BL/6J and heterozygous tg-MTHFR mice. Overall, the effects of folate deficiency were not exacerbated by age in C57BL/6J mice. Brain methylation metabolites differed significantly depending on the method of sacrifice. Specifically, folate deficiency decreased SAM in mice sacrificed by microwave radiation and increased SAH in mice sacrificed by asphyxiation. Folate deficiency reduced dopamine and serotonin turnover in several brain regions although the levels of the parent neurotransmitters were unaffected. Choline and acetylcholine levels were reduced by folate deficiency in the mid-brain. Folate deficiency impaired open field behavior, but did not have any significant effect on spatial memory. These studies help to further our understanding of the mechanisms involved in folate deficiency on CNS function in the aging brain.
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.
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.
Folding, dynamics and interaction studies of the Neuropeptide Y family.
(2011-12-19) Hegefeld, Wendy A.; Jas, Gouri Sankar.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Detailed molecular knowledge of the complex dynamics of biological processes such as folding and the interaction with cellular membranes may greatly advance the treatment of human disease. The Neuropeptide Y family of peptides are highly conserved peptides found in nature, consist of 36 amino acids, and are widely distributed in the central nervous system of mammals. A detailed study of thermodynamics, kinetics, membrane translocation, and receptor interaction of human neuropeptide Y (NPY) and the thermodynamics and detailed molecular modeling of human peptide YY (PYY) were investigated. These studies employed a combination of experiment and simulation to characterize the two most important members of the neuropeptide Y family. The formation of secondary structures is one of the most fundamental processes in protein folding. To obtain a detailed understanding of protein folding it is useful to study peptide models that provide well defined stable structures in solution. NPY and PYY are composed primarily of two important secondary structural elements: the α-helix and hairpin-like structure. Therefore, further analysis of the three most prevalent helical secondary structures found in nature (α-, 3₁₀-, π-helix) and the β-hairpin structure were carried out with carefully designed peptide models to characterize their individual structures and formation. These studies suggest two different mechanisms of formation for helical and β-hairpin structures.
Gamma Hydroxybutyrate (GHB): mechanisms of central nervous system toxicity.
(2006-07-31T01:14:52Z) Lyng, Eric E.; Bottiglieri, Teodoro, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Gamma Hydroxybutyrate (GHB) is an endogenous metabolite of gamma-aminobutyric acid (GABA) and a putative neurotransmitter found in mammalian brain. The illicit use of GHB is a growing health care concern in the U.S. Low doses have euphoric and stimulatory effects while high doses act as a CNS depressant and can cause respiratory failure. In addition to fatalities and drug facilitated rape, in 2004 over 7000 GHB overdoses were reported in the U.S. Gamma Butyrolactone (GBL) and 1,4-Butanediol (1,4-BD), precursors of GHB, can cause similar effects after being converted to GHB in the body. While GHB is a Schedule I compound, recently it was given a Schedule III classification as a drug for treatment of cataplexy associated with narcolepsy. Individuals affected by the inherited disorder succinic semialdehyde dehydrogenase (SSADH) deficiency have significant elevation of GABA and GHB in body tissues, and a range of neurological complications. Currently there is no treatment option for a GHB overdose situation or for SSADH patients. GHB has been shown to inhibit striatal dopamine release leading to sedation and loss of locomotor activity in rodents. However, GHB’s mechanism of action is poorly understood. This dissertation investigated acute and chronic effects of GBL, a precursor to GHB, on locomotor function and monoamine neurotransmitter metabolism in mice. Dose response studies were performed to characterize the effects of GBL. Compounds aimed at increasing central dopaminergic activity or antagonism of GABAergic activity were tested for their ability to antagonize the locomotor effects induced by GBL. In total, eight different compounds were studied of which pergolide and nomifensine were successful at antagonizing the loss of locomotor activity when administered either prior to or after GBL. Chronic administration of GBL over the course of 14 days was evaluated in mice using a rotarod system. These studies did not reveal any long term detrimental effect of GBL on locomotor function. This dissertation is the first report showing the ability of pergolide and nomifensine to antagonize the loss of locomotor activity induced by GBL. These studies provide insight into treatment options for GHB toxicity or overdose as well as for patients with SSADH deficiency.
Gene expression profiling to understand the alterations in the monocyte compartment of pediatric systemic lupus erythematosus.
(2008-06-11T17:41:33Z) Patel, Pinakeen Shankarbhai.; Pascual, Virginia.; Banchereau, Jacques.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Blood monocytes from SLE patients display DC function, as they are able to induce the proliferation of allogeneic T cells. Furthermore, sera from SLE patients induce healthy monocytes to differentiate into DCs. This DC-inducing property is in part due to the presence of type-I IFNs in SLE sera, as well as other, yet uncharacterized factors. To understand these alterations, we performed a thorough phenotypic analysis and gene expression profiling of monocytes from children with active, newly diagnosed and untreated disease. Phenotypic analysis of freshly isolated SLE blood monocytes revealed a modest expansion of CD14highCD16+ cells and an otherwise lack of expression of molecules related to DC function. Further characterization of a fraction of SLE monocytes inducing allogeneic T cell proliferation revealed that upon contact with T cells, SLE monocytes secrete proinflammatory cytokines such as IL-1 and IL-6 and do upregulate expression of innate immunity receptors involved in DC differentiation and molecules responsible for antigen presentation. To recapitulate the initial events leading to monocyte differentiation in this disease, we studied the effects of SLE serum on healthy monocyte at the trasncriptional and protein levels. These studies revealed the upregulation of expression on these cells of chemokine receptor such as CX3CR1 and CCR7, which may lead to the migration of blood monocytes to inflammed tissues and/or secondary lymphoid organs respectively in vivo. There, contact with T cells would lead to the acquisition of antigen presenting function and skewing from tolerogenic to immunogenic responses.
Growth inhibition of human multiple myeloma cells by a conditional-replicative, oncolytic adenovirus armed with the CD154 (CD40-ligand) transgene.
(2007-03-08T15:37:14Z) Rodrigues, Margret S.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Human multiple myeloma (MM) remains incurable despite successes in induction therapy. Based on our previous findings that treatment with the recombinant CD40L protein inhibited the growth of CD40+ human myeloma cell lines, we examined the targeting of the myeloma cell surface receptor, CD40 by its natural ligand, CD40L, using a gene transfer approach. For targeted delivery of CD40L, we have constructed a conditional-replicative adenoviral for delivery of the CD40L transgene (AdEHCD40L). AdEHCD40L incorporates tumor/tissue specific promoters limiting transgene expression and viral replication to HIF (hypoxia-inducing factor)-1α and/or estrogen overexpressing cancer cells. Conditional expression of the early adenoviral E1A gene and the CD40L transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.7% and 30%, respectively). Treatment with AdEHCD40L resulted in pronounced growth inhibition (RPMI 8226: 95.5 ± 2.1%; Kas-6/1: 80.5 ± 9.8%, mean ± SD), and was more effective than the parental construct without the CD40L transgene (AdEHNULL)(p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells and IMR-90, normal fibroblast cells (2.8 ± 0.3%). We also observed a 53% reduction in myeloma xenograft growth, following AdEHCD40L intratumoral injections (4.8 ± 0.9 mm, vs. 10.5 ± 1.2 mm in mock-treated animals; p=0.002), which was more effective than AdEHNULL (7.6 ± 1.1 mm; p=0.03). Evidence of adenoviral hexon and CD40L transgene expression within the treated tumors was demonstrated. In the study of cellular events leading to myeloma growth inhibition, we observed increased apoptotic activity at 72 hrs following treatment with AdEHCD40L infection (21.3 ± 6.5% vs. 7.7 ± 1.9% in untreated; p=0.007) which was accompanied by increased S phase accumulation (68.0 ± 2.4%, vs. 54.0 ± 5.5% in AdEHNULL and 48.9 ± 4.6% in untreated culture; p<0.003). High throughput gene array expression analysis identified pro-apoptotic TNF superfamily members, Fas and TRAIL and cytokines, IL-8 and RANTES to be uniquely upregulated by AdEHCD40L. These findings confirm the growth inhibitory activity of AdEHCD40L, through the induction of apoptosis and is a promising approach for the experimental gene therapy of human multiple myeloma.
Humanized mice to test vaccination against influenza virus via dendritic cells.
(2008-06-11T14:19:03Z) Yu, Chun-I, 1975-; Palucka, Karolina.; Banchereau, Jacques.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Critical to the development of human vaccines is the availability of in vivo models of the human immune system that permit testing of vaccine efficacy. Here,we used NOD-SCID-β²m-/- immunodeficient mice which, when engrafted with human CD34⁺ hematopoietic progenitors, develop all subsets of human dendritic cells(DCs) and B cells. T cells and their subsets were reconstituted by adoptive transfer. We found myeloid DCs, plasmacytoid DCs and monocytes in the bone marrow, spleen, and peripheral tissues including skin and lungs. To test DC biologyin vivo, we first used live influenza A/PR8/34 (H1N1) virus. Upon intranasal inoculation, all subsets of human antigen presenting cells were activated. Matured DCs were found accumulated in mediastinal lymph nodes. To evaluate the value of these mice for testing human vaccines, humanized mice were immunized with1) ex vivo-generated DCs, 2) seasonal influenza vaccines and 3) protein antigens fused to anti-DC receptor. Upon vaccination with ex vivo-generated DCs pulsed with heat-inactivated influenza virus, mice developed influenza-specific immunity, i.e. influenza-specific immunoglobulins (Igs) in the serum and influenza virus matrix protein 1 (FluM1)-specific CD8⁺ T cells in the blood, spleen and lungs. Influenza-specific Igs were protective as sera from vaccinated mice inhibited influenza virus-induced hemagglutination in vitro and offered passive protection in vivo. Upon vaccination with seasonal influenza vaccines, i.e. live attenuated trivalent vaccine (LAIV) or killed trivalent vaccine (TIV), humanized mice developed both humoral and cellular immunity. Plasma cells differentiation and the secretion of specific Igs were dependent on the reconstitution with CD45RA⁻CD27⁺CD4⁺ central memory T cells. CD8⁺ T cells specific to two influenza antigens, i.e. FluM1 and NS1, were detected in mice vaccinated with LAIV. TIV-vaccinated mice showed the expansion of FluM1, but not NS1, specific CD8⁺ T cells. Antigen-specific CD8⁺ T cells produced IFN-γ and expressed surface CD107a consistent with the acquisition of effector function. Finally, upon vaccination with anti-DC receptor (DCIR)-FluM1 fusion protein and poly I:C as an adjuvant, DCs efficiently cross-presented FluM1 and expanded antigen-specific CD8⁺T cells. Therefore, humanized mice might be valuable model for testing human vaccines against influenza virus.
Identification of dysregulated miRNA targets in human prostate cancer.
(2010-02-02T19:49:42Z) Chen, Po-hsun.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
MicroRNAs (miRNAs) are small, non-coding RNAs (18~24 nt) that regulate diverse cell functions in mammalian cells, including proliferation, differentiation, metabolism, stress resistance, and death. There is increasing evidence that altered expression and function of regulatory miRNAs contribute to the uncontrolled growth of human cancers. We examined the hypothesis that miRNA expression profiles can distinguish malignant from normal tissues. With the use of high throughput, liquid phase hybridization analyses (mirMASA) were carried out with 40 Stage 1c/2 prostate cancer specimens, using 114 miRNA-specific probes. We identified 5 miRNAs (miR -23b, -100, -145, -221, and -222) that were significantly downregulated in malignant prostate tissues as compared with their normal counterpart from the same patient. Decreased expression was confirmed by quantitative real-time PCR (qRT-PCR) analyses. The pathophysiological role of these downregulated miRNAs was further characterized in the prostate cancer LNCaP cell line, which exhibited similarly reduced expression of miR- 23b, -145, -221, and -222. Ectopic expression of mature miR-23b and miR-145 reduced LNCaP cell growth by >40%. In contrast, transfection with a miRNA (miR-141) that was upregulated did not markedly affect cancer cell growth. The altered expression of miR- 145 was of particular interest, in view of its similarly reduced expression in breast, cervical, colon, and lung carcinomas. In silico search (TargetScan) yielded 396 eligible targets for miR-145, of which 6 were markedly upregulated in LNCaP cells. These include the prostaglandin F receptor (PTGFR), transforming growth factor (TGFBR2), p21-activated kinase 7 (PAK7), SLIT-ROBO Rho GTPase activating protein 2 (SRGAP- 2), kinesin family member 3A (KIF3A), and Ras association (RalGDS/AF-6) domain family 2 (RASSF2). miR-145 was uniformly downregulated in all prostate caner lines tested (LNCaP, 22Rv1, PC-3, Du-145, as compared with the nonmalignant prostate line RWPE-1) This miRNA may be differentially expressed in androgen dependent (AD) and androgen independent (AI) prostate cancer cell lines according to qPCR analysis. Mean expression level of miR-145 was approximately 6-fold lower in AD (LNCaP, 22Rv1) than AI (PC3, Du-145) lines. qPCR analysis suggests that PTGFR and RASSF2 were upregulated in 3 of 4 prostate cancer cell lines, whereas MYCN was only upregulated in androgen independent cell lines. The expression of CCND2, MAP3K3 and MAP4K4 were not significantly upregulated by qPCR analysis. These findings suggest that altered miR-145 expression may impact prostate cancer growth through its loss of regulation of PTGFR and/or RASSF2 activities. qPCR analysis of miR-145 treated prostate cancer cells showed decreased expression of PTGFR and RASSF2 indicating these two genes are potential targets of miR-145 modulation. Direct binding only showed moderate reduction of RASSF2 and a more pronounced reduction of PTGFR. These studies suggest that miR-145 may modulate prostate cancer cell growth by modulating PTGFR activity.

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