UTMB Health SHARED (Univ. of Texas Medical Branch at Galveston)
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Innate immunity in Zika Virus infection and neurogenesis deficits
No full textZika virus (ZIKV) outbreaks and their strong link to microcephaly have raised public health concerns globally. Clinical and animal studies have shown that miscarriage and brain malformation are more frequent when infection occurs during early pregnancy. ZIKV-associated microcephaly is most likely due to the high susceptibility of neural stem/progenitor cells (NS/PCs), the origin of the brain, to ZIKV. Microglia are the first and major innate immune defenders of the brain. They arise exclusively from the progenitor cells in the embryonic yolk sac (YS) and migrate to the fetal brain during a restricted time window. Microglia from the brain are able to transmit the virus to NS/PCs and induce apoptosis in vitro. Toll-like-Receptor 3 (TLR3) is an important innate immune receptor responsible for the initial recognition of ZIKV. ZIKV depletes NS/PCs through TLR3 activation, and impedes neuronal differentiation in a cell-strain-dependent manner, which is well correlated with the upregulation of the innate immune genes transcription profile. Although significant progresses have been made in the past years, it remains largely unknown how ZIKV transports from the mother to the fetal brain, and how ZIKV targets NS/PCs to cause neurogenesis reduction. In this project, I developed a mouse model to explore the role of microglia in ZIKV dissemination to the fetal brain, and used human NS/PC (hNS/PC) lines to determine the role of innate immune genes in neurogenesis reduction after ZIKV infection. In this dissertation, I will briefly discuss ZIKV and the innate immunity in ZIKV infection. Next, I will describe my findings that show 1) YS-derived microglial progenitors can act as “Trojan horses” to bring ZIKV from the mother to the fetal brain in a restricted time window, ablation of microglial progenitors reduced the viral load in embryonic mouse brains; and 2) ZIKV infects NS/PCs and overactivates innate immune responses to inhibit neurogenesis, whereas experimentally inhibiting the overactivated innate immune responses ameliorates neurogenesis deficits. Finally, I will summarize and discuss the data integrating the role of innate immunity in ZIKV infection and neurogenesis abnormalities
Psychosocial Work Environment Stressors, Health, and Health Disparities: Findings from the 2002 to 2014 General Social Surveys
No full textIn the U.S. poorly understood changing patterns of work limit our ability to determine if the prevalence of factors threatening worker safety and health are changing as well as shifting in their distribution across occupations and worker demographics such as sex and race/ethnicity. The National Institute for Occupational Health & Safety (NIOSH) recognizes the burden job stress imposes across several aspects of well-being, increased healthcare usage, and lost productivity. Concomitantly, the U.S. is relatively unusual among industrialized countries in that it reports the health status of its population based on race/ethnicity while most other countries focus on social class differences. One of the overarching goals of the U.S. Department of Health and Human Services (DHHS) Healthy People initiatives has been addressing health disparities; for 2020, it aimed to achieve health equity, eliminate disparities, and improve the health of all groups. Health disparities have been shown to result from of a variety of causes, including those attributable to a society’s values and attitudes, its legal and political systems, and social institutions. Unfortunately, few U.S. researchers utilize information on works role in the existence or perpetuation of health disparities. This is somewhat due to the data challenges faced by researchers, at least relative to the data collected by many European nations. The General Social Survey (GSS) NIOSH Quality of Worklife (QWL) data enables us to investigate and contribute new information to both NIOSH and DHHS research priorities. Using the 2002, 2006, 2010, and 2014 GSS QWL data, pursuing this dissertation’s aims produced the following findings (a) psychosocial work stressor exposures were consistent in the years studied and associated with respondents’ occupation, (b) respondents’ sex was not associated with work stressor exposures while non-Hispanic blacks reported higher levels than non-Hispanic whites, (c) increasing work stressor exposure is associated with poorer mental and physical and self-rated health, and (d) work stressor exposure was not a factor in mediating sex or race/ethnicity health disparities. These results contribute evidence pertaining to priority research areas of multiple U.S. government agencies and suggest the need for continued examination of the impact of psychosocial work environment factors on health
Neuroplasticity, Neurorehabilitation, and New Hope for Brain-Injured Persons
No full textIn the last twenty-five years, treatment for Traumatic Brain Injury (TBI) has stagnated, which has relegated many TBI survivors to permanent disability or premature death. The reasons for this stagnation are arguably historical. In the mid nineteenth century, Paul Pierre Broca established localization theory, which holds that certain cognitive functions are located within specific areas of the brain. The cultural adoption of localization theory ultimately led to brain mapping in the late nineteenth and early twentieth century, still in use today, which attempts to physically map the purported area of the brain where certain cognitive functions occur. In the twentieth century, a nihilistic attitude developed among physicians concerning the possibility of successful treatment of TBI, which is chronicled in first person TBI patient narratives analyzed in this dissertation. Therapeutic nihilism toward TBI continued and was exacerbated when the evidence-based medicine paradigm was developed and culturally adopted by the medical establishment and clinical researchers in the 1990s. In 2017, the Lancet Neurology Commission argued that the culture of evidence-based medicine and a lack of recognition of the complexity and heterogeneity of TBI by medical professionals and researchers has led to the stagnation of TBI treatment in recent decades. The Commission has advocated for a precision-based medicine (individualized) and multidisciplinary approach to TBI treatment. In the last half of the twentieth century, evidence began to emerge validating neuroplasticity, the idea that the human brain can heal after insult or injury, and treatments can now be individualized based upon neuroplastic principles that provide hope for the brain injured
Functionally selective receptor signaling and endocytosis by dopamine D1 receptor agonists
No full textThe dopamine D1 receptor (D1R) is a G protein-coupled receptor that signals through Gs and Golf G proteins. D1R agonists increase cAMP production and recruit β-arrestin to the plasma membrane, which may lead to receptor desensitization and/or induce D1R endocytosis. In addition, the D1R is essential for voluntary movement, cognition and reward processes. As such, the D1R is a validated drug target for a multitude of neurological disorders including Parkinson’s disease and neuropsychiatric disorders. Furthermore, D1R functionally selective agonists, which activate one D1R signaling pathway over another have been recently described. Functionally selective agonists, also known as biased agonists, may fine-tune receptor signaling to improve therapeutic efficacy and/or reduce adverse effects. However, the mechanism-of-action for D1R biased agonists is not clearly defined and the function of Gs/olf and β-arrestins in D1R signaling and biased agonism requires further investigation. This research project tests the hypotheses that β-arrestins facilitate agonist-induced D1R endocytosis and that D1R agonists will induce receptor endocytosis based on their ability to recruit β-arrestins. In Aim 1 of this project, the fundamental function of D1R Gs/olf and D1R β-arrestin signaling are examined using CRISPR/Cas9 genome editing to delete either Gs/olf or β-arrrestin1/2 in cells. These studies determined that Gs/olf are essential for D1R cAMP signaling but these G proteins do not have a role in agonist-induced D1R endocytosis. In contrast, this project demonstrates that β-arrestin1/2 are important for agonist-induced D1R endocytosis and this pathway contributes to D1R desensitization. Aim 2 of this project examined the pharmacology of existing and recently discovered D1R agonists and fully defined their signaling via Gs/olf/cAMP, β-arrrestin recruitment and D1R endocytosis. Structurally distinct catechol and non-catechol D1R agonists induced G protein biased signaling and had reduced or absent recruitment of β-arrestins. Interestingly, A-77636 is a β-arrestin super-agonist. Additionally, biased agonism influences D1R endocytosis such that G protein biased agonists do not induce D1R endocytosis. Together, these studies indicate that β-arrestin is important for agonist-induced D1R endocytosis. Moreover, biased agonists that do not recruit β-arrestin also do not induce D1R endocytosis. This dissertation demonstrates a clear function for β-arrestin in D1R endocytosis and extends these findings to biased D1R agonists. The mechanism-of-action for the G protein biased D1R agonists includes reduced β-arrestin recruitment and subsequent D1R endocytosis. The range of functionally selective D1R agonists characterized here provide valuable tools to investigate D1R signaling, trafficking and therapeutic potential
Regulation of signaling and function of the voltage-gated sodium channel complex by protein:protein interactions
No full textAs fundamental determinants of neuronal function, voltage-gated Na+ (Nav) channels are important targets for therapeutic development against a wide range of health conditions. Dysfunction of Nav channels in the CNS is associated with disorders ranging from neurological (i.e., epilepsy, neurodegeneration) to psychiatric (i.e., major depression disorder, schizophrenia). Unfortunately, commercially available drugs targeting Nav channels are directed against highly conserved domains across Nav isoforms, giving rise to severe side effects such as cardiotoxicity and movement disorders. Thus, there is an unmet need for discovering new probes and pathways that regulate Nav channels that could potentially help designing new medications.
Recent evidence suggests that protein:protein interactions (PPI) between Nav channels and their accessory proteins play a key role in regulating neuronal firing, and that minimal disturbances to these tightly controlled PPI can lead to persistent maladaptive plasticity. These PPI interfaces are highly specific and provide ideal targets for drug development, especially in the CNS where selectivity and specificity are vital for limiting side effects. However, for the most part how these protein:channel interactions are regulated in the cell is still poorly understood, and methods for assessing these interactions are lacking. Therefore, the goal of the present study was to develop robust assays to reconstitute the Nav channel complex in cells and identify cellular pathways and small molecules regulating PPI interfaces with the Nav channel complex.
Specifically, we focused on the PPI between Nav1.6 and its regulatory protein, fibroblast growth factor 14 (FGF14). Using a newly developed assay we screen cellular pathways followed by biophysical validation, we discovered a mechanism by which the JAK2 tyrosine kinase might directly influence neuronal firing through phosphorylation of FGF14. Furthermore, we conducted a high-throughput screening of ~45,000 small molecules and identified potent modulators of the FGF14:Nav1.6 complex that are functionally active and predicted to be permeable to the blood-brain barrier. While providing a robust in-cell screening platform that can be adapted to search for any channelopathy-associated regulatory protein, these results lay the potential groundwork for a new class of drugs targeting Nav channels with a broad range of applicability for CNS disorders
Spike Processing and Protease Usage Effects on SARS-CoV-2 Pathogenesis
No full textThe emergence of SARS-CoV-2 and its subsequent variants have ignited a multitude of studies on the spike protein and its domains. While most studies focus on the impact of receptor binding domain changes, mutations in the C-terminus of S1 (CTS1) have largely been overlooked. The SARS-CoV-2 CTS1 contains various features that are unusual to sarbecoviruses – the furin cleavage site (FCS) and the QTQTN motif. In this dissertation, we demonstrate that the CTS1 is more complex than previously thought. We have established the aspects of cleavage site efficiency, differential protease usage, loop length, and post translational modifications that all contribute to SARS-CoV-2 pathogenesis. Using our reverse genetics system, we generated several infectious clones to examine the various features of the CTS1: ΔQTQTN demonstrated the importance of loop length and glycosylation. PQQA established that the integrity of the FCS is necessary. Omicron CTS1 mutants YKH (H655Y, N679K, and P681H) and N679K demonstrated the interplay between mutations with different contributions and expanded on the impact of post translational modifications. These studies show that alterations to any of these aspects greatly affect the pathogenicity of the virus. Together, we demonstrate that the mutations in the CTS1 are key determinants of coronavirus pathogenicity and should be especially considered in surveillance for the next coronavirus outbreak
HCV Core Protein affects Lipid Metabolism in a Genotype-Dependent Manner
No full textChronic HCV infection is the leading cause of steatosis (fatty liver disease) and hepatocellular carcinoma (HCC). The virus establishes a chronic infection in 70% of patients and infects approximately 71 million people worldwide. Genotypes (gt) 1 and 3 are the most prevalent, with gt3 HCV being associated with more severe disease. It is known that core protein plays a role in the development of steatosis, but the precise mechanism is not yet understood. In this study, we investigate the role of genotypes in core-mediated differential regulation of lipid metabolism. Prior studies described the lipid induction by HCV infection or ectopic expression of core derived from gt1a and 3a HCV qualitatively. The goal of this thesis is to define the gt1a and 3a HCV core-mediated lipid regulation in a quantitative manner. Using FACS analysis, and immunofluorescence analysis by using confocal microscope, we found that gt3a core protein induces larger lipid droplet formation, although the quantity of lipids remains similar to that induced by gt1a core protein. We then attempted to determine the difference in fatty liver-associated gene expression levels induced by gt1a and gt3a core proteins. To do this, we utilized microarray to analyze the gene expression in cells transfected with different HCV core proteins. The analysis showed that both gt1a and gt3a core upregulated SOCS3; this upregulation has been shown to influence the liver response to previously used antiviral therapies, as well as the induction of insulin resistance. Additionally, genes that were downregulated by gt1a and gt3a core are largely associated with insulin resistance, which plays a role in the development of diabetes. Interestingly, more genes appear to be downregulated by gt3a than gt1a core protein, which could suggest that gt3a core protein induces insulin resistance and steatosis in a different manner, or to a more severe degree. Overall, these results present a potential mechanism for the relationship between insulin resistance and lipid metabolism deregulation induced by gt1a and gt3a core proteins
Structural Basis For Poliovirus Cloverleaf-Mediated Regulation Of Genome Replication
No full textPoliovirus is a member of the Enterovirus genus, members of which are characterized by small, non-enveloped capsids containing short, positive-sense single-stranded RNA genomes. During the viral replication cycle, it is critical that the virus generates the appropriate amounts of viral proteins and genomic RNA. Upon entry in the cell, the viral genome is first used as a template for translation of viral genes and then as a template for genome replication. Regulation of these two processes is mediated by a series of six stem-loops in the 3’ untranslated region of the genomic RNA. Stem-loops II-VI comprise an internal ribosomal entry sequence (IRES), which makes cap-independent translation possible. Stem-loop I, also known as the cloverleaf, serves as a switch to shut off translation, and promote negative strand RNA synthesis. The cloverleaf has previously been shown to form a three-component complex with the viral protein product 3CD (a fusion of 3C protease and 3D RNA polymerase) and host-protein Poly-C binding Protein 2. The formation of this complex is critical for the initiation of negative-strand RNA synthesis. In order to investigate the function and role of the cloverleaf, we report the X-ray crystal structure of the poliovirus cloverleaf RNA. Additionally, we report binding studies and conceptual modeling to characterize the interaction between the cloverleaf RNA, 3CD and Poly-C Binding Protein 2
Description of interactions of Ebola virus with a putative reservoir species using next-generation sequencing
No full textEbola virus (EBOV) causes a severe, often fatal disease in humans and nonhuman primates. Recently, EBOV has caused two very large outbreaks, one of which is ongoing in the Democratic Republic of the Congo. Bats are the likely reservoir of EBOV, but little is known of their relationship with the virus. Next-generation sequencing has become an extremely powerful and flexible tool in virology over the past decade as new library preparation techniques have been developed that permit the selective sequencing of small RNAs, and the characterization of entire viral populations at incredible levels of detail. For this work, I exploited this technology to explore two aspects of the bat/virus nexus; namely the small RNA profile of infection, and the evolution of the virus in bat cells. The biology of the virus in human cells was used for comparison. Here I describe a new class of small noncoding RNAs produced by EBOV during infection of bat and human cells that resemble microRNAs, but are not associated with the microRNA machinery, and lack any discernable RNAi function. I also describe the evolution of EBOV in an experimental passage series in bat and human cells. This work led to the discovery of a potential role for host RNA editing enzymes in the evolution of EBOV in bats, and identified loci within the viral genome that appear to be associated with adaptation to human cells
Handwriting Lab: An Occupational Therapy-Led Program to Foster Teacher Readiness Toward Evidence-Based Practices
No full textBackground. The gap between evidence-based recommendations for handwriting instruction
and current classroom practices demands attention to the context of teachers as primary
stakeholders in successful translation of the vast knowledge on handwriting. Existing
handwriting programs are not feasible without consideration of the teacher implementing the
program. Methods. Kindergarten teachers participated in an 8-week handwriting lab aimed at
improving handwriting intervention. The teachers engaged with the occupational therapist (OT)
on campus in pre- and post- intervention knowledge- and perspective-sharing, including
coaching on ready-made materials. The teachers were then monitored weekly across the
implementation of a motor learning-based and a structured set of systematic daily steps for
teaching the lowercase letters. Data was also collected on changes in students’ letter
writing. Results. Two consenting kindergarten teachers completed the program. Teacher
outcomes indicated favorable results toward the success and feasibility of the program’s
methods. Teachers experienced positive changes in their commitment to evidence-based
handwriting instruction. Quantitative data on student outcomes also revealed positive changes
in students’ ability to write lowercase letters. Discussion. As seen through this program, the
lack of application of evidence-based knowledge on handwriting can be feasibly addressed by
supporting teacher readiness throughout the intervention. Conclusion. The specific teacher-focused
methods of the Handwriting Lab translated to positive changes for teachers and
students. Translation of knowledge on handwriting into the complex clinical arena of the school
setting is a critical area for the ongoing application of the holistic lens of occupational therapy