Necroptosis is a pro-inflammatory mechanism of programmed cell death. It has been implicated in many diseases such as inflammatory diseases, neurodegenerative diseases, cancer and during viral infections. The focus of this research work was to establish the relationship between poxvirus pathogenesis and necroptosis, and the translation implications of necroptosis in oncolytic virotherapy. Vaccinia virus (VACV) is the currently used vaccine for smallpox and it has also been developed as a vaccine vector for several pathogens. E3L is one of the key innate immune evasion genes of VACV and it encodes E3 protein composed of dsRNA binding domain in the C-terminus and Z-NA-binding domain (Z-NA BD) in the N terminus. Both domains are necessary for type 1 interferon resistance and pathogenesis. Recently, it has been shown that in in vitro, the N-terminus of E3 is necessary to inhibit necroptosis occurring through the host-encoded cellular proteins RIP3 and Z-NA-binding protein DAI interaction leading to phosphorylation of MLKL, the key executioner step in the pathway. The research work presented here clearly demonstrates that in a mouse model, the N-terminus of VACV E3 is necessary to inhibit necroptosis during pathogenesis in mice. Another poxvirus belonging to the same family as VACV is monkeypox virus (MPXV) and is an emerging human pathogen. MPXV contains a natural truncation in the N-terminus of its E3 homologue, F3. The results indicate that during MPXV infection in mice, pathogenesis was higher only in DAI knockout mice and not in MLKL knockout mice, suggesting that DAI is possibly activating other proteins not leading to necroptosis. The characterization of VACV as an oncolytic virus was carried out with a focus on future clinical trials. In this study, a pan screening was conducted in various cancer cell lines as many cancers downregulate necroptotic proteins. The results reveal that the N-terminal deletion mutant of VACV selectively replicates in cancer cell lines with a deficient necroptotic pathway and thus, can be used as a potential treatment against specific tumors and evidently, provides abundant scope for future studies.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by out-of-frame mutations in the dystrophin gene, and the absence of a functional dystrophin protein ultimately leads to instability of the sarcolemma, skeletal muscle necrosis, and atrophy. While the structural changes that occur in dystrophic muscle are well characterized, resulting changes in muscle-specific gene expression that take place in dystrophin’s absence remain largely uncharacterized, as they are potentially obscured by the characteristic chronic inflammation in dystrophin deficient muscle.

The conservation of the dystrophin gene across metazoans suggests that both vertebrate and invertebrate model systems can provide valuable contributions to the understanding of DMD initiation and progression. Specifically, the invertebrate C. elegans possesses a dystrophin protein ortholog, dys-1, and a mild inflammatory response that is inactive in the muscle, allowing for the characterization of transcriptome rearrangements affecting disease progression independently of inflammation. Furthermore, C. elegans do not possess a satellite cell equivalent, meaning muscle regeneration does not occur. This makes C. elegans unique in that they allow for the study of dystrophin deficiencies without muscle regeneration that may obscure detection of subtle but consequential changes in gene expression.

I hypothesize that gaining a comprehensive definition of both the structural and signaling roles of dystrophin in C. elegans will improve the community’s understanding of the progression of DMD as a whole. To address this hypothesis, I have performed a phylogenetic analysis on the conservation of each member of the dystrophin associated protein complex (DAPC) across 10 species, established an in vivo system to identify muscle-specific changes in gene expression in the dystrophin-deficient C. elegans, and performed a functional analysis to test the biological significance of changes in gene expression identified in my sequencing results. The results from this study indicate that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract disease progression. Furthermore, these findings allow for the identification of transcriptome changes that potentially serve as both independent drivers of disease and potential therapeutic targets for the treatment of DMD.

Traumatic brain injury (TBI) consists of the primary mechanical forces to the head followed by secondary inflammatory cascades. This inflammatory cascade consists of neuroinflammation characterized by microglial activation as the first line of defense. Another component of secondary inflammation comprises of activation of peripheral immune cells that can infiltrate the compromised blood brain barrier and susceptible organs such as the lungs. Acute inflammatory processes in the lungs include a disruption of the epithelial barriers allowing infiltration of neutrophils, and edema build up in the alveoli. This is known as acute lung injury (ALI) and it dampens respiratory function in approximately 20-25% of TBI patients necessitating an intervention. Remote ischemic conditioning (RIC) is an intervention consisting of repeated intervals of cessation and reperfusion of blood flow to a distal limb and has treated ALI, myocardial infarction, and neurological injury. TBI was hypothesized to induce ALI through degradation of alveolar-capillary membrane and infiltration of peripheral leukocytes. Furthermore, RIC was hypothesized to protect the integrity of the alveolar-capillary membrane, reduce infiltration of peripheral immune cells, and reduce microglial activation in the brain through myokine recruitment. Male CD1 mice were subject to either midline fluid percussion or sham injury and further randomized into 4 groups: sham, sham RIC, TBI, TBI RIC. RIC was administered on proximal thigh for 4x5 minutes, with 5-minute reperfusion one hour prior to TBI. One-hour post-injury, brain, lung, BAL fluid, and blood were collected. Lung histopathology showed RIC reduced hydrostatic edema in the alveoli by protecting the alveolar capillary membrane. BAL findings revealed TBI mice had increased neutrophil counts, RIC lowered neutrophil counts. In the brain, RIC increased cortex microglial endpoints were observed with no other significant differences in microglial morphology as well as plasma myokine levels across all sham, sham RIC, TBI, and TBI RIC animals. While underlying mechanisms still have to be further studied, this current study provides evidence that RIC can be used as a therapeutic intervention to ameliorate TBI-induce ALI.

Immediate early genes (IEGs) are rapidly activated in response to an environmental stimulus, and most code for transcription factors that mediate processes of synaptic plasticity, learning, and memory. EGR3, an immediate early gene transcription factor, is a mediator of biological processes that are disrupted in patients with schizophrenia (SCZ). A microarray experiment conducted by our lab revealed that Egr3 also regulates genes involved in DNA damage response. A recent study revealed that physiological neuronal activity results in the formation of DNA double-stranded breaks (DSBs) in the promoters of IEGs. Additionally, they showed that these DSBs are essential for inducing the expression of IEGs, and failure to repair these DSBs results in the persistent expression of IEGs. We hypothesize that Egr3 plays a role in repairing activity- induced DNA DSBs, and mice lacking Egr3 should have an abnormal accumulation of these DSBs. Before proceeding with that experiment, we conducted a preliminary investigation to determine if electroconvulsive stimulation (ECS) is a reliable method of inducing activity- dependent DNA damage, and to measure this DNA damage in three subregions of the hippocampus: CA1, CA3, and dentate gyrus (DG). We asked the question, are levels of DNA DSBs different between these hippocampal subregions in animals at baseline and following electroconvulsive stimulation (ECS)? To answer this question, we quantified γ-H2AX, a biomarker of DNA DSBs, in the hippocampal subregions of wildtype mice. Due to technical errors and small sample size, we were unable to substantiate our preliminary findings. Despite these shortcomings, our experimental design can be modified in future studies that investigate the role of Egr3 in activity-induced DNA damage repair.

Damage to DNA can affect the genes it encodes; if this damage is not repaired, abnormal proteins may be produced and cellular functions may be disturbed. DNA damage has been implicated in the initiation and progression of a variety of diseases. Conversely, DNA damage has also been discovered to contribute to beneficial biological processes. Madabhushi and colleagues (2015) determined that activity-dependent DNA double strand breaks (DSBs) in the promoter region of immediate early genes (IEGs) induced their expression. EGR3 is an IEG transcription factor which regulates the expression of growth factors and synaptic plasticity-associated genes. In a previously conducted microarray experiment, it was revealed that EGR3 regulates the expression of genes associated with DNA repair such as Cenpa and Nr4a2. These findings inspired us to investigate if EGR3 affects DNA repair in vivo. Before conducting this experiment, we sought to standardize and optimize a method of inducing DNA damage in the hippocampus. Electroconvulsive stimulation (ECS) is utilized to induce neuronal activity. Since neuronal activity leads to the formation of DNA DSBs, we theorized that ECS could be used to induce DNA DSBs in the hippocampus. We predicted that mice that receive ECS would have more DNA DSBs than those that receive the sham treatment. Gamma H2AX, a biomarker for DNA damage, was utilized to quantify DNA DSBs. Gamma H2AX expression in the dentate gyrus, CA1 and CA3 regions of the hippocampus was compared between mice that received the sham treatment and mice that received ECS. Mice that received ECS were sacrificed either 1 or 2 hours post-administration, constituting treatment conditions of 1 hr post-ECS and 2 hrs post-ECS. Our results suggest that ECS has a statistically significant effect exclusively in the CA1 region of the hippocampus. However, our analyses may have been limited due to sample size. A power analysis was conducted, and the results suggest that a sample size of n=4 mice will be sufficient to detect significant differences across treatments in all three regions of the hippocampus. Ultimately, future studies with an increased sample size will need to be conducted to conclusively assess the use of ECS to induce DNA damage within the hippocampus.

Cocaine use remains a prevalent problem, yet there are no effective pharmacological treatments against cocaine use disorders. Cocaine is known to affect serotonin neurotransmission in the brain. Previous data has shown the modulatory role of CP 94,253, a serotonin 1B receptor (5-HT1BR) agonist on cocaine self-administration at different periods of the use-abstinence-relapse cycle. CP 94,253 facilitates cocaine self-administration in rats during the use maintenance phase, where rats are receiving daily intake of cocaine, yet attenuates it after a period of abstinence, when drug delivery is discontinued and rats are placed in home cages. Here we study the therapeutic potential of 5-HT1BR agonist pre-treatment on cocaine self-administration during these different time periods. Male and free-cycling female rats were trained to lever-press for cocaine (0.75 mg/kg i.v.) or sucrose pellets, until they met stable performance for total number of infusions on a fixed ratio 5 schedule of reinforcement. Rats were then tested with either the FDA-approved but less selective 5-HT1BR agonist zolmitriptan (3, 5.6, and 10 mg/kg s.c.; in descending order) prior to a period of abstinence or the more selective 5-HT1BR agonist CP 94,253 (5.6 mg/kg s.c.) after a period of prolonged abstinence and relapse (i.e. resumption of daily cocaine self-administration after a period of abstinence). Each session ran for 2 hours during which the training dose was available for the 1st hour and a low dose of cocaine (0.075 mg/kg i.v.) for the 2nd hour. Zolmitriptan was found to attenuate cocaine self-administration measures at a dose of 3 and 5.6 mg/kg when testing at the low dose of cocaine and at all three doses (3, 5.6, and 10 mg/kg) when testing at the training dose of cocaine. Zolmitriptan at the doses effective at attenuating cocaine intake did not alter sucrose self-administration. CP 94,253 (5.6 mg/kg s.c.) was found to have significant attenuative effects on self-administration measures both after a period of prolonged abstinence and after a period of relapse. Overall, these experiments showed that zolmitriptan decreased cocaine reinforcement without altering sucrose reinforcement as well as that CP 94,253 attenuates cocaine intake even after a period of relapse. These findings support the therapeutic potential of 5-HT1BR agonists as pharmacological treatments for cocaine use disorders.

Development of the cerebral cortex requires the complex integration of extracellular stimuli to affect changes in gene expression. Trophic stimulation activates specialized intracellular signaling cascades to instruct processes necessary for the elaborate cellular diversity, architecture, and function of the cortex. The canonical RAS/RAF/MEK/ERK (ERK/MAPK) cascade is a ubiquitously expressed kinase pathway that regulates crucial aspects of neurodevelopment. Mutations in the ERK/MAPK pathway or its regulators give rise to neurodevelopmental syndromes termed the “RASopathies.” RASopathy individuals present with neurological symptoms that include intellectual disability, ADHD, and seizures. The precise cellular mechanisms that drive neurological impairments in RASopathy individuals remain unclear. In this thesis, I aimed to 1) address how RASopathy mutations affect neurodevelopment, 2) elucidate fundamental requirements of ERK/MAPK in GABAergic circuits, and 3) determine how aberrant ERK/MAPK signaling disrupts GABAergic development.

Here, I show that a Noonan Syndrome-linked gain-of-function mutation Raf1L613V, drives modest changes in astrocyte and oligodendrocyte progenitor cell (OPC) density in the mouse cortex and hippocampus. Raf1L613V mutant mice exhibited enhanced performance in hippocampal-dependent spatial reference and working memory and amygdala-dependent fear learning tasks. However, we observed normal perineuronal net (PNN) accumulation around mutant parvalbumin-expressing (PV) interneurons. Though PV-interneurons were minimally affected by the Raf1L613V mutation, other RASopathy mutations converge on aberrant GABAergic development as a mediator of neurological dysfunction.

I therefore hypothesized interneuron expression of the constitutively active Mek1S217/221E (caMek1) mutation would be sufficient to perturb GABAergic circuit development. Interestingly, the caMek1 mutation selectively disrupted crucial PV-interneuron developmental processes. During embryogenesis, I detected expression of cleaved-caspase 3 (CC3) in the medial ganglionic eminence (MGE). Interestingly, adult mutant cortices displayed a selective 50% reduction in PV-expressing interneurons, but not other interneuron subtypes. PV-interneuron loss was associated with seizure-like activity in mutants and coincided with reduced perisomatic synapses. Mature mutant PV-interneurons exhibited somal hypertrophy and a substantial increase in PNN accumulation. Aberrant GABAergic development culminated in reduced behavioral response inhibition, a process linked to ADHD-like behaviors. Collectively, these data provide insight into the mechanistic underpinnings of RASopathy neuropathology and suggest that modulation of GABAergic circuits may be an effective therapeutic option for RASopathy individuals.

Of the 2.87 million traumatic brain injuries (TBI) sustained yearly in the United States, 75% are diffuse injuries. A single TBI can have acute and chronic influences on the neuroendocrine system leading to hypothalamic-pituitary-adrenal axis (HPA) dysregulation and increased affective disorders. Preliminary data indicate TBI causes neuroinflammation in the hippocampus, likely due to axonal damage, and in the paraventricular nucleus of the hypothalamus (PVN), where no axonal damage is apparent. Mechanisms regulating neuroinflammation in the PVN are unknown. Furthermore, chronic stress causes HPA dysregulation and glucocorticoid receptor (GR)-mediated neuroinflammation in the PVN. The goal of this project was to evaluate neuroinflammation in the HPA axis and determine if GR levels change at 7 days post-injury (DPI).

Adult male and female Sprague Dawley rats were subjected to midline fluid percussion injury. At 7 DPI, half of each brain was post-fixed for immunohistochemistry (IBA-1) and half biopsied for gene/protein analysis. IBA-1 staining was analyzed for microglia activation via skeleton analysis in the hypothalamus and hippocampus. Extracted RNA and protein were used to quantify mRNA expression and protein levels for GRs. Data indicate increased microglia cell number and decreased endpoints/cell and process length in the PVN of males, but not females. In the dentate gyrus, both males and females have an increased microglia cell number after TBI, but there is also an interaction between sex and injury in microglia presentation, where males exhibit a more robust effect than females. Both sexes have significant decreases of endpoints/cell and process length. In both regions, GR protein levels decreased for injured males, but in the hippocampus, GR levels increased for injured females. Data indicate that diffuse TBI causes alterations in microglia morphology and GR levels in the hypothalamus and hippocampus at 7 DPI, providing a potential mechanism for HPA axis dysregulation at a sub-acute time point.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, diagnosed late in

the disease by a series of motor deficits that manifest over years or decades. It is characterized by degeneration of mid-brain dopaminergic neurons with a high prevalence of dementia associated with the spread of pathology to cortical regions. Patients exhibiting symptoms have already undergone significant neuronal loss without chance for recovery. Analysis of disease specific changes in gene expression directly from human patients can uncover invaluable clues about a still unknown etiology, the potential of which grows exponentially as additional gene regulatory measures are questioned. Epigenetic mechanisms are emerging as important components of neurodegeneration, including PD; the extent to which methylation changes correlate with disease progression has not yet been reported. This collection of work aims to define multiple layers of PD that will work toward developing biomarkers that not only could improve diagnostic accuracy, but also push the boundaries of the disease detection timeline. I examined changes in gene expression, alternative splicing of those gene products, and the regulatory mechanism of DNA methylation in the Parkinson’s disease system, as well as the pathologically related Alzheimer’s disease (AD). I first used RNA sequencing (RNAseq) to evaluate differential gene expression and alternative splicing in the posterior cingulate cortex of patients with PD and PD with dementia (PDD). Next, I performed a longitudinal genome-wide methylation study surveying ~850K CpG methylation sites in whole blood from 189 PD patients and 191 control individuals obtained at both a baseline and at a follow-up visit after 2 years. I also considered how symptom management medications could affect the regulatory mechanism of DNA methylation. In the last chapter of this work, I intersected RNAseq and DNA methylation array datasets from whole blood patient samples for integrated differential analyses of both PD and AD. Changes in gene expression and DNA methylation reveal clear patterns of pathway dysregulation that can be seen across brain and blood, from one study to the next. I present a thorough survey of molecular changes occurring within the idiopathic Parkinson’s disease patient and propose candidate targets for potential molecular biomarkers.

Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known.

MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.

I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.