Glioblastoma Multiforme (GBM) remains as one of the most aggressive and lethal cancer types, often resulting in poor prognosis. Currently, Temozolomide (TMZ) is the standard chemotherapy for combating GBM. However, GBM’s upregulation of O-6-Methylguanine-DNA Methyltransferase (MGMT) mitigates the alkylating effects of TMZ treatment, generating a dire new need for novel or adjuvant therapy. U138MG is a TMZ-resistant GBM cell line used for the development of new chemotherapy. Here, we investigated whether inhibition of proton sensing GPR68 would decrease MGMT expression and sensitize U138MG cells to TMZ treatment. Using various genetic, protein, and cell-based assays we determined that inhibition of GPR68 may be decreasing MGMT protein expression and sensitizing U138MG to TMZ via the Gq/NFkB pathway. Furthermore, we identified that co-administration of TMZ and OGM resulted in a synergistic decrease in cell growth compared to OGM treatment alone.

Mammalian circadian rhythms are driven by a network of neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN exhibits daily (24-hour) rhythms in spontaneous action potential (AP) firing rate that encodes a time-of-day signal that coordinates the timing of circadian physiological and behavioral processes. Large-conductance Ca2+-activated K+ (BK) channels have a major role in driving the diurnal patterns of spontaneous firing in SCN neurons. BK K+ currents are larger at night, correlating with reduced neuronal excitability. The diurnal variation in BK current in the SCN is required for setting the day-night difference in firing frequency. BK currents undergo multi-level regulation by genetic and posttranslational mechanisms as well as functional coupling to Ca2+ channels. Intracellular Ca2+ (Ca2+i) is required for BK channel activation and previous studies have shown BK current is predominantly coupled to two types of Ca2+ sources in the SCN: L-type Ca2+ channels (LTCCs), and ryanodine receptors (RyRs). Circadian rhythms in Ca2+i have also been identified in SCN neurons. However, the Ca2+ channels involved in generating both AP and Ca2+i rhythms have not been clearly identified. First, to determine which Ca2+ channels are involved in AP rhythms, this study measured the impact of Ca2+ channel agonists and antagonists on the circadian parameters of spontaneous AP activity from organotypic SCN slice cultures grown on multi-electrode arrays. Next, to determine which Ca2+ channels are involved in Ca2+i rhythms, this study tested the effects of the same Ca2+ channel pharmacology on the circadian parameters of Ca2+i measured from SCN slice cultures transfected with a fluorescent Ca2+ sensor. Lastly, this investigated a potential mechanism by which LTCCs contribute to firing rate in SCN neurons by examining their ability to activate BK channels under controlled conditions. This study provides insight into the roles of specific Ca2+ sources in neural coding of the circadian time signal in the SCN.

Background: The solid organ transplant population has an elevated risk of cancer compared with the general population. Excess risk is largely due to immunosuppression. As this population grows, understanding long-term health risks such as cancer is critical. Population-based estimates of cancer mortality are needed since they measure the downstream outcome following a cancer diagnosis. Furthermore, quantifying deaths attributable to cancer can inform priorities to reduce the cancer burden. Methods: Linked transplant and cancer registry data were used to identify incident cancers and deaths among solid organ transplant recipients in the United States (1987-2014). Population-attributable fractions (PAFs) of deaths due to cancer and corresponding cancer-attributable mortality rates were estimated. Cancer-attributable mortality rates computed using the PAF were compared to cancer-specific mortality rates computing using cause of death (COD). The life-years lost (LYL) to cancer were estimated using two methods: an approach using matching to construct a cancer-free cohort and an approach using Cox proportional hazards regression models. Results: Among 221,962 transplant recipients, 15,012 developed cancer. Thirteen percent of deaths (PAF=13.2%) were attributable to cancer, corresponding to a cancer-attributable mortality rate of 516 per 100,000 person-years. Lung cancer was the largest contributor to mortality (PAF=3.1%), followed by non-Hodgkin lymphoma (NHL, PAF=1.9%), colorectal cancer (PAF=0.7%), and kidney cancer (PAF=0.5%). Overall, the cancer-specific mortality rate lower, 368 per 100,000 person-years. Within 10 years post-transplant, the mean LYL was 0.16 years per transplant recipient and 2.7 years per cancer. Cancer accounted for 1.9% of the total LYL expected in this population. Lung cancer was the largest contributor, accounting for 24% of all LYL, and NHL had the next highest contribution (15%). Conclusions: Cancer is a substantial cause of mortality among solid organ transplant recipients resulting in excess deaths and a shortened lifespan. Lung cancer and NHL are major contributors to the cancer burden including LYL to cancer, highlighting opportunities to reduce cancer mortality through prevention and screening.

Older adults using opioids in the US increased annually from 1999 to 2011. This is a significant public health concern because prescription opioid use increases fall-risk, the most common cause of traumatic brain injury (TBI) among older adults and a leading cause of disability and mortality. No studies have investigated the association between prescription opioid use and TBI. This dissertation characterized prescription opioid use in Medicare beneficiaries aged ≥65 years and examined the relationship between opioid use and incident TBI. Using Medicare administrative claims data from 2010 – 2015, I assessed how older adult prescription opioid use has changed over time, as well as changes in opioid prescriber specialties and pre-opioid diagnoses/ procedures. I also used these data to estimate risk for TBI associated with prescription opioid use. Next, I used R Adams Cowley Shock Trauma Center Registry (STR) data from 2015 – 2019 to explore the relationship between prescription opioid use and TBI injury mechanisms and severity. I found that the percent of older adults using prescription opioids decreased from 35.4% to 32.9% (p<0.001). Primary care physicians prescribed the most opioids, but their share of prescriptions dropped from 59.0% to 52.8% (p<0.001). Back pain remained the most common diagnosis among older adult opioid users, unchanged over time (24.1% to 25.3%, p=0.594). Opioid use increased TBI risk (odd ratio: 1.34, 95% confidence interval: 1.28 - 1.40) among older adults, regardless of opioid dosage and duration differences. Compared to non-users in the STR data, older adult opioid users who sustained TBI were 85% more likely to be injured in a fall compared to a motor vehicle incident (OR 1.85, 95% CI 1.20 – 2.86). Opioid use was associated with a 39% increase in sustaining more severe TBIs. (OR 1.39, 95% CI 1.09 – 1.79). This dissertation found opioid prescriptions decreasing among older adults. This parallels reductions in primary care opioid prescribing as well as changes in opioid-related diagnoses and procedures. These are the first studies to provide evidence that prescription opioids raise TBI risk and severity in older adults. Future studies could refine the association between opioid and TBI using data with definite dosing details

Thyroid hormones control many aspects of physiology such as metabolism and thermogenesis. Because thyroid hormones control such crucial bodily functions, their levels in the body are tightly regulated. Hypo- and hyperthyroidism can result when the level of thyroid hormone is too low or too high, respectively, with potentially serious pathophysiological consequences. µ-Crystallin is an NADPH-regulated thyroid hormone binding protein. The protein is minimally expressed in the skeletal muscle of most people; however, some individuals express relatively high baseline levels of µ-crystallin. We generated a transgenic mouse, the Crym tg mouse, that expresses high levels of µ-crystallin in its skeletal muscle, in order to explore the consequences of expressing high levels, comparable to those seen in some humans. The Crym tg mouse expresses mouse µ-crystallin at levels 2.6-147.5-fold higher than control mice in their skeletal muscle. Consequently, intramuscular triiodothyronine (T3) levels are elevated ~190-fold in the tibialis anterior, while serum thyroxine levels are decreased by 1.2-fold. Crym tg mice have a decreased respiratory exchange ratio that corresponds to a 13.7% increase in fat utilization as an energy source. Female Crym tg mice gained weight faster on high fat or high simple carbohydrate diets. Gene ontology enrichment analysis of transcriptomic and proteomic data revealed alterations to the expression of genes associated with metabolism and fiber type, while the fiber sizes of Crym tg soleus muscle are significantly smaller than those of controls. Taken together, these results suggest that µ-crystallin may play a role in regulating metabolism, perhaps through the control of thyroid hormone in muscle. Thus, humans who naturally express higher levels of µ-crystallin in their skeletal muscle may have an altered metabolism, comparable to the Crym tg mice.

Thymine DNA glycosylase (TDG) helps maintain genomic integrity by removing thymine from G·T mispairs arising via deamination of 5-methylcytosine (mC). TDG employs strict regulation for both the opposing guanine, as well as the base downstream of the target thymine, in order to limit removal of thymine from canonical A·T pairs, as erroneous removal of thymine from A·T pairs is mutagenic and cytotoxic. TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidation products of mC generated by ten-eleven translocation (TET) enzymes during active DNA demethylation. Remarkably, using single-turnover kinetics reactions to determine the maximum rate of substrate removal, k¬max, we find that TDG activity for fC and caC shows little dependence on the opposing base or the downstream base, revealing a major difference in specificity for excision of fC and caC relative to T. Using a novel 19F NMR approach to determine the flipping equilibrium for thymine into the TDG active site, we establish that specificity during thymine excision manifests largely by modulating the stability for thymine flipping in the active site. Structure-function analysis employing a variety of opposing bases reveals that both the thermodynamic stability (ΔH) of A·T pairs, as well as direct contacts between TDG and the opposing base, contribute to opposing base specificity. The differences in specificity observed for thymine versus fC or caC are likely explained by interactions between these substrates and the TDG active site. Structural information obtained from x-ray crystallography, combined with TDG mutational studies, identified several TDG active site residues that form stabilizing interactions with fC and caC, helping to both stabilize base flipping into the active site, as well as enhance the chemical steps of base excision. Conversely, two conserved residues in TDG, A145 and H151, limit stability of thymine in the active site, and destabilize thymine as a leaving group. As a result, additional contacts between both the opposing guanine, as well as the guanine downstream of the target thymine, appear necessary to orient thymine in a manner which produces stable, productive flipping into the active site.

Improving the efficacy of T cell therapies for solid tumors and leukemias could improve clinical outcomes. In leukemia, allogeneic hematopoietic cell transplantation and donor lymphocyte infusions can be potentially curative, however, tumor evasion of the graft-versus-leukemia effects still limit their efficacy. In the solid tumor microenvironment, immune suppressive myeloid cells inhibit T cell activation which can be detrimental to anti-tumor T cell responses. Toll-like Receptor/MyD88 signaling in cytotoxic T cells can provide a strong co-stimulation signal to improve T cell activation, function, and efficacy to counteract evasion mechanisms in hematopoietic and solid tumors. Unfortunately, TLR agonists lack specificity to T cells and may induce hyper inflammation or induce pro-tumor effects. Rather than provide TLR agonist therapy in leukemia and solid tumors, there exists potential to modify or engineer T cells to provide a direct MyD88 co-stimulus. Our research group developed a CD8α:MyD88 T cell co-receptor that mimics TLR co-stimulation in conjunction with T cell receptor activation. Our goal was to first determine whether this engineered T cell co-receptor could enhance graft-versus-leukemia responses in allogeneic hematopoietic cell transplantation and donor lymphocyte infusion therapies. In a suppressive solid tumor, we asked if MyD88 co-receptor could improve T cell activation and function in a suppressive tumor microenvironment. Using multiple experimental transplant models, we found that MyD88 co-stimulation in donor CD8+ T cells could improve the graft versus tumor response with some non-lethal increases in graft-versus-host disease. Looking further, we found that the CD8α:MyD88 co-receptor increased donor cytotoxic T cell proliferation, survival, and function in vivo. Donor CD8α:MyD88 T cells were able to directly kill tumor better than transduced controls. In the second part of this project we found that cytotoxic T cells receiving a synthetic MyD88 co-stimulation maintained strong basal activation in the presence of myeloid suppression. In the suppressive B16-GMCSF melanoma model, CD8α:MyD88 T cell were able to control tumor growth and reduce populations of suppressive myeloid cells in the tumor. These data show that augmented MyD88 co-stimulation in cytotoxic T cells could benefit patients undergoing both autologous and allogeneic T cell therapies.

Pathogens transmitted by mosquitoes are responsible for illnesses that cause nearly 700 million cases every year. A significant proportion of this morbidity is due to malaria, a disease caused by Plasmodium parasites and spread through the bites of infected Anopheles mosquitoes. While we have seen a reduction in malaria mortality rates thanks to the development of antimalarial therapeutics and entomological controls, there is a potential of malaria resurgence associated with the emergence and spread of antimalarial and insecticide resistance, highlighting the need for additional malaria control strategies. Malaria transmission occurs only if one Plasmodium parasite develops through key stages in the mosquito, including passing through the midgut, which harbors a microbial community that can influence Plasmodium transmission. Despite the opportunities they present for novel interventions, the development of Plasmodium sporozoites and the factors that shape the microbiota in mosquitoes are incompletely understood. Here, I sought to provide new insights into the microbial variations of wild-caught mosquitoes and the transcriptional regulatory programs of Plasmodium sporozoites. To this end, I simultaneously characterized the bacterial composition of 665 individual field-caught Anopheles mosquitoes in addition to their species, insecticide resistance genotype, blood-meal status, and infection status. My analyses revealed that mosquito collection site is the main driver of the microbial diversity, while other factors showed marginal or non-significant contribution. I also generated scRNA-seq data from 36,958 sporozoites of three Plasmodium species and collected from multiple anatomical sites of the mosquito and developmental stages of the parasite in an effort to better understand parasite developmental processes critical for malaria transmission. I identified transcriptional variations among salivary gland sporozoites of different Plasmodium species, patterns of gene regulation accompanying the journey of Plasmodium berghei sporozoites, and novel candidates potentially critical for mechanisms involved in sporozoite maturation. In addition, my analyses highlighted novel extensive transcriptional heterogeneity among sporozoites isolated from the same anatomical site, indicating asynchronous sporozoite development in the mosquito, that is regulated by intrinsic and environmental factors. Altogether, my findings improve our understanding of factors that influence malaria parasite transmission and lay the groundwork for identifying key transmission components, to inform development of novel intervention strategies.