Patients with chronic kidney disease (CKD) develop hyperhomocysteinemia (HHcy), have increased inflammatory monocytes (MC) and 10-times higher cardiovascular mortality than the general population. Here, we investigated HHcy-related MC differentiation in CKD. Twenty seven CKD and CVD, and 14 healthy subjects were recruited. CD40 was selected as a CKD-induced MC activation marker by mining for CKD-MC-mRNA screen database. We found that CD14++CD16+ MC, often denoted as inflammatory subset, soluble CD40 ligand (sCD40L), and TNFα/IL-6 levels were augmented in CVD and CKD subjects. CD40hiCD14++CD16+ MC, plasma homocysteine (Hcy) and S-adenosylhomocysteine (SAH) levels were increased in CVD and further elevated in CKD subjects. In cultured human peripheral blood mononuclear cells, CKD patient serum, Hcy, CD40L and TNFα/IL-6 induced CD40hiCD14++CD16+ MC differentiation, which was prevented by Hcy-lowering folic acid and neutralizing antibodies against TNFα and IL-6. Interestingly, CD14++CD16+ and CD40hiCD14++CD16+ MCs were negatively correlated with plasma S-adenosylmethionine/SAH (SAM/SAH) ratios, an indicator of methylation status, in CKD and CVD subjects. In white blood cells (WBC) isolated from CKD and CVD subjects with lower SAM/SAH ratios, hypomethylation was identified on the CG pair of NFκB consensus element in the core promoter located at the CpG island of CD40 gene by DNA methylation mapping using bisulfite converting pyrosequencing. Moreover, Hcy inhibited DNA methyltransferase-1 activity in cultured human blood MC. In conclusion, HHcy induces CD14++CD16+ and CD40hiCD14++CD16+ MC differentiation, at least in part, via sCD40L induction and CD40 DNA hypomethylation in CKD and CVD subjects. To study the role of CD40 in the development of kidney pathology and vascular disease, we then established mouse model of CKD-induced CVD (5/6 nephrectomy CKD model plus left carotid artery ligation) in CD40-/- mice. Bone marrow (BM)-derived cells were traced by the transplantation of BM cells from enhanced green fluorescent protein (EGFP) transgenic CD40+/+ mice after sublethal irradiation of the recipient CD40-/- mice. We demonstrated here that CKD accelerated carotid artery atherosclerosis, exacerbated metabolism, increased spleen weight and circulating CD40+ inflammatory MC, and further increased differentiation of mononuclear phagocytic cells (MPC); CD11b+F4/80- MC, CD11b+F4/80+ macrophage (Mϕ) and CD11c+CD11b+F4/80+ bone marrow-derived dendritic cell in the kidney and aorta, which were abolished by CD40-/- mice. We also found that CKD kidney elevated CD40 expression and induced MC chemotactic signals; CCL2, CCL12, and CCL5 chemokines, which were abolished in CD40-/- mice. In conclusion, our results suggest that CD40 induction in the chronic kidney disease mediates kidney chemokine production, which in turn contributes to acceleration of myeloid cell infiltration, MPC differentiation, and carotid artery atherosclerosis. / Pharmacology

Endoplasmic reticulum (ER) stress, which results from the aggregation of misfolded proteins in the ER, has been implicated in many forms of kidney injury, including hypertensive nephrosclerosis. ER stress induction increases levels of active TGFβ1, a pro-fibrotic cytokine, which can lead to epithelial-to-mesenchymal transition (EMT) in renal proximal tubular cells. EMT occurs when epithelial cells undergo phenotypic changes, which can be prevented by inhibiting ER stress. Further, the ER stress protein TDAG51 is essential for the development of TGFβ1-mediated fibrosis. The low molecular weight chemical chaperone 4-phenylbutyrate (4-PBA) can protect against ER stress-mediated kidney injury. It acts directly on the kidney, and can prevent ER stress, renal tubular damage, and acute tubular necrosis. In a tunicamycin-mediated model of kidney injury, this damage is prevented primarily through repression of the pro-apoptotic ER stress protein CHOP. Along with providing renoprotective effects, 4-PBA can inhibit endothelial dysfunction and elevated blood pressure in a rat model of essential hypertension. In addition to lowering blood pressure, 4-PBA reduces contractility, augments endothelial-dependent vasodilation, and normalizes media-to-lumen ratio in mesenteric arteries from spontaneously hypertensive rats. Further, ER stress leads to reactive oxygen species generation, which is reduced with 4-PBA. Dahl salt-sensitive rats given 4-PBA are protected from hypertension, proteinuria, albuminuria, and renal pathology. Rats provided with vasodilatory medications demonstrate that lowering blood pressure alone is not renoprotective. In fact, endothelial dysfunction, as demonstrated by an impaired myogenic response, is culpable in the breakdown of the glomerular filtration barrier and subsequent renal damage. As such, alleviating ER stress using 4-PBA serves as a viable therapeutic strategy to preserve renal function and prevent ER stress-mediated endothelial dysfunction, renal fibrosis, glomerular filtration barrier destruction, and progression of hypertensive nephrosclerosis. / Thesis / Doctor of Philosophy (PhD) / Chronic kidney disease is characterized by progressive loss of kidney function, and is a major public health problem. Kidney cells make proteins that help the kidney function properly. However, if the proteins are made improperly, the kidney does not function as well. This can lead to poor filtration and protein in the urine, damage to important kidney structures, and kidney scarring. High blood pressure, a risk factor for kidney disease, is often accused of causing kidney damage. This thesis shows that malfunctioning blood vessels can cause kidney injury, and lowering blood pressure may not prevent this. However, there are pharmacological molecules that can protect the kidney from damage. These molecules help the cells make proteins properly, preventing blood vessel malfunction and kidney damage. Our findings suggest that helping blood vessels and kidney cells create properly functioning proteins is more protective for the kidney than lowering blood pressure alone.

Juvenile rainbow trout were chronically exposed to cadmium in hard water, soft water, and in calcium-supplemented soft water in order to understand the effects of long term cadmium exposure in freshwater fish. A particular goal was to characterize changes in gill cadmium burden and the cadmium-binding properties of the gills during chronic sublethal exposures, so as to examine the applicability of the acute gill surface metal binding model or Biotic Ligand Model to trout chronically exposed to cadmium. Trout were exposed for 30 days to sublethal concentrations of cadmium in: a) moderately hard, Hamilton tap water (Ca = 1000 μM), b) synthetic soft water (Ca = 130 μM), or c) calcium-supplemented soft water (Ca = 260, 470, 770, and 1200 μM Ca). For both the hard and soft water cadmium exposures, no effects were observed on growth, swimming performance, and whole body ions. Growth and whole body and plasma Ca²⁺ concentrations were similar for all treatments in the calcium-supplemented soft water experiment; however, swimming performance was significantly reduced for the 470 μM Ca + Cd exposed fish. Acclimation to cadmium occurred in the hard water and lower concentrations of calcium-supplemented exposures but not in the soft water exposure. Cadmium accumulation was greatest in kidneys and gills and was directly related to cadmium exposure concentration. Tissue metal burdens were reduced with increased water calcium concentrations. Affinity of the gill for cadmium and the number of binding sites for cadmium decreased at higher water calcium concentrations. Affinity of the gill for Cd decreased with chronic cadmium exposure but binding site numbers increased with chronic Cd exposure. The acute gill binding model or Biotic Ligand Model, originally developed in soft water, was successfully applied to fish in both hard and soft water; however, complications arose when extending the model to fish chronically exposed to cadmium at various water calcium concentrations. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

This thesis examines the processes by which the self-concept is shaped by chronic illness in later life. This study contributes to our understanding of the development of the self in later life by examining patterns of continuity and change. Twenty-four women diagnosed with osteoarthritis (0A) were interviewed. The participants were community dwelling females, aged between 67 to 85 years. They were selected to reflect a range of OA, from mild to severe forms of the disease. A qualitative approach, more specifically, the grounded theory method was adopted for this study. The findings illustrate that OA in later life presents a source of change for the self concept. However, the influence of these processes of change vary considerably. In particular, the significance of change for the self-concept is shaped by the individual's perception of the impact of chronic illness for the self. These are shaped by several factors. The impact of OA on the self is influenced by the ways in which the person's multiple identities are affected. And, the person's perception of continuity and change for her "overall" or global sense of self must be considered. Finally, the type of process mechanisms that are drawn upon shape the person's perception of continuity and change. Such processes include: shifting and non-shifting prominence hierarchies, the ability to adjust and accommodate to one's limitations and the extent to which interactions and valued identities are modified, social comparison, reflected appraisal, and the dialogue between the past, the present, and the future self. The data show that older people do reflect some of the same issues that have previously been revealed by studies of chronic illness which do not focus on later life. However, experiences of chronic illness in later life also present unique variations. / Thesis / Master of Arts (MA)

The purpose of this study was to identify what high school principals indicate are effective strategies and interventions to reduce chronic student absenteeism. Research has shown chronic student absenteeism is highest among high school students (Stronge and Associates, 2019; U.S. Department of Education, n.d.), and effects may include low academic achievement, possible high school dropout, and poor outcomes in adulthood (Elias, 2019; Ready, 2010; Stronge and Associates, 2019; Virginia Department of Education [VDOE], n.d.). Chronic student absenteeism is one measure of school performance in Virginia and therefore, a responsibility of the school principal to monitor, maintain, or improve (VDOE, n.d). This study sought to answer the following research questions: 1. What strategies and interventions do high school principals indicate they utilize and implement to reduce chronic student absenteeism? 2. What are the perceptions of high school principals regarding the effectiveness of strategies and interventions they utilized and implemented in order to reduce chronic student absenteeism? This study included a survey of 8 Virginia high school principals whose school experienced a reduced rate of chronic absenteeism between 2016-2017, 2017-2018, and 2018- 2019. The Virginia Department of Education (VDOE) reported chronic absenteeism as a measure of school accountability beginning in 2016. Additionally, this study included semi-structured interviews with 3 of the high school principals. The survey and interview questions aimed to determine various strategies and interventions high school principals implement to reduce chronic student absenteeism, along with the effectiveness of each. Principals in this study reported communication, involving school stakeholders, creating a positive school culture, and utilizing accountability practices as means to reduce chronic student absenteeism. Principals perceived communication and engaging instruction to be effective strategies or interventions utilized in order to reduce chronic student absenteeism, while data collection and management were considered to be least effective. The study suggests principals could engage in those practices perceived as effective in reducing chronic student absenteeism, but also school divisions could provide job embedded professional development to enhance the knowledge and skills of principals related to the topic. / Doctor of Education / The purpose of this study was to identify what high school principals indicate are effective strategies and interventions to reduce chronic student absenteeism. This study also explored how effective principals perceived each strategy or intervention was in regard to reducing chronic student absenteeism. The study included Virginia high school principals whose school experienced a reduced rate of chronic student absenteeism between 2016-2017, 2017- 2018, and 2018-2019. Principals participated in a survey and individual interview; the survey and interview instruments were designed by the researcher (see Appendix G and Appendix H). The study results indicated high school principals are utilizing communication as a strategy or intervention to reduce chronic student absenteeism. Additionally, principals also reported involving various school stakeholders, creating a positive school culture, and utilizing accountability practices in order to reduce chronic student absenteeism. Principals perceived communication and engaging instruction to be effective strategies or interventions, while data collection and management was perceived to be least effective. Future actions could include principals' continued efforts to engage families in practices to reduce chronic absenteeism. Principals could also monitor and support teacher instruction for student engagement. Additional implications and future research to decrease chronic student absenteeism are shared.

Subclinical endotoxemia (low levels of circulating bacterial endotoxin) has been observed in patients suffering from chronic inflammatory diseases such as atherosclerosis, diabetes, and obesity. However, the link between this condition and chronic inflammation is poorly understood. Previous work from our lab has shown that chronic exposure to super-low doses of bacterial endotoxin (LPS) aggravates atherosclerosis resulting in increased plaque size and instability in a macrophage-dependent manner in a mouse model of atherosclerosis. Further, we showed that super-low dose LPS (SLD-LPS) treatment was able to inhibit lysosomal fusion in immortalized macrophages. However, this was done under more acute treatment conditions. The aim of this project was to examine the molecular mechanisms by which chronic SLD-LPS may polarize macrophages to a non-resolving pro-inflammatory state consistent with chronic inflammation. This was carried out in two projects, the first a more broad phenotypic paper showing the disruption in homeostasis by chronic SLD-LPS in immortalized macrophages, while the second uses primary bone marrow-derived mouse macrophages to identify specific molecular signaling pathways used by chronic SLD-LPS. Here we show that chronic SLD-LPS led to the novel upregulation of pro-inflammatory mediators p62 and ccl2 with simultaneous downregulation of homeostatic mediators Nrf2 and slc40a1 in immortalized wild-type mouse macrophages. Further we showed this effect was reversed using the homeostatic restorative agent sodium phenylbutyrate (4-PBA), a newly reported activity for this reagent in mouse macrophages. This indicated that a disruption in homeostasis, possibly involving autophagy, may be responsible for the non-resolving pro-inflammatory polarization of macrophages. Therefore, in our second project, we further explored the effect of chronic SLD-LPS treatment on the homeostatic arm of the response by focusing on the Nrf2 inhibitor Keap1. Here we show that chronic SLD-LPS results in an accumulation of Keap1 in mouse bone marrow-derived macrophages, an effect specific to chronic SLD-LPS, as high doses of LPS failed to induce Keap1. We suggest that this effect may be related to a disruption in lysosomal fusion as evidenced by accumulation of autophagy flux markers MLKL and p62. Further, we show that these effects are dependent on the non-traditional TLR4 adaptor TRAM, suggesting an alternative dose-dependent signaling pathway for LPS. Together this work identifies novel signaling mechanisms involved in non-resolving pro-inflammatory polarization of murine macrophages, providing new insight behind how chronic super-low dose LPS exposure may lead to chronic inflammation. / Doctor of Philosophy / Inflammation is the body's natural response to injury or insult and can be beneficial in certain contexts such as pathogen clearance. However, left un-checked, chronic inflammation can exacerbate or even lead to disease pathology, such as is the case with modern diseases such as atherosclerosis, obesity, diabetes, etc. Despite the high prevalence of these diseases, effective treatments and therapies are still lacking. Recently it was discovered that many patients suffering from chronic inflammatory diseases had low levels bacterial endotoxin (LPS) in their circulation, a condition referred to as subclinical endotoxemia. However, possible links between this condition and chronic inflammatory disease remain poorly understood. Using a mouse model of atherosclerosis, previous research from our lab showed that persistent exposure to super-low doses of bacterial endotoxin (similar to those observed in humans) lead to aggravated atherosclerosis with both increased plaque size and instability. Further, we showed that this effect was primarily mediated by pro-inflammatory polarized immune cells called macrophages, but the molecular mechanism behind this polarization is still unclear. Further research into these molecular mechanisms may provide better targets for the development of future chronic inflammatory disease treatments. Here using a combination of mouse cell line and primary cell cultures, we discuss how chronic exposure to super-low doses of bacterial endotoxin leads to the chronic non-resolving pro-inflammatory polarization of macrophage immune cells, with particular emphasis on the distinct molecular signaling mechanisms induced by chronic super-low dose LPS.

In writing this autoethnography, I invite you to enter into my world. It is not a world that is easy, or altogether happy. In the end, though, it is a story of survival and of perseverance. Trauma touches almost every person in some way. War, sexual abuse, physical and emotional abuse, death, and difficult life events color how we make sense of the world. Trauma may happen in one blinding moment or slowly eat away at us for years. Writing is often a way to cope with that trauma. This dissertation represents a small portion of my traumatic lived experiences that led me up to the doctoral process, and those that occurred in the ten years it took me to complete my dissertation. / Doctor of Philosophy / In writing this autoethnography, I invite you to enter into my world. It is not a world that is easy, or altogether happy. In the end, though, it is a story of survival and of perseverance. Trauma touches almost every person in some way. War, sexual abuse, physical and emotional abuse, death, and difficult life events color how we make sense of the world. Trauma may happen in one blinding moment or slowly eat away at us for years. Writing is often a way to cope with that trauma. This dissertation represents a small portion of my traumatic lived experiences that led me up to the doctoral process, and those that occurred in the ten years it took me to complete my dissertation.

The nutritional needs of the renal patient vary as the renal function decreases. Little information exists concerning energy requirements for patients on hemodialysis. Renal failure has been called a wasting disease as evidenced by decreased body weight, body fat, arm circumference and serum proteins. This research was designed to help precisely define energy requirements for the hemodialysis subject. Resting energy expenditure (REE) was measured by indirect calorimetry in 17 male hemodialysis subjects whose mean age was 55 years at the Veterans Administration Medical Center in Hampton, Va. The Beckman MMC Horizon System, a portable device which permits the determination of heat production from gas exchange, oxygen consumption and carbon dioxide production was used. By regression analysis, the measured REE was compared to the basal energy expenditure (BEE): the ideal weight based on the Metropolitan Life Insurance tables, the current weight taken on day of indirect calorimetry, a non-dialysis day, and the post dialysis weight taken immediately after dialysis. A correlation analysis of the dependent variable, IEE, with the current, ideal and post dialysis BBE variable resulted in correlation coefficients of .3783, .0003, and .3946 respectively. None of these correlation coefficients were significantly correlated with the REE. The post dialysis variable had the highest correlation coefficient, and thus the strongest relationship to the REE. While any of the weights studied could be used to determine energy needs for the hemodialysis subject, post dialysis weight may be the most desirable choice. / M.S.

Wildlife diseases often occur under quantifiable and consistent patterns, which can be understood to statistically predict their occurrence and spread across landscapes. Chronic wasting disease (CWD) is a neurodegenerative disease in the deer family Cervidae caused by a prion, a pathogenic and misfolded variant of a naturally occurring protein. Managing and controlling CWD is imperative for conservation of ecologically and economically important cervid species, but unclear transmission mechanisms within landscapes complicate evidence-based management. Gaps of information in the landscape ecology for CWD are particularly pronounced for areas with recent disease emergence and spread, such as within the CWD cluster in the Mid-Atlantic United States. Thus, I identified current gaps in information and sought to fill neglected areas of research, specifically focusing on landscape determinants for CWD occurrence and spread in the state of Virginia. In chapter 2, I conducted a scoping study that collected and synthesized decades of CWD research and identified trends with respect to statistical and mathematical modeling methods used, connectivity within the CWD research community, and the geographic areas from which studies were performed. In chapter 3, I investigated landscape determinants for CWD in Virginia using remote sensing landscape data and an epidemiological dataset from Virginia Department of Wildlife Resources (DWR) using diverse algorithms and model evaluation techniques. Finally, in chapter 4, I modeled landscape connectivity between confirmed CWD cases to examine potential paths and barriers to CWD spread across landscapes. My results indicate that landscape ecology was rarely incorporated throughout CWD's 50+ year history. I provide evidence that remotely-sensed landscape conditions can be used to predict the likelihood of CWD occurrence and connectivity in Virginia landscapes, suggesting plausible CWD spread. I suggest areas of future work by explicitly identifying gaps in CWD research and diagnostic methods from which models are based, and encourage further consideration of host's ecology in modeling. By integrating remotely-sensed data into my modeling framework, the workflow should be easily adaptable to new study areas or other wildlife diseases. / Master of Science / Understanding why diseases occur in some locations and not others can be a critical challenge for disease ecologists. One disease that has received significant attention from the media and scientific community is chronic wasting disease (CWD), which is caused by a misfolded protein called a prion. Virginia Department of Wildlife Resources (DWR) has identified a stark increase in the number of CWD cases since first discovered in 2009, which threatens white-tailed deer populations and a 500 million dollar industry used for conservation of Virginia wildlife species. Previous research found that CWD does not occur randomly on the landscape, but otherwise little is known about the landscape ecology of CWD. To provide insight on Virginia's CWD outbreak, I assessed methods used to investigate other CWD outbreaks in both space and time. Also, I used landscape data collected from satellites and data from CWD cases in Virginia, and applied statistical tools to identify patterns in the landscape that were linked with CWD cases. My results suggest that landscapes were rarely examined to understand CWD, and instead, researchers focused on understanding how populations will respond to the disease. I also provide evidence that, at least in Virginia, researchers can use satellite information with disease data to predict CWD on the landscape and estimate its spread. This information can be used by wildlife managers to control the disease. For example, disease surveillance can be increased in areas where CWD has been predicted, or herd sizes can be reduced in areas likely to promote disease spread. This information could also be used to tailor wildlife health regulations aimed to minimize the risk of other deer populations acquiring the disease. Ultimately, the landscape plays an important role in CWD, but research on this topic is limited; therefore, additional research is needed to understand and eventually control this disease affecting ecologically and culturally important game species.