From the years of 1956 through 1977, Archaeologist Robert H. Dyson, Jr., directed the excavations of the archaeological site of Tepe Hasanlu, located in the West Azerbaijan region of Iran. Several archaeological reports of the finds include the overview of over 400 skeletons discovered in Hasanlu’s Lower Mound cemetery, though only 97 were ever retained for osteological analysis. Totaling 113 burials examined, 88-individuals were derived from the Low Mound and 25 individuals were from the High Mound. The excavated artifacts and skeletons are now split between The Pennsylvania Museum and The National Museum of Iran in Tehran. The Upper and Lower Mound burials of Tepe Hasanlu were examined through bioarchaeological, osteological, and archaeological (burial connotations) methodologies in an attempt to reveal the lifestyle, burial practices, and economy of the famous ancient city. It was hypothesized that there is a correlation between pathological conditions, sex, and the perceived economic/social status of the Bronze through Seluco-Parthian burials based on the associated grave goods and sociocultural characteristics viewed and examined within the burial space. This hypothesis was tested through skeletal and archaeological analysis, and it was found that individual pathological conditions correlate with the burial goods classified in Levels 1 and 2, however, Level 0 is more variable. The variations found among the level classifications and the frequencies of their individual pathological conditions reveal a high rate among Level 1 and a low rate among Level 2 burials. Additionally, it is possible that the different associated economic classes at Hasanlu exhibited dissimilar biological susceptibilities, nutrition, health, and lifestyles relating to their pathological conditions. However, this could also be a result of differential sampling, or the burials were excavated from a class-based cemetery. Age was found to be a major factor in the presentation of age-related degenerative pathological conditions, which was especially evident in the dentition of the sampled Hasanlu population. Furthermore, the High and Low Mounds appear to be drastically different in the associated economic class of their burials, as the LM appeared to contain individuals of higher classes (Level 2), and the HM exhibited a preference for burials with little (Level 1) to no grave goods (Level 0). As a result of such, it is theorized that the LM and HM were exhibiting different social structures and values, possibly representing separate societal class structures (egalitarian/non-egalitarian) influenced by economic opportunities or Hasanlu’s influence on the region. From the data examined, it appears that the social preferences exhibited by the Hasanlu peoples was evident in nearly every aspect of the burial space dictated by age, sex, and perceived economic class (Levels 0-2) through the burial characteristics of side, position, goods location, grave construction, interments, and possibly orientation. It is with this data that there are proven differences in the various subjects of archaeology and osteological features within the sampled burial population likely influenced by wealth, lifestyles, and/or various burial preferences.

Obesity is associated with insulin resistance, dyslipidemia, and cardiovascular disease. The current obesity epidemic is the result of surplus energy consumption. Excess energy is stored in expanding adipose tissue. Adipose tissue growth entails the enlargement of existing adipocytes, the formation of new fat cells from preexisting progenitors, and the coordinated development of supporting vasculature. Identifying adipocyte progenitors and the mechanism of adipose tissue expansion is crucial for the development of new strategies to combat obesity and its complications. Though important progress has been made towards understanding the developmental origin of adipocytes, the identities of adipocyte progenitors are still not completely known. The main objective of this study is to determine whether endothelial cells of the adipose tissue can give rise to new adipocytes. Our results indicate that murine endothelial cells of adipose tissue are pluripotent and can potentially give rise to preadipocytes. Lineage tracing experiments using the VE-Cadherin-Cre transgenic mouse reveal localization of reporter genes in endothelial cells, preadipocytes and white and brown adipocytes. Moreover, capillary sprouts from human adipose tissue, which have predominantly endothelial cell characteristics, are found to express Zfp423, a preadipocyte determination factor. In response to PPARγ activation, endothelial characteristics of sprouting cells are progressively lost, and cells form structurally and biochemically defined adipocytes. Taken together, our data support an endothelial origin of a population of adipocytes. The ability of the vascular endothelium to give rise to adipocytes may explain how angiogenesis and adipogenesis can be temporally and spatially coordinated. Analysis of BAT and WAT revealed that adipose depots have distinct compositions of adipocyte progenitors. Of the CD45-CD29+Sca1+CD24+ progenitor population, only 17% and 52% express VE-Cadherin in WAT and BAT, respectively. Our data show that the number of these specific progenitors in BAT and WAT are highly variable and suggest that a considerable number of adipocytes progenitors may have a non-endothelial cell origin. Differences in composition and types of adipocyte progenitors may explain the differences in the adipocytes phenotypes that we observe in discrete depots. In brief, we find that the vascular endothelium gives rise to a population of brown and white fat cells, and that the number of endothelial-derived adipocyte progenitors residing in BAT and WAT is highly variable. These results expand our current understanding of adipose tissue growth, and, we hope, will accelerate the development of treatments for obesity-related complications.

Objectives: Contemporary data are lacking with respect to the incidence rates of, factors associated with, and impact of cardiac arrest from ventricular fibrillation or tachycardia (VF-CA) on hospital survival in patients admitted with an acute coronary syndrome (ACS). The objectives of this multinational study were to characterize trends in the magnitude of in-hospital VF-CA complicating an ACS and describe its impact over time on hospital prognosis. Methods: The study population consisted of 59,161 patients enrolled in the Global Registry of Acute Coronary Events Study between 2000 and 2007. Overall, 3,618 patients (6.2%) developed VF-CA during their hospitalization for an ACS. Incidence rates of VF-CA declined over time, albeit in an inconsistent manner. Patients who experienced VF-CA were on average older and had a greater burden of cardiovascular disease, yet were less likely to receive evidence-based cardiac therapies than patients in whom VF-CA did not occur. Hospital death rates were 55.3% and 1.5% in patients with and without VF-CA, respectively. There was a greater than 50% decline in the hospital death rates associated with VF-CA during the years under study. Patients with a VF-CA occurring after 48 hours were at especially high risk for dying during hospitalization (82.8%). Conclusions: Despite reductions in the magnitude of, and short-term mortality from, VF-CA between 2000 and 2007, VF-CA continues to exert a significant adverse effect on survival among patients hospitalized with an ACS. Opportunities exist to improve the identification and treatment of ACS patients at risk for VF-CA to reduce the incidence of, and mortality from, this serious arrhythmic disturbance.

The purpose of this work was to examine biological mechanisms and symptom outcomes of illness uncertainty and psychological stress in Parkinson’s disease (PD). Parkinson’s disease is a chronic, progressive neurodegenerative disorder characterized by complex symptoms that fluctuate in onset, severity, level of disability, and responsiveness to treatment. In addition to characteristic motor symptoms of tremor, rigidity, bradykinesia, and postural instability, a considerable number of individuals with PD also experience debilitating pain, fatigue, and medication-induced motor complications of dyskinesia, dystonia, and on-off phenomena. The unpredictable nature of PD symptoms and motor complications coupled with the inability to halt or slow disease progression may result in uncertainty and psychological stress. Evidence is lacking regarding biological mechanisms and symptom outcomes of uncertainty and psychological stress in PD. As such, 80 men and women diagnosed with PD after the age of 49 were recruited to participate in this study. Data specific to characteristics that may contribute to uncertainty and psychobehavioral measures of uncertainty, appraisal, psychological stress, and symptom outcomes of motor symptoms, pain, and fatigue were collected. Biological measures of neuropeptide Y (NPY) and cytokines were obtained. The results revealed that participants perceived a moderate level of illness uncertainty. Uncertainty correlated significantly with motor symptoms, pain severity, and pain interference and predicted more severe pain severity and pain interference. Psychological stress correlated significantly with motor symptoms, pain severity, pain interference, and fatigue and predicted more severe symptoms across all outcomes. NPY was positively correlated with threat appraisals and psychological stress. Cytokines were below the level of detection in this sample, and not used beyond descriptive analyses. In summary, this study found uncertainty and psychological stress contributed to more severe symptom outcomes in PD. This knowledge may be used to guide future studies aimed at further elucidating biobehavioral symptom and health outcomes of uncertainty and psychological stress in PD. It will also facilitate the development of interventions specifically targeted to uncertainty and psychological stress for the ultimate purpose of improving symptom management, health outcomes, and disease progression in PD.

The opportunistic fungus, Aspergillus fumigatus, is a leading cause of morbidity and mortality among the immunocompromised population. Experimental and clinical findings have established that phagocytic defenses are critical in the recognition and clearance of A. fumigatus. Previous studies found that Toll-like receptors (TLRs), specifically TLR2 and TLR4, were essential in the detection of the mold. Furthermore, one study found that mice deficient in TLR9 lived longer than their wild-type counterparts following challenge with A. fumigatus. We sought to determine the role of TLR9 during A. fumigatus infection. Our results show that A. fumigatus contains unmethylated CpG DNA, the natural ligand of TLR9. Furthermore, A. fumigatus DNA stimulates a potent pro-inflammatory response in mouse bone marrow derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells (pDCs). A genome wide analysis showed that A. fumigatus DNA contains 87 human and 23 mouse putative immunostimulatory motifs. The response to A. fumigatus DNA is TLR9-dependent, as BMDCs from TLR9-/- mice were unresponsive to the fungal DNA. In addition, HEK293 cells cotransfected with human TLR9 and NFκB driven Luciferase conferred responsiveness to A. fumigatus CpG-rich sequences found in the fungal DNA. Our results show that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines. While pDCs secrete IFNα in response to A. fumigatus DNA, these cells have been mainly described to play critical roles in the antiviral responses. The role of pDCs during fungal infections remains to be elucidated. Our data show that CD304+ peripheral blood pDCs challenged with A. fumigatus hyphae secrete large concentrations of IFNα and TNFα in response to infection. Furthermore, the response appears to be TLR9- independent. However, pDCs spread over the hyphae and inhibit fungal growth. Furthermore, pDCs undergo cell lysis upon incubation with A. fumigatus. The antifungal activity of the pDCs was retained in the cell lysates, suggesting that this response was mediated by an intracellular factor. Addition of exogenous Zn2+, but not Fe3+, partially restores hyphal growth. In addition, western blot of pDC lysates show that these cells have the Zn2+-binding protein calprotectin. Over 60% cell death is observed in the pDC population following a 2 hour incubation with A. fumigatus. The observed pDC cell death can be partially attributed to gliotoxin, as pDCs challenged with A. fumigatus stains deficient in production of the mycotoxin result in decreased pDC cytotoxicity. Furthermore, pDC cell death occurs independent of contact with the mold, confirming that pDC cell death is mediated by a secreted fungal factor. In addition, our results show that pDCs are required for the host response against A. fumigatus. Mice depleted of their pDCs are more susceptible to A. fumigatus infection than the control counterparts, suggesting that pDCs play a role in the antifungal response. Also, we observe a 5-fold increase in the pDC population in the lungs of infected mice. Therefore, the possibility of these cells playing a role in recruiting and communicating with other immune cells cannot be eliminated. Upon maturation, pDCs acquire characteristics of conventional DCs (cDCs) such as upregulation of major histocompatability complex (MHC) and becoming more phagocytic. Whether mature pDCs are involved in the detection of and responses against fungal pathogens remains to be determined. Here we show that mature pDC secrete IFNα and TNFα in response to A. fumigatus conidia as early as 6 hours post-challenge. While cytokine secretion of mature pDCs against A. fumigatus does not require opsonization, it requires for A. fumigatus being alive and growing. Furthermore, supernatants from conidial growth induced cytokine secretion by the mature pDCs. The work presented in this thesis establishes that the nucleic acids in A. fumigatus serve as a pathogen associated molecular pattern (PAMP) that can induce a TLR9- dependent response. Furthermore, I show that pDCs secrete cytokines and induce an antifungal response against A. fumigatus conidia and hyphae. While the pDC population in the blood appears to be small, our work shows that these cells could be intimately involved in the antifungal responses against A. fumigatus.

Background:Lower back pain is one of the most common health-related complaints in the adult population. Thirty percent of Americans 65 years and older reported symptoms of lower back pain in 2004. With an aging population, the proportion of people over the age of 65 is expected to reach 20% by the year 2030. Because of this increase in older adults, lumbar spinal stenosis (LSS) associated with arthritic changes will also likely increase. In older adults, lower back pain is most often caused by degenerative lumbar spinal stenosis. Stenosis is the narrowing ofthe spinal canal, causing pressure on the nerve roots and is frequently treated surgically. Lumbar spinal stenosis is one of the most common reasons for back surgery in patients 65 years and older 2. However, risks associated with surgery increase with age 3-5 and older patients may choose non-surgical treatment for their lower back pain, including injection treatment. Injection treatment, usually consisting of anti-inflammatory medications and analgesics, has improved since the mid-1990's when fluoroscopic guidance was developed. Information about injection treatment for lower back pain is limited, especially in the older population. An extensive review of published literature regarding injection treatment revealed a paucity of information about older adults diagnosed with lumbar spinal stenosis. In this study, three aims were designed to gain more information about the effectiveness of injection treatment in older patients with lumbar spinal stenosis. In the first (retrospective) study, information about receipt of second injections and time between injections was collected to examine injection usage. In the second and third (prospective) studies, information about pain relief and functional return following injection treatment was collected to examine the effectiveness of injection treatment in patients age 60 and older diagnosed with lumbar spinal stenosis. To our knowledge, such results have not been repolted for this population in the literature. Objective:Injection treatment is a commonly used non-surgical procedure to alleviate lower back pain in older adults. However, older patients do not have enough information about how long pain relief will last after treatment or the amount of pain relief and functional return they will experience. These studies focused on three topics: 1) usage of injection treatment; 2) effectiveness of injection treatment on pain relief; 3) effectiveness of injection treatment on functional return. In addition, the variations of the effectiveness were examined by selected patient attributes. Methods:In a retrospective study, medical records of patients aged 60 years or older from a high volume dedicated spine center at the University of Massachusetts Memorial Hospital were retrospectively reviewed. This study included those diagnosed with degenerative LSS, who had not received an injection for lower back pain within six months, and whom were treated between June I, 2006 and May 31, 2007. In two prospective studies, patients scheduled for lumbar injection treatment between January 1 and June 30, 2008 were selected from the University of Massachusetts Memorial Hospital Spine Center. Selection criteria included patients age 60 and over, diagnosed with degenerative lumbar spinal stenosis and no previous lumbar injection within 6 months or lumbar surgery within 2 years. The Pain sub-score of the SF-36 questionnaire was used to measure pain at baseline and at one and three months post injection. The Physical Component Score (PCS) of the SF-36 questionnaire and the Oswestry Disability Index (ODI) were used to measure function at baseline and at one and three months post injection. Variations in longitudinal changes in scores by patient characteristics were analyzed in both unadjusted (univariate) analyses using one-way analysis of variance (ANOVA), and adjusted (multiple regression) analyses using linear mixed effects models. Results: In the retrospective cohort, the mean age of the cohort was 68, 64% were female, 59% were married, with a mean Body Mass index of 32 kg/m2. Of 92 eligible patients, 57% returned for a second injection within six months of the first. The mean number of months between injections was 4.8 for all patients, ranging from 1 to 22 months. When patient characteristics were examined, the only variable that showed a statistically significant difference was age. Patients aged 70 years and older were found to be 67% less likely to return for a second injection when compared to patients age 60-69 (OR=0.33 (0.12 - 0.94)p In the prospective cohort, information was collected on 62 patients. Mean Pain scores improved significantly from baseline to one month (14.1 points), and from baseline to three months (8.3 points). Post injection changes in Pain scores varied by Body Mass Index (BMI) and baseline emotional health. Based on a linear mixed effects model analysis, higher baseline emotional health, as measured by the SF-36 Mental Component Score (MCS>50), was associated with greater reduction in pain over three months when compared to lower emotional health (MCS Conclusion: Patients over age 70 do not return for repeat injection as frequently as patients age 60-69. In addition, each year a patient ages over age 60, they are 10% less likely to return for a repeat injection. Lower back pain in older adults with LSS is clinically significantly alleviated after injection treatment. In addition, injection treatment for LSS is associated with return of lost function needed for daily living activities in older adults. Pain relief and functional return varies by patient personal and clinical characteristics. Higher emotional health was associated with more pain relief and more functional return experienced over three months following injection treatment. Additional information is needed about why older patients do not return for second injections at the same rate as younger patients and how emotional health affects response to injection treatment in older adults.

Host cell attachment by pathogenic bacteria can play very different roles in the course of infection. The pathogenic spirochetes Borrelia hermsii and Borrelia burgdorferi sensu lato which cause relapsing fever and Lyme disease, respectively, are transmitted by the bite of infected ticks. After transmission, these spirochetes can cause systemic infection. Relapsing fever spirochetes remain largely in the bloodstream causing febrile episodes, while Lyme disease will often colonize a variety of tissues, such as the heart, joint and nervous system, resulting in a chronic multisystemic disorder. Borrelia species have the ability to bind to various cell types, a process which plays a crucial role in pathogenesis and may influence spirochetal clearance from the bloodstream. Colonization of multiple tissues and cell types is likely promoted by the ability to bind to components found in target tissues, and many B. burgdorferi adhesins have been shown to promote attachment to a wide variety of cells and extracellular matrix components. Different Lyme disease strains have been shown to preferentially colonize certain tissues, although the basis of this tissue tropism is not well understood. In this study we found that among different Lyme disease strains, allelic variation of the adhesin DbpA contributes to variation in its in vitro binding activities raising the possibility that this variation contributes to tissue tropism in vivo. In studying B. hermsii infection, we found evidence by both histological and fluorescence in situ hybridization (FISH) analysis of tissues that indicated that red blood cells were removed by tissue resident macrophages in infected mice. Spirochetes in the spleen and liver were often visualized associated with RBCs, lending support to the hypothesis that direct interaction of B. hermsii spirochetes with RBCs leads to clearance of bacteria from the bloodstream. Our findings indicate that host cell attachment play a key role in the establishment of Lyme disease infection, and in contrast contributes to the clearance of relapsing fever infection.

Chronic intake of alcohol can result in a range of pathology in the liver. Whilst the earliest changes observed with chronic ethanol, including the accumulation of lipid, or steatosis, are readily reversible upon cessation of alcohol consumption, longer exposure to ethanol may achieve more complex disease states including steatohepatitis, fibrosis, and cirrhosis that can cause irreversible damage and progress to fulminant hepatic failure. A key concept in the pathogenesis of alcoholic liver disease is that chronic ethanol primes the liver to increased injury through an interplay between hepatocytes and non-parenchymal cells, chiefly immune cells, of the liver. These relationships between hepatocytes and non-parenchymal cell types in alcoholic liver disease are reviewed in Chapter 1A. The Hypoxia Inducible Factors are a set of transcription factors that classically have been described as affecting a homeostatic response to conditions of low oxygen tension. Alcoholic liver disease is marked by increased hepatic metabolic demands, and some evidence exists for increased hepatic tissue hypoxia and upregulation of hypoxia-inducible factor mRNA with chronic alcohol. However, the biological significance of these findings is unknown. In Chapter 1B, we review the literature on recent investigations on the role of hypoxia inducible factors in a broad array of liver diseases, seeking to find common themes of biological function. In subsequent chapters, we investigate the hypothesis that a member of the hypoxia inducible- factor family, HIF1α, has a role in the pathogenesis of alcoholic liver disease. In Chapter 2, we establish a mouse model of alcoholic liver disease and report data confirming HIF1α activation with chronic ethanol. We demonstrate that HIF1α protein, mRNA, and DNA binding activity is upregulated in ethanol-fed mice versus pair-fed mice, and that some upregulation of HIF2α protein is observable as well. In Chapter 3, we utilize a mouse model of hepatocyte-specific HIF1α activation and demonstrate that such mice have exacerbated liver injury, including greater triglyceride accumulation than control mice. Using cre-lox technology, we introduce a degradation resistant mutant of HIF1α in hepatocytes, and after four weeks of ethanol feeding, we demonstrate that mice with the HIF1α transgene have increased liver-weight to body weight ratio and higher hepatic triglyceride levels. Additionally, several HIF1α target genes are upregulated. In Chapter 4, we examine the relationship between HIF1α activation and hepatic lipid accumulation using a recently published in vitro system, in which lipid accumulation was observed after treating Huh7 cells with the chemokine Monocyte Chemoattractant Protein-1 (MCP-1). We report that MCP-1 treatment induces HIF1α nuclear protein accumulation, that HIF1α overexpression in Huh7 cells induces lipid accumulation, and finally, that HIF1α siRNA prevents MCP-1 induced lipid accumulation. In Chapter 5, we use mouse models to investigate the hypothesis that suppression of HIF1α in hepatocytes or cells of the myeloid lineage may have differing effects on the pathogenesis of alcoholic liver disease. We find that ethanol-fed mice expressing a hepatocyte-specific HIF1α deletion mutant exhibit less elevation in liver-weight body ratio and diminished hepatic triglycerides versus wild-type mice; furthermore, we find that challenging these mice with lipopolysaccharide (LPS) results in less liver enzyme elevation and inflammatory cytokine secretion than in wild-type mice. In Chapter 6, we offer a final summary of our findings and some directions for future work.

Title: Treatment of CMV Vitritis in a Preterm Newborn Author’s Section: Remil Simon1, Darshan Shah1, Peter Blosser1, Demetrio Macariola1, Jeffrey Carlsen2 1.Department of Pediatrics, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 2.Johnson City Eye Clinic, Johnson City, TN Body: Cytomegalovirus (CMV) infection in the neonate is an infrequent occurrence in the developing world, and observing the symptoms of ocular CMV infection such as vitritis is rare. Treating CMV infection promptly is necessary to prevent mortality and potential neurological deficits including blindness and hearing loss. We encountered a preterm infant presenting with CMV sepsis immediately after birth. Our question was: will the current standard of treatment for CMV sepsis prevent CMV ocular infection? With our method of treatment, we followed the current standard of treatment for CMV infection by administering intravenous Gancyclovir for 6 weeks and oral Valgancyclovir for 6 months. Despite using the standard treatment to prevent neurological sequelae, the patient developed CMV vitritis and retinitis bilaterally. Although the treatment did not prevent CMV ocular infection, the severity of CMV retinitis and vitritis improved with treatment, and full resolution of vitritis was noted by day of life 61.

Scrub typhus is a potentially fatal infection that threatens one billion persons in the Asia-Pacific region and is caused by the obligate intracellular bacterium, Orientia tsutsugamushi. How this organism facilitates its intracellular survival and pathogenesis is poorly understood. Intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) into the host cell to modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank libraries as well as Type 1 and Type 4 secretion systems (T1SS and T4SS), which are expressed during infection. In silico analyses of the Anks’ C-termini revealed that they possess characteristics of T1SS secretion signals. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks. In addition to infecting endothelial cells, O. tsutsugamushi infects professional phagocytes. To better understand why these innate immune cells are unable to eliminate O. tsutsugamushi, we addressed the activity of host NF-κB proinflammatory transcription factor. Screening of O. tsutsugamushi infected cells at an MOI of 1 revealed inhibition of NF-κB nuclear accumulation as early as 8 hours in HeLa and bone-marrow derived macrophage cells. When stimulating infected cells with TNF-α, IκBα degradation still occurs, however NF-κB dependent gene transcription remains downregulated. Immunofluorescence microscopic analysis of TNF-α treated cells ectopically expressing all O. tsutsugamushi Anks revealed that two nuclear trafficking Anks, Ank1 and Ank6, result in a significant decrease in NF-κB nuclear accumulation. Additionally, these Anks also significantly inhibited NF-κB dependent gene transcription. Co-immunoprecipitation experiments revealed that both Anks interact with importin-β1, exportin-1, and the p65 NF-κB subunit. Treating cells with importazole significantly reduces the nuclear accumulation of Ank1 and Ank6. Finally, treating infected cells or cells ectopically expressing Ank1 or Ank6 with leptomycin B resulted in restoration of NF-κB nuclear accumulation. With these data, we propose that O. tsutsugamushi secretes Ank1 and Ank6 to initially interact with importin-β1, which permits their nuclear entry where they then interact with NF-κB and subsequently exportin-1 to prevent NF-κB nuclear accumulation.