Malaria is a re-emerging infectious disease with approximately half of the world's population at risk. In the US, since the 1950’s the Center for Disease Control (CDC) has been reporting between 1,000 and 1,500 cases of malaria every year. A majority of these cases were among US travellers and were attributed to Plasmodium falciparum. In August 2002, two cases of human malaria due to Plasmodium vivax were reported in Loudoun County, Northern Virginia. The Center for Disease Control and Prevention concluded that these cases were acquired locally. This was because of an absence of other risk factors such as international travel, and blood transfusion. Pools of Anopheles quadrimaculatus and Anopheles punctipennis collected in Loudoun County, Northern Virginia in 2002 tested positive for P. vivax subtype 210 indicating local transmission of malaria in the area. The purpose of this study is two-fold: 1) to determine the abundance of blood-fed Anopheles mosquitoes in the three sites close to the 2002 local transmission of human malaria in Loudoun County, Northern Virginia, and 2) to determine the infection status of the blood-fed Anopheles mosquitoes collected in the area. We observed a significant difference in the total abundance of Anopheles quadrimaculatus and Anopheles punctipennis at all the three sites, with Anopheles quadrimaculatus being more abundant. We also found a significant difference in the total abundance of Anopheles quadrimaculatus across the years at each of the three sites. All the pools collected in 2009 and 2010 tested negative for human Plasmodium parasites. The pools collected in 2010 tested negative for avian Plasmodium and Haemoproteus parasites. However, in 2009, 20 (28%) pools out of 71 tested positive for avian Plasmodium and Haemoproteus parasites. Four (20%) pools tested positive for avian Plasmodium, three of which were composed of Anopheles quadrimaculatus and were collected at Algonkian Regional Park while, one pool of Anopheles punctipennis was collected at Youngs Cliff. In addition, one (5%) pool composed of Anopheles quadrimaculatus and collected from Algonkian Regional Park tested positive for Haemoproteus. Eleven (55%) pools tested positive for both Haemoproteus and Plasmodium. Of these, four pools were composed of Anopheles quadrimaculatus and were collected from Algonkian Regional Park, two pools composed of Anopheles quadrimaculatus and one pool composed of Anopheles punctipennis were collected from Youngs Cliff. Lastly, four pools collected at Potomac Drive were composed of Anopheles quadrimaculatus. In summary, Algonkian Regional Park showed a high number of mosquitoes with malarial parasites. The maximum likelihood estimation (MLE) of mosquito infection rates based on the Biggerstaff (2006) method showed that among the three sites Youngs cliff had the overall highest infection rate (74.50%) for Anopheles mosquitoes compared to Algonkian Regional Park (53.86%) and Potomac drive (25.01%). An. quadrimaculatus had a higher infection rate compared at Algonkian Regional Park (57.95%) and Potomac drive (32.56%) compared to An. puncipennis (0%), while at Youngs cliff An. puncipennis had a higher infection rate (182.88%) compared to An. quadrimaculatus (56.06%).

While most cells of the central nervous system (CNS) express opioid receptors, the role of the endogenous opioid system in CNS development remains poorly understood. Identification of opioid functions during brain maturation is particularly crucial in light of the increasing trend in opioid abuse and the use of opioid drugs during pregnancy. New substitution therapies in pregnant opioid addicts include buprenorphine, a mu opioid receptor partial agonist and kappa opioid receptor antagonist. However, while clinical studies demonstrated buprenorphine efficacy in reducing neonatal withdrawal symptoms, there is a lack of information on the potential effects of this drug on brain development. Previous work from our laboratory has shown that perinatal exposure to buprenorphine induces dose-dependent alterations in rat brain myelination. These time-specific effects suggested that both therapeutic and supra- therapeutic doses of the drug could alter the normal pattern of oligodendrocyte development. In support of this hypothesis, this thesis work has now found that buprenorphine exerts direct actions on the oligodendrocytes that are highly dependent on both the drug dose and stage of cell differentiation. When exposed to buprenorphine, oligodendrocyte progenitors isolated from 3-day-old rat brain exhibit increased cell proliferation. In contrast, treatment of more mature oligodendrocytes obtained from 9-day-old rat brain induces dramatic dose- specific effects on cell process network extension and membrane outgrowth. These later effects are accompanied by significant parallel changes in the expression of the four major splicing isoforms of myelin basic protein (MBP), a critical component of the myelin membrane and mature myelinating oligodendrocytes. Furthermore, similar dose-specific effects on MBP expression are also elicited by methadone, a mu opioid receptor agonist already approved for the treatment of pregnant opioid addicts. Experiments with CTOP, a highly selective antagonist of the mu opioid receptor, further contribute to the idea that this receptor subtype plays an important function in controlling oligodendrocyte maturation. These findings underscore the potential effects of opioid exposure during brain maturation and further indicate an important regulatory role of the endogenous opioid system in the control of oligodendrocyte development and myelination.

Amphibian declines have been occurring world wide over the past several decades. Infectious disease and environmental changes have been implicated as the causative agents in these declines. Fish stocking from hatcheries provides a unique opportunity for organisms, including infectious diseases, to travel over long distances. Ranavirus was previously found in two ponds at Harrison Lake National Fish Hatchery in Charles City County, Virginia. Therefore, the primary objective of this study was to determine if ranavirus is present in tadpoles from four warm water fish hatcheries in Virginia. A secondary objective of this study was to determine if there are relationships between environmental variables and the proportion of tadpoles that test positive for ranavirus. Ranavirus was detected via PCR in three of the four warm water fish hatcheries, Harrison Lake National Fish Hatchery, Front Royal Fish Hatchery, and King and Queen Fish Hatchery, but was not detected at Vic Thomas Fish Hatchery. Temperature and the length of time a pond is filled with water were significant predictors of the proportion of tadpoles that tested positive for ranavirus, as determined by logistic regression analysis. Results from this study indicate that ranavirus is present in Virginia warm water fish hatcheries and can be found over multiple years. Precautions should be taken to ensure that ranavirus is not spread when fish are transferred from one hatchery to another or to the wild.

Periodontal disease is a chronic oral disease that is triggered by bacteria. One of these bacteria is Porphyromonas gingivalis. Some strains of P. gingivalis produce capsule, however, so far the role of capsule in the interactions with host cells in P. gingivalis is not well understood. Here, we investigated the contribution of capsule to triggering host response as well as its protective role on bacterial internalization by host cells with subsequent killing. qRT-PCR analysis showed more upregulation of expression of various groups of genes in macrophages challenged with the non-capsulated strain than in those challenged with the capsulated one with ratios as high as 8.4:1. Cytokine quantification of IL-6 using ELISA indicated that the non-capsulated strain produced more IL-6 in macrophages at 1 hr post-infection and drastically more at 8 hrs post-infection than the capsulated strain with a 4-fold difference. Maturation markers were induced at two fold higher rate in dendritic cell challenged with the non-capsulated strain at 4 hrs compared to dendritic cells challenged with the capsulated strain. The rate of phagocytosis of the non-capsulated form of P. gingivalis by both dendritic cells and macrophages was 5-6 fold higher, respectively. On the contrary, survived of the non-capsulated P. gingivalis was drastically reduced compared to the capsulated strain. Our results indicate that the Porphyromonas gingivalis capsule plays an important role in aiding the evasion of the host immune system activation as well as promoting survival of the bacterium within host cells. As such it is a major virulence determinant of P. gingivalis.

Head and Neck Squamous Cell Carcinoma (HNSCC) is an epithelial skin cancer of the upper aerodigestive tract, and is the sixth most common malignancy in the U.S. HNSCC patients undergoing chemotherapy commonly develop anemia, a condition in which the body lacks mature red blood cells (RBCs). Erythropoietin (EPO) is a systemically circulating hormone in the body that regulates the production of RBCs and is applied to treat anemia. Recently, several studies implicated shortened life expectancy of cancer patients by EPO administration. It may be due to an unexpected activation of survival and proliferation pathways of cancer cells by EPO because of the presence of ectopically expressed erythropoietin receptor (EPOR) on the surface of cancer cells. The current study tests the biological effects of EPO and EPOR in HNSCC. A shRNA-mediated knockdown of the EPOR gene was applied to two specific cell lines, HN4 and its metastatic derivative HN12. Knockdown of EPOR decreased proliferation in both HN4 and HN12 cells. To our surprise, application of EPO to HN12 control cell line downregulated proliferation in these high EPOR-expressing cells. Conversely, EPO increased proliferation in a breast cancer cell line, MCF-7. Although EPO greatly impacted HNSCC proliferation, no significant difference was found in migration of these cells upon its application. It was indicated that the pathway responsible for proliferative effects in HNSCC from EPOR association could be due to activation of the PI-3/Akt pathway, judged by its phosphorylation of AKT. However, we need to further elucidate the contradictive biological mechanisms of downregulation of HNSCC and upregulation of MCF-7 proliferation. We also need to expand the number of screened cell lines and confirm the relevance of our observation. Collected together, these findings confirm the hypothesis that EPO and EPOR can impact HNSCC tumor progression, and that their effects may vary among cancer types. These results draw attention to the possible detrimental use of EPO in cancer treatment, and its administration therefore, should be reevaluated.

Head and neck squamous cell carcinoma (HNSCC) is one of the ten most common cancers worldwide, with a survival rate of less than 50%. A class of mutant p53, known as gain of function (GOF) mutant p53, has been found to be expressed in tumors in these patients. GOF mutant p53 not only loses the wild type tumor suppressor functions, but also gains aberrant functions that have been linked to tumorigenesis. In this current study, we utilized a model system consisting of cells derived from HNSCC tumors in order to investigate our hypothesis that GOF mutant p53 enhances, and p63 inhibits, EPS8 and CXCL5 expression and promoter activity. We found decreased EPS8 expression, CXCL5 expression, and cellular migration associated with the loss of GOF mutant p53. This indicates an enhancing role of GOF mutant p53 in cellular migration and expression of these target genes. The loss of GOF mutant p53 was also associated with decreased EPS8 and CXCL5 promoter activity, indicating upregulation of these target gene promoters by GOF mutant p53. We found increased EPS8 expression,CXCL5 expression, and cellular migration with the loss of p63 in cell expressing high levels of p63. This indicates an ixinhibitory role of p63 on the expression of these target genes and cellular migration. Loss of p63 was also associated with increased EPS8 and CXCL5 promoter activity, indicating p63 may be downregulating these target gene promoters. Based on our knowledge of EPS8 and CXCL5 in tumorigenesis, our findings suggest that GOF mutant p53 and p63 play opposing rolesin HNSCC tumorigenesis. Additional studies are needed to further elucidate the mechanism by which GOF mutant p53 and p63 regulate EPS8 and CXCL5 expression and promoter activity.

Top-loading This study compared the effects on oxygen (O2) transport of four fluids: 5.9% human serum albumin (HSA) a non-O2 carrying iso-oncotic solution (volume control);HBOC-201 (Hemopure, Biopure Corp., Cambridge, MA); MP50, HBOC-201 with a P50 of 18 mmHg; and lastly LP50, an HBOC-201 with P50 of 17 mmHg and higher viscosity (4 cP). It has been proposed that HBOC with a higher O2 affinity and a viscosity closer to that of whole blood will cause less vasoconstriction and thus a lower MAP than Hemopure (P50 = 40 mmHg and 2.2 cP). Intravital microscopic measurements were made on the spinotrapezius muscle of anesthetized, male Sprague-Dawley rats. Interstitial PO2 was measured using phosphorescence quenching microscopy at baseline and following four top-loading infusions, in which increasing concentrations of HBOC-201 were infused to reach target plasma concentrations of 1, 10, 100 and 300 μM. Both HBOC-201 and MP50 increased PO2 by about 10%, but PO2 was unchanged with LP50. LP50 was more hypertensive (160±5mmHg) than HBOC-201 (144±5) and MP50 (141±6). Arteriolar diameters were not significantly different among the three HBOCs. The lower P50 HBOCs did not lead to higher PO2 compared with HBOC-201 and the higher viscosity HBOC led to higher MAP. Hemorrhage and Resuscitation This study compared the efficacy of three resuscitation fluids in a model of hemorrhage and resuscitation: HBOC-201, a hemoglobin-based oxygen carrier ([Hb] = 13 ± 1 g/dl; Biopure Corp.), HBOC-201 with 92 μg/ml nitroglycerin (NTG), and 5.9% human serum albumin, a non-oxygen carrying colloid solution. Intravital microscopic measurements were made on the spinotrapezius muscle of anesthetized, male Sprague-Dawley rats. Interstitial fluid (ISF) oxygen tension (PO2) was measured using phosphorescence quenching microscopy at baseline, post-hemorrhage and three post-resuscitation time points. Following 40% blood volume withdrawal, animals were maintained in this condition for 30 minutes before resuscitation was begun. Baseline ISF PO2 (65 ± 2 mmHg) decreased during hemorrhage (3.6 ± 0.4 mmHg) and all resuscitation fluids increased ISF PO2 towards baseline. HBOC-201 with nitroglycerin produced the highest PO2 throughout the experimental time course, and the findings are consistent with the NO scavenging theory because adding NO alleviated known side effects such as increased vascular resistance and hypertension that are associated with HBOC-201 infusions.

Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, cause MLC20 dephosphorylation and muscle relaxation via inhibition of myosin light chain (MLC) kinase and activation of MLC phosphatase. Hydrogen sulfide (H2S) produced as a byproduct by luminal sulfate-reducing commensal bacteria or as an endogenous signaling molecule synthesized from L-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) regulates muscle contraction. However, the role of H2S in the regulation of MLC phosphatase activity and MLC20 phosphorylation is not known. The aim of the present study was to examine the expression of CSE and CBS in smooth muscle cells and to elucidate the molecular mechanism of H2S-induced muscle relaxation. Expression of CSE and CBS was determined by RT-PCR and western blot in muscle cells. The effect of H2S on CCh-stimulated RhoA/Rho kinase pathway and muscle contraction was examined using an endogenous activator of CSE (L-cysteine) and an exogenous H2S donor (NaHS). Isometric contraction in isolated muscle strips and scanning micrometry in isolated muscle cells was measured. Rho kinase activity was measured by immunokinase assay. Expression of CSE, but not CBS was detected in smooth muscle cells of stomach and colon from mouse, rabbit and human. Carbachol-induced contraction in muscle strips and in freshly dispersed muscle cells was inhibited by L-cysteine and NaHS in a concentration-dependent manner (1 to 100 mM). Glibenclamide, an inhibitor of KATP channels and a known target of H2S, had no effect on the inhibition of contraction by H2S. L-cysteine (10 mM) or NaHS (1 mM) inhibited carbachol-induced Rho kinase activity, and the inhibition by L-cysteine was blocked in cells transfected with CSE-specific siRNA. We conclude that both endogenous and exogenous H2S induce muscle relaxation, and the mechanism is inhibition of RhoA/Rho kinase activity and stimulation of MLCP activity leading to MLC20 dephosphorylation.

Nowadays we have tremendous amount of genetic data needing to be interpreted. Somatic mutations, copy number variations and methylation are example of the genetics data we are dealing with. Discovering driver mutations from these combined data types is challenging. Mutations are unpredictable and have broad heterogeneity, which makes our goal hard to accomplish. Many methods have been proposed to solve the mystery of genetics of cancer. In this project we manipulate those above mentioned genetics data types and choose to use and modified an existing method utilizing Markov Chain Monte Carlo (MCMC). The method introduced two properties, coverage and exclusivity. We obtained the data from The Cancer Genome Atlas (TCGA). We used MCMC method with three cancer types: Glioblastoma Multiform (GBM) with 214 patients, Breast Invasive Carcinoma (BRCA) with 474 patients and Colon Adenocarcinoma (COAD) with 233 patients.

The enteric nervous system controls the gut through the release of specific neurotransmitter and neuromodulators at specific sites such as mucosal secretory cell or smooth muscle cell. In the present study, we have examined the response to one of these neurohumoral agents, Neurotensin, in the rat distal colon. Neurotensin is a paracrine and endocrine modulator of the digestive tract. Even though these effects have been seen in colonic preparations, there are very few functional studies of the effects of Neurotensin in the rat colon, especially the distal colon. In the current study we propose the following hypothesis that Neurotensin will lead to contractile effect on basal tone and phasic contraction in the distal rat colon and will mediate this process primarily through the NT1 receptor. This hypothesis is based on evidence from the mixed action of Neurotensin in other regions of the gut and the more widespread distribution of the NT1 receptor. We have identified two specific aims to investigate this hypothesis. Aim 1 is to investigate the role of Neurotensin in tonic contraction and phasic contraction of the distal rat colon. In this aim, we will expose distal rat colon strips to varying doses of Neurotensin and record changes in basal tone and phasic activity. For our second aim, we will investigate the receptors mediating these responses to Neurotensin. In this aim, we will introduce NT1, NT2, and nonspecific inhibitors to distal rat colon and observe modulation in Neurotensin effects. We will also determine the existence of the receptors via Western Blot. The rat distal colon did respond in a dose-response fashion to varying doses of Neurotensin, but elicited different effects dependent on the strip preparation. When the mucosa was intact, circular muscle responded with an inhibitory effect to phasic activity, but there was little to no change in tonic activity. When the mucosa was removed, the circular muscle responded to Neurotensin by eliciting an increase in tonic activity, but had no effect on phasic activity. The use of SR48692, a specific NT1 receptor inhibitor, showed that the effects that were observed due to Neurotensin were not mediated through the NT1 receptor. With the use of SR142948, a non-selective NT1/NT2 inhibitor, the effects of Neurotensin was completely abolished. This led us to believe that the observed effects were mediated through a Neurotensin receptor and that receptor is likely the NT2 receptor. This was confirmed by the use of the specific NT2 receptor antagonist, levocabastine. The existence of the receptor in rat colon had to be confirmed in order to ensure that the effects observed were mediated through the NT2 receptor and not from an outside mediator. Western Blot analysis confirmed the existence of the NT2 receptor within the mucosa, within the muscle, and within the intact preparation of the distal rat colon. Although these results conflict with our hypothesis, it provides for an interesting template and avenue of exploration.