Hemoglobin-based oxygen carriers provide a promising future as an alternative to human blood transfusions. Hemoglobin-based oxygen carriers, HBOCs, provide a lowcost, easy to maintain, and safe solution. They require no refrigeration and are universally compatible, and the required transfusion volume is less than that of a normal transfusion. HBOCs have been known to have adverse side effects such as renal toxicity, gastrointestinal dismotility, and hypertension. Many of these problems stem from the lack of a membrane, which protects the hemoglobin from dissociating and extravasating into the blood vessel wall. Extracellular hemoglobin, like that found in HBOCs, has a greater affinity for nitric oxide, NO, than oxygen due to the lack of a protective membrane like that of a red blood cell. This NO scavenging effect has been used to explain the increase in mean arterial pressure, MAP, as well as any other smooth muscle dysfunction. NO has been accepted as the endothelial derived relaxing factor which serves to dilate or maintain the vascular tone of the arterioles. It is theorized that the drop in NO due to scavenging by hemoglobin causes an increase in MAP. In this study, using the mesentery of rats as a model to observe the microcirculation, various doses of HBOC 201, HBOC 205 (MW 400), and HBOC 205 (MW 600) were infused into the rats. Measurements of MAP and arteriolar diameter were taken in response to increasing doses of each HBOC to establish a dose-response relationship. Chemicals such as sodium nitrite, NaNO2, and Nw- Nitro-L-arginine methyl ester hydrochloride, L-NAME, a NOS inhibitor, were used to chemically alter the levels of NO. The purpose was to see if modifying the levels of NO could alter the changes in MAP due to the HBOCs. MAP rose in response to the increasing doses of HBOCs, but the arterioles failed to show any vasomotor responses. NaNO2 showed an ability to reduce the increase in MAP as a result of the HBOC, but again had no affect on arteriolar diameter. L-NAME was able to reproduce changes in MAP similar to that of the HBOCs, but again had no effect on diameter. The results support the theory of NO scavenging by the HBOC leading to an increase in MAP. The lack of activity in arterioles indicates that NO may not be a major factor in controlling vascular tone in this tissue. The results support that HBOC’s side effects are a result of NO scavenging, but further work is needed to better understand vasoactivity in the mesentery.

Studies of gene flow in natural plant populations often focus on either historical or abiotic dispersal methods (e.g. wind, water, gravity), but there is little information available on contemporary, animal-mediated pollen dispersal patterns. Emerging molecular laboratory techniques allow unprecedented insights into spatial patterns of pollen-mediated gene flow. However, to date, technical challenges have limited their widespread application. The genome of a pollen grain can be amplified via whole genome amplification (WGA) prior to traditional amplification via polymerase chain reaction (PCR) to prevent the stochastic effects associated with low copy number amplification. Even still, WGA can suffer from low success rates or poor repeatability. The present study examined the extent to which WGA can be used to aid in understanding insect-mediated pollen flow in Cornus florida (flowering dogwood) within Virginia Commonwealth University’s Inger and Walter Rice Center for Environmental Life Sciences. Initial amplification of DNA isolated from frozen grains was successful, until the pollen had been stored longer than 120 days at -20ºC. After this time point, the PCR targets failed to amplify. The percent success of downstream PCR amplification on fresh pollen grains varied from 20% to 100%, with an average of 62% success. The addition of a common molecular crowder, polyethylene glycol, produced consistent amplification, regardless of input DNA concentration and eliminated the need for triplicate samples. Successful pollination and subsequent reproduction of flowering plants has a substantial ecological and agricultural importance that warrants increased understanding into how insects move pollen across the landscape. Determining the haploid profiles of a single pollen grain will allow scientists to elucidate dispersal patterns of pollen grains and track the movement and efficiency of biotic pollinators.

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.