Ramphogordius sanguineus (Rathke 1799) is a gregarious nemertean with a worldwide distribution and found mainly on hard substrates associated with mussels, oysters and other organisms of the fouling community. Asexual reproduction occurs by spontaneous fragmentation and only anecdotal accounts of sexual reproduction exist. This is the first phylogeographic study of R. sanguineus as well as the first species delimitation analyses employing DNA markers. Analysis of the mitochondrial gene nad6 and nuclear ISSR markers showed little diversity among geographically widespread populations, but AMOVA analyses of both markers revealed moderate to high genetic differentiation. Populations from Maine and Massachusetts exhibited the highest level of differentiation. These findings are consistent with predictions for invertebrates lacking a planktonic larval stage. Results of the nad6 tree-based delimitation analysis were in agreement with modern morphological and histocompatibility observations, suggesting that R. sanguineus is a single species and that a former division into four separate species was solely based on geographic location.

The thesis contains two manuscripts- 1) Developmental changes in pectoral muscle fiber diameter and number in the Blue Catfish, Ictalurus furcatus and 2) Reduction in pectoral spine and girdle in domesticated Channel Catfish, Ictalurus punctatus is likely caused by the absence of fish predators. The first study showed that fiber diameter increased linearly with fish size, whereas fiber number increases non linearly with fish size in the Blue Catfish correlated with dietary shift. The second study showed shorter spines in domesticated Channel Catfish as compared to wild Channel Catfish was a result of reduction in selection pressure during domestication in the absence of fish predators.

The contractility of smooth muscle in inflammatory bowel disease and experimental colitis is reduced due to inhibition of neurotransmitter release and a decrease in the response of smooth muscle to contractile agonists. We and others have shown that inflammation induced by TNBS treatment alters the expression and/or activity of signaling molecules involved in the regulation of Ca2+ mobilization, MLC20 phosphorylation and contraction in colonic smooth muscle. Although, thin filament- associated proteins such as calponin, caldesmon, tropomyosin and smoothelin do not directly participate in contraction, they regulate acto-myosin interaction and thus, muscle contraction. Calponin, caldesmon and tropomyosin inhibit actomyosin interaction and the inhibition is relieved upon phosphorylation of these proteins. Recent studies have shown that visceral smooth muscle from smoothelin knockout mice exhibited decreased contraction. However, the effect of inflammation on the expression of thin filament- associated proteins is not known. The aim of the present study is to determine the changes in the expression of calponin, caldesmon, tropomyosin, and smoothelin in colonic circular smooth muscle from TNBS- and DSS-induced colitis in mice. The animals were euthanized on day 3 and a segment of inflamed distal colon was removed. Colonic muscle strips from colitis mice and control mice were dissected for western blot and real-time RT-PCR analysis; contraction was measured by scanning micrometry in cells isolated from the muscle strips. Contraction in response to acetylcholine in muscle cells isolated from colonic muscle strips derived from mice with TNBS colitis was significantly inhibited compared with the response of cells derived from untreated colon or colon treated with ethanol. Expression of α-actin, γ-actin calponin, caldesmon, smoothelin-A and tropomyosin mRNA in muscle strips from TNBS or DSS colitis was significantly increased compared to control animals. Similarly, expression of α-actin, calponin, caldesmon, smoothelin-A and tropomyosin protein as determined by western blot was significantly increased compared to control animals. We conclude that the expression of α-actin, γ -actin calponin, caldesmon, smoothelin-A and tropomyosin is upregulated in colonic circular smooth muscle from TNBS or DSS colitis. Increase in the expression of calponin, caldesmon and tropomyosin, which act to inhibit acto-myosin interaction, could contribute to decrease in smooth muscle contraction.

Transient intracellular elevations of Ca2+ are common signaling mechanisms used to allosterically regulate proteins. One potential target of Ca2+ is Ca2+/calmodulin dependent protein kinase type II (CaMK-II). CaMK-II is a multi-functional protein kinase known to influence cellular pathways such as cell motility and cell cycle progression. Within the cell cycle, CaMK-II promotes the expression of the regulator protein cyclin D1, which is necessary for cell cycle progression. To further understand CaMK-II’s role in cyclin D1 expression, the binding partners of cytosolic CaMK-II were studied using mass spectrometry. Several proteins were identified including β actin, β tubulins, tropomodulin- 3 and Flightless-I. Flightless-I was of the most interest because of its role as a transcriptional co-activator of β-catenin-dependent genes such as cyclin D1 and its ability to translocate out of the nucleus following serum stimulation or CaMK-II activation. For this study, I sought to determine whether the interaction between CaMK-II and Flightless-I mediates transcription of cyclin D1. First, Flightless-I was linked to green fluorescent protein (GFP) and live cell imaging was performed. Under serum stimulation or constitutive CaMK-II expression, GFP-Flightless-I was cytosolic. Serum starvation or inhibition of CaMK-II expression resulted in the localization of GFP-Flightless-I to the nucleus. Next, a luciferase based reporter gene assay was used to evaluate the effect of Flightless-I and CaMK-II on β-catenin-dependent transcription of cyclin D1. Over-expression of Flightless-I or inhibition of CaMK-II both resulted in decreased β-catenin-dependent transcription; whereas suppression of Flightless-I by siRNA enhanced transcription. Taken together, these results suggest a novel mechanism whereby the interaction between CaMK-II and Flightless-I influences gene transcription necessary for cell cycle progression.

Parkinson’s disease (PD) is a devastating neurodegenerative disorder characterized by progressive loss of dopaminergic neurons located in the substantia nigra. Accumulating evidence indicates that microglia-driven neuroinflammation contributes significantly to chronic neurodegeneration in PD. The matrix metalloproteinases (MMPs) play an important role in several neuroinflammatory paradigms; however, their relationship to dopaminergic neurodegeneration in PD remains relatively unexplored. To address this, the temporal relationship between MMP-2, MMP-3, MMP-9, and MMP-13 expression and dopaminergic neurodegeneration was compared in the 6-hydroxydopamine (6-OHDA) and lipopolysaccharide (LPS) models of PD. In dopaminergic SH-SY5Y cells, 6-OHDA treatment significantly increased MMP-13 mRNA expression; however, examination of 6-OHDA-lesioned rats demonstrated that MMP-13 protein expression does not change over the neurodegenerative time course, indicating that MMP-13 likely is not a major factor in neurotoxin-mediated neurodegeneration. MMP-3 and MMP-9 were not detected in either 6-OHDA model, but an increase in proMMP-2 expression was detected in nigral tissue samples 5 days post-surgery. This holds potential significance; however, active MMP-2 was not detected at any time point, suggesting that MMP-2-mediated proteolysis is not involved in 6-OHDA-induced neurodegeneration. In contrast to the 6-OHDA models, LPS triggered dramatic increases in MMP-2 and MMP-3 expression and activation that correlated with the neurodegenerative phase of this model. A significant increase in proMMP-9 was associated with this model, while no change in proMMP-13 was detected. In addition to MMP characterization, connective tissue growth factor (CTGF/CCN2), a protein that is transcriptionally and post-translationally regulated by MMPs was examined. In the LPS model, CTGF/CCN2 expression increased 5-fold and a protein fragment potentially representing cleaved CTGF/CCN2 was detected. The changes in CTGF/CCN2 occurred during peak increases in MMP-2 and MMP-3 activity. These experiments illustrate a strong temporal relationship between increased MMP expression and activation, elevated CTGF/CCN2 expression, and inflammation-induced dopaminergic neurodegeneration. There are clear differences between the 6-OHDA and LPS models that could hold significance for the pathogenesis of PD. Future studies aimed at interrupting MMP and CTGF function in the LPS model are required to further establish their role in inflammation-induced neurodegeneration and to assess their potential as a therapeutic target for the development of novel neuroprotective therapies for PD.

There are few published studies of larval fish assemblages from unregulated, tidal freshwater rivers. Patterns in the spatial and temporal distribution of larval fishes in the Mattaponi River were examined. Sampling took place on a weekly basis from February through August, 2006 and 2007. Larval fishes were categorized by taxa, reproductive guild, and residency guild. Group comparisons using multi-response permutation procedures (MRPP) indicated significant spatial and temporal differences in assemblage composition on multiple scales. Differences in assemblage composition were analyzed using non-metric multidimensional scaling (NMS). Interannual differences were attributable to anadromous and semi-migratory species. Seasonal differences were attributable to herrings, perches, and minnows. Both interannual and seasonal differences in assemblage composition may have been a result of changes in discharge. Spatial (i.e. longitudinal) variation of the larval fish assemblage differed by tidal regime. NMS and MRPP identified a distinct tidal freshwater larval fish assemblage. Tidal freshwater habitats may act as ecotones between marine and riverine ecosystems.

Hemoglobin-based oxygen carriers (HBOCs) offer a safe, more plentiful and long term alternate to blood banks. However, they have been found to increase blood pressure which can be attributed to an increase in total peripheral resistance (TPR). Lumenal nitric oxide (NO) scavenging by these HBOCs seems to be responsible for this hypertensive effect. In addition, it is believed that hemoglobin (Hb) tetramers and dimers may extravasate and consume additional nitric oxide in the perivascular and interstitial space. The purpose of the present study was to elucidate the role of NO scavenging and to confirm extravasation as a contributor to HBOC vasopressor effects in the spinotrapezius muscle. The present study investigated the vessel reactivity and mean arterial pressure response to three HBOCs: HBOC 201, HBOC 205 MW 400, and HBOC 205 MW 600. These varied in molecular weight (MW) and percentage of tetramers and dimers. It was found that larger polymers of HBOC showed no significant decrease in vasoactivity. Although larger polymers are less likely to extravasate, the remaining tetramers and dimers seem sufficient to contribute to the observed vasoactivity. Using NaNO2, a NO donor, in conjunction with the HBOCs almost completely abolished this hypertensive effect at higher concentrations. Further examination utilizing a nitric oxide synthase (NOS) inhibitor to mimic the HBOC vasopressor effects demonstrated that lower concentrations of NaNO2 were able to abolish the hypertensive effect. In vitro studies only further supported these results by demonstrating that NO consumption increases with HBOC dose. HBOC labeled with TRITC showed conclusive evidence that extravasation also plays a role in NO scavenging, even when minimal amounts of tetramers and dimers are present. In conclusion, the present study offers strong evidence that NO scavenging is responsible for the observed vasopressor effects. It also offers evidence supporting the theory that HBOC extravasation may be contributing to these vasopressor effects as well.

Schizophrenia is a debilitating disorder caused by the interaction of genetic and environmental factors. In this study, we identified candidate genes and single nucleotide polymorphisms from two genome-wide association studies, GAIN and CATIE. Nine SNPs representing four candidate genes were selected for replication studies with our Irish samples: Irish Case-Control Study of Schizophrenia (ICCSS), the Irish Study of High-Density Schizophrenia Families (ISHDSF), and the Irish Trio Study of Schizophrenia (ITRIO). In the ITRIO sample, rs4704591 (CMYA5 gene) showed nominal significance (p = 0.0447947). Combining ICCSS, ISHDSF, and ITRIO samples for rs4704591 increased sample size and power and yielded a p-value of 0.00388. This marker remained significant after Bonferroni correction for 9 markers genotyped in this study. CMYA5 gene binds to dysbindin protein in muscle. The dysbindin gene may influence glutamatergic neurotransmission, which has been suspected of being a mechanism by which the pathophysiology of schizophrenia is manifest. Our data suggest CMYA5 gene may be associated with schizophrenia in Caucasian subjects.

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.