Nectria haematococca mating population (MP) VI has a cytochrome P450 which confers tolerance to the pea phytoalexin pisatin. This enzyme, termed pisatin demethylase (PDA), detoxifies pisatin and is a virulence factor on pea. PDA is on a 1.6 Mb conditionally dispensable chromosome, and PDA is in a cluster of three other genes involved in pea pathogenicity. Analysis of the PEP cluster suggests it may have been acquired by horizontal gene transfer (HGT). Isolates lacking PDA are still more tolerant of pisatin than other closely related fungi and a "nondegradative" tolerance mechanism was demonstrated previously that might be responsible for this tolerance.ABC transporter, NhABC1, was identified as the gene responsible for this tolerance, fulfilling the major goal of this dissertation. NhABC1 is induced by pisatin and NhABC1 mutants are reduced in virulence on pea to a similar degree as PDA mutants. However, isolates lacking both PDA and NhABC1 are essentially non-pathogenic on pea and are more sensitive to pisatin than either single mutant, demonstrating these two proteins are the major mechanisms responsible for pisatin tolerance. A second ABC transporter in N. haematococca (NhADP1) was also shown to be involved in virulence on pea, however its function in planta remains unknown. The final part of this dissertation concerns a partial analysis of the genome sequence of N. haematococca MPVI. The genome encodes 68 ABC transporters, some of which were in multiple copies when compared to other fungal genomes. This finding led to a whole genome approach to identify extra copies of genes, which are in single copies in the most closely related sequenced fungus, Gibberella zeae. When a comparison between the orthologs found in both genomes and the unique genes found in N. haematococca was made, the results suggest HGT may have shaped the genome of N. haematococca. Several lines of evidence supports this: the large genome size of N. haematococca, the unexpected phylogenetic relationship of the extra copies of genes, the enrichment of the unique genes on dispensable portions of the genome, and a difference between codon usage and GC content of the orthologs versus the unique genes.

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Grain crops are often afflicted with a disease commonly referred to as root rot or foot rot. Because root rot diseases are very variable in their symptom expression, they are frequently overlooked as the abnormal condition is often attributed to drought, wind, frost or other causes. For these reasons it is difficult to estimate the damage caused exclusively by root rots. It is generally agreed that the fungus Helminthosporium sativum P.K. & B. is of prime importance in the root rot complex affecting barley and wheat. By plating tests of seed samples of barley and wheat grown in representative areas of Canada, Greaney (1944) showed that the most important pathogen present was H. sativum. Oswald (1950) in California found the four primary pathogenic fungi attacking barley and wheat, in their order of apparent economic importance, to be Helminthosporium sativum, Fusarium roseum f. cerealis, Ophiobolus graminis and Fusarium nivale. Simard and Ludwig (1946) found that, in the years 1942 and 1943, 65 per cent of the Quebec grown barley seed samples examined by them carried H. sativum. The significance or Helminthosporium sativum in the barley and wheat growing areas is indicated by estimates of the damage caused by this fungus. Maohaeek (1943) concluded from the results of a three year survey in Manitoba that barley and wheat yields are reduced by as much as 12 per cent because of root rot.[...]

Chemokines are central to the migration of leukocytes around the body, during both inflammatory and homeostatic conditions. Chemokines mediate their effects by binding to chemokine receptors found on the migrating cell’s surface. Chemokine binding to the chemokine receptor results in signaling, which allows the cell to migrate towards the epicenter of chemokine production. In addition to ‘classical’ chemokine receptors which are involved in leukocyte migration, a discrete family of chemokine receptors exist which are considered to be ‘atypical’, as binding to their cognate ligands does not result in classical signaling as detected by calcium flux assays. One of these atypical chemokine receptors is the chemokinescavenging receptor D6, which can bind to and internalize at least 14 inflammatory CC chemokines in vitro. In addition, an analysis of D6 function in vivo has shown that D6 is important for the resolution of the inflammatory response. D6 KO mice treated with phorbol ester to the shaved dorsal skin developed an inflammatory skin pathology that resembled the human condition psoriasis in many respects. In contrast, WT mice treated with phorbol ester developed a very mild inflammatory response, which quickly resolved. These data suggested that a loss of D6 expression ‘primed’ the mouse to develop a psoriasisform pathology, requiring only minor irritation/trauma to develop the pathology. Similarly, histologically normal (uninvolved) skin from a psoriatic patient has a propensity to developing inflammatory lesions upon minor trauma, and could also be suggested to be ‘primed’ for lesion development. Collectively, these data led us to the following hypothesis, ‘A loss of D6 expression in uninvolved psoriatic skin is associated with the development of psoriatic lesions’. To test this hypothesis, D6 expression in clinical samples from psoriasis patients was analysed. Full thickness biopsies from psoriasis patients were taken from a histologically normal site (uninvolved psoriatic skin), in addition to an elliptical biopsy covering the skin directly adjacent to the psoriatic lesion (peri-lesional psoriatic skin), in addition to the lesion itself (lesional psoriatic skin). D6 expression was analysed in these biopsies by QPCR and immuno-staining. It was observed that D6 expression was significantly elevated in psoriatic skin compared to healthy control skin. In particular, in uninvolved psoriatic skin D6 was significantly increased compared to healthy control skin, or peri-lesional 3 psoriatic skin or lesional psoriatic skin. The increase in D6 expression in uninvolved psoriatic skin localised to the epidermis and the LVs. A significant increase in PBMC-D6 expression was also noted in psoriatic patients compared to healthy control PBMCs. These data suggest that at sites not directly involved in the pathology, D6 is elevated in an attempt to limit inflammation-induced damage. Further immuno-staining showed the inflammatory CC chemokines CCL2 and CCL5 (both high affinity D6-binding ligands) were detected in uninvolved psoriatic epidermis, but were apparently unable to mediate their function due to the lack of significant leukocyte infiltration into the tissue. These data gave rise to the idea that D6 in uninvolved psoriatic skin was significantly elevated in an attempt to block the release of inflammatory CC chemokines into the dermis, and subsequent migration of inflammatory leukocytes into the tissue, and the onset of lesion formation. Interestingly, D6 expression on the epidermis was strongest towards the lower layers of the epidermis, which suggested a role for epidermal- D6 in ‘barrier function’, preventing the uncontrolled release of inflammatory CC chemokines into the dermis. In addition to inflammatory CC chemokines, a variety of inflammatory cytokines have been previously detected in uninvolved psoriatic skin. Several of these cytokines were shown to increase D6 expression in vitro in this study. Therefore, it is possible the significant increase in D6 expression in uninvolved psoriatic skin is partly mediated by cytokine stimulation. A loss of D6 expression was observed when comparing uninvolved psoriatic skin and perilesional psoriatic skin. These data suggested that a loss of D6 expression occurs directly before the onset of lesion formation. It was also shown in this study that a loss of D6 expression could occur after micro-trauma to uninvolved psoriatic skin, which suggests a possible mechanism of how D6 expression is lost in peri-lesional psoriatic skin. To analyze whether the increase in D6 expression in psoriatic skin was disease specific, or a generic response to cutaneous inflammation, D6 expression in eczema skin was studied. It was found that D6 expression in eczema skin is elevated compared to healthy control skin, but less so compared to psoriatic skin. Collectively these data suggest that increased D6 expression may be a feature of inflammatory skin diseases.

The purpose of this thesis is to explore the diagnostic potential of Hewson's ratios in evaluating the performance of selected subjects on the Wechsler scales, the Kahn Intelligence Test: Experimental Form was administered and analyzed in an attempt to develop some meaningful method of utilizing this test in diagnosis of brain disorders.

Nosocomial (hospital-acquired) pneumonia (HAP) continues to be an important cause of morbidity and mortality in the hospital. HAP is the third most common nosocomial infection after urinary tract and surgical wound infections. In addition, HAP is the nosocomial infection with the highest mortality rate. These infections are often difficult to treat, because most are caused by Gram-negative bacilli (GNB) that may be highly resistant to antimicrobial agents. HAPs frequently occur in intensive care patients with underlying lung and/or systemic diseases. Many patients are intubated and are on assisted ventilation. Several sources of infection associated with ventilators or respirators have been described in the past. Most of these sources have been eliminated by improvement in techniques used in the disinfection and cleaning of ventilator equipment. Today, the focus of concern is microbial contamination of the breathing circuit of the ventilator. The Centers for Disease Control (CDC) recommend that the ventilator breathing circuits be changed every 24 hours. The very limited epidemiological and microbiological data from one medical center demonstrate that it may not be necessary to change these circuits as often as every 24 hours. However, before changing this conservative recommendation, more data are needed to establish the safety of changing circuits at longer intervals. The approximate cost of the ventilator circuit is $15. It is estimated that changing ventilator breathing circuits at 48 hours rather than 24 hours would amount to $50,000 per year in savings at the Medical College of Virginia. On a national scale the savings would amount to millions of dollars. Most patients who are placed on ventilatory assistance are supported by continuous volume respirators. Air is humidified when it is passed through a cascade, or wick humidifier. After passage through the humidifier, the gases are delivered to the patient by the inspiratory tubing in the breathing circuit. The inspiratory tubing is connected to the endotracheal tube of the patient by a Yconnector and swivel adaptor. Expired gases from the patient are conducted away by the expiratory tubing which connects to the other limb of the Y-connector. Condensate frequently collects in the respiratory breathing circuit. The warm moist environment of the respiratory circuit is conducive to growth of any microorganisms that may enter the circuit. When the respiratory circuit is contaminated with microorganisms, there is the potential for delivery of bacteria or fungi to the patient's lower respiratory tract. Whether or not infection takes place is determined by one or a combination of several factors including the virulence of the organisms, the size of the inoculum, the presence of foreign bodies in the respiratory tract and the status of host defenses.

There are two morphologically distinct cell types in the normal urothelium: umbrella cells and basal/intermediate cells. Immunohistochemical studies from our group suggest that there may be more than one urothelial progenitor. Bladder cancer is the fifth most common cancer in the United States and the second most prevalent genitourinary malignancy. Urothelial carcinoma accounts for 90% of bladder cancers. Based on clinical and histological studies, urothelial carcinomas are thought to develop through two independent pathways and are classified into two main phenotypic variants: low-grade tumors (usually papillary and "superficial" with high recurrence), and high-grade tumors (usually flat carcinoma <italic>in situ</italic> lesions that are often associated with and progress to muscle invasion). MicroRNAs (miRNAs) are single-stranded non-coding RNA molecules, approximately 21-23 nucleotides in length, that regulate gene expression. Since their discovery in 1993, they have emerged as major mediators of cellular functions and tissue homeostasis. Importantly, distortion of their normal function is commonly observed in human malignancies, suggesting that they act as a new class of tumor suppressors and oncogenes. Despite the strong links reported between miRNAs and the pathogenesis of numerous human cancers, there are few studies centering on their characterization in normal urothelium and there is little consensus on which miRNAs contribute to urothelial tumor initiation and progression. Through a series of studies, we profiled the expression of miRNAs in distinct compartments of the normal bladder, including umbrella and basal-intermediate urothelial cells, as well as the muscularis propria; and bladder carcinoma <italic>in situ</italic> (CIS) lesions. We discovered and validated the expression of miR-133a and miR-139-3p in umbrella cells, and miR-142-3p in basal-intermediate cells. This study represents the first molecular characterization of miRNA expression in the normal urothelium. Strikingly, we found that miRNA expression levels of CIS most closely resembled the miRNA profile of umbrella cells. Finally, we examined well-established umbrella and basal-intermediate cell immunohistochemical biomarkers in an independent series of CIS samples. Once more, this analysis revealed that CIS lesions shared a common phenotype with umbrella cells through the expression of umbrella-specific markers. Mechanistic studies were performed in parallel to further delineate the potential role of two critical miRNAs involved in cell invasion that were previously unassociated with urothelial carcinomas: miR-198 and miR-126. Overexpression of miR-198 increased cell invasion in non-invasive bladder cancer cells, an effect that was magnified with concurrent down-regulation of the miR-200 family. In contrast, elevated levels of miR-126 suppressed cell invasion in invasive bladder cancer cells, possibly through regulation of gene expression of the matrix metalloproteinase ADAM9. Correspondingly, knock-down studies of ADAM9 in invasive bladder cancer cells also inhibited cell invasion. We further demonstrated preferential expression of ADAM9 in muscle-invasive bladder tumors compared to non-muscle invasive tumors, and that ADAM9 expression significantly correlated with a poor prognosis in patients with urothelial carcinoma. Our studies represent a comprehensive and accurate description of the different miRNAs expressed in distinct urothelial cellular compartments and CIS tumors. This study is also the first to provide evidence of the possible origin of CIS lesions from umbrella cells. Additionally, important translational results of our studies support the use of miR-198, miR-126, and ADAM9 as clinical biomarkers of disease progression, and provide a rationale for the therapeutic inhibition of ADAM9 in aggressive urothelial carcinomas. Overall, the findings reported here indicate that several miRNAs are differentially regulated in urothelium development and tumorigenesis, and may form a basis for clinical development of new biomarkers for urothelial carcinoma.

Though mutant proteins are broadly expressed in neurodegenerative diseases, only some neuronal subsets are vulnerable. In patients with amyotrophic lateral sclerosis (ALS), most motor neurons degenerate but those innervating extraocular muscles, pelvic sphincters and slow limb muscles exhibit selective resistance. Previous work in our lab used laser capture microdissection followed by microarray analysis of vulnerable lumbar level (L5) motor neurons and the resistant motor neuron populations that innervate the extraocular muscles (III) and the pelvic sphincter muscles (DL) to identify hundreds of molecular markers of each subset. By looking at the genes that had > 10 fold differential expression and that were selectively and strongly expressed in motor neurons into adulthood, we identified matrix metalloproteinase-9 (MMP-9) as a potential susceptibility gene. We first examined the expression of MMP-9 in wild-type mice and found that it is expressed only after postnatal day 5 in fast motor neurons, which are selectively vulnerable in ALS. Further, MMP-9 was the only secreted member of the MMP family constitutively expressed in this population. In mutant SOD1 mice, MMP-9 expression was strongly positively correlated with cell loss at end-stage of the disease and with early induction of endoplasmic reticulum stress, as measured by p-EIF2á. To test whether MMP-9 is a driver of disease, we crossed Mmp9 null mice with mSOD1 mice and found that both partial reduction and complete ablation of MMP-9 levels delayed muscle denervation, prolonged survival, and improved motor function, measured both behaviorally and electrophysiologically. Importantly, even acute knock-down in motor neurons with AAV6 gene therapy or central inhibition of MMP-9 after symptom-onset were able to protect muscle innervation of the fast hindlimb muscle, TA. Further, MMP-9 expressed by motor neurons is required for full ER stress activation, suggesting it may be a very early intermediate in the disease pathway triggered by mutant SOD1. However, virally introducing MMP-9 into resistant pools does not confer susceptibility, implying that there is an additional factor (or factors) also present in fast motor pools that is necessary to induce axonal die-back. These studies suggest that MMP-9 is a promising candidate therapeutic target for ALS. Importantly, these data support the study of neuronal diversity as a potential way to define novel therapeutic strategies for the treatment of neurodegenerative diseases.