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.

Legionella pneumophila was first recognized as a cause of human pneumonia in 1976 . Since then, much has been learned about the microbiology, pathophysiology and epidemiology of this organism. The features which permit one strain but not another to invade human lung tissue and produce disease remain incompletely understood. This study e valuated several attributes of a virulent and an avirulent strain of L. pneumophila in an attempt to identify characteristics which would distinguish the two. Evaluation of a new medium, buffered egg yolk agar, showed that virulence was maintained after 26 passages, which was the same as the buffered charcoal yeast extract agar used for comparison. However, growth appeared earlier and was heavier on the charcoalcontaining medium. Morphologically, the avirulent strain produced greater numbers of filamentous forms and was found to be encapsulated more frequently. Treatment with polymixin B produced morphologic changes similar to those previously reported but failed to alter the virulence of either strain. No plasmids were found in either strain nor were consistent differences in protein content demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Both strains reacted less intensely as cultures aged with a monoclonal but not a polyclonal antibody in a direct fluorescent antibody assay . This change was more pronounced when the virulent organism was tested. Chemotactic assays showed similar tendencies when human or guinea pig mononuclear cells were compared to two estuarine species (hogchoker and spot) and one freshwater species (golden shiner minnow) of fish. In vivo results were also similar when two of the three species of fish were tested, suggesting that either the spot or the minnow may be used in evaluation of certain characteristics of L. pneumophila. Organisms were isolated from apparently healthy fish up to 15 days after inoculation in some instances. This suggests that fish may be a possible additional environmental reservoir for Legionella pneumophila.

The highly polymorphic DlS80 locus has no known genetic function. This variable number of tandem repeat (VNTR) has been valuable in forensic identification. We have obtained allelic and genotypic frequencies for five African populations (Benin, Cameroon, Egypt, Kenya and Rwanda), which could be employed as databases to identify individuals. The polymerase chain reaction, followed by vertical polyacrylamide gel electrophoresis and silver staining was our method of analysis. Allele frequencies were used to infer genetic associations using Phylip 3.5, Principal Component and G-test statistical programs. Tests for Hardy-Weinberg equilibrium were employed. Fst estimates and power of discrimination values were also determined for each of our populations. Our analyses of 28 additional populations demonstrated that the D1 S80 locus alone provided for the discrimination of major racial groups. Genetic homogeneity between the African groups was observed. We have generated a database useful for human differentiation and phylogenetic studies.

The primary aims of this study were to examine acquired genetic and epigenetic changes that occur in individuals with increasing age and to determine how these changes are influenced by genetic/environmental factors. Acquired genetic changes were assessed by determining the frequency and chromosomal contents of spontaneously occurring micronuclei in identical and fraternal twins. A total of 115 individuals (48 twin pairs and 19 singletons) were evaluated, ranging in age from 7 to 85 years. As expected, micronuclei frequencies, which are indicative of genomic damage, significantly increased with age (p<0.0001, r=0.446). The majority of micronuclei (32%) contained sex chromosomes and the frequency of sex chromosome-bearing micronuclei significantly increased with age (p<0.0001). The frequency of autosome-containing micronuclei was not significantly influenced by age or gender. However, some autosomes were seen more (chromosomes 4, 8, and 9) or less (chromosomes 17 and 22) frequently than expected by chance (p<0.05). An evaluation of the numerical contents of the sex chromosome-containing micronuclei and their corresponding binucleates showed that the majority of the binucleates had an abnormal chromosomal complement (either hypodiploid or hyperdiploid), with the subset of binucleates having a normal chromosomal complement decreasing with age for both the Y chromosome in males and the X chromosome in females. Model fitting, implemented in Mx, showed the variation in the frequency of micronuclei to be best explained by either additive genetic and unique environmental components, or common and unique environmental factors. Specific environmental exposures and health conditions that were shown to influence micronuclei frequencies, included: multivitamins, leafy green vegetables, fruit, vitamin E supplements, arthritis, heart disease, allergies, and alcohol. To assess acquired epigenetic changes, global methylation profiles of two identical twin pairs were compared and found to differ, indicating that individuals do develop alterations in their methylation profiles with age. Furthermore, the twin pair having a significant difference in their micronuclei frequencies and environmental exposures had more differences in their methylation pattern compared to the twin pair whose micronuclei frequencies and environmental factors did not differ. Overall, genetic and epigenetic changes were shown to occur with age and to be influenced by genetic and lifestyle factors.

<p>Breast cancers are the most common source of metastases to bone of which cancer-induced bone pain is a frequent pathological feature. Cancer-induced bone pain is a unique pain state with a multiplicity of determinants that remains to be well understood and managed. Current standard treatments are limited by dose-dependent side effects that can depress the quality of life of patients. Glutamate is a neurotransmitter and bone cell-signalling molecule that has been found to be released <em>via</em> the system x_C^-cystine/glutamate antiporter on cancer cells of types that frequently metastasize to bone, including breast cancers. This project examines the hypothesis that limiting glutamate release from cancer cells metastasized to bone will reduce bone tissue disruption and cancer-induced bone pain. A mouse model of cancer-induced bone pain was established with intrafemoral human breast cancer cells (MDA-MB-231), and behavioural measurements were taken for weight bearing and induced paw withdrawal thresholds. The system x_C^- inhibitors sulfasalazine and (S)-4-carboxyphenylglycine both attenuated glutamate release from cancer cells in a dose-dependent manner <em>in vitro</em>. Treatment with sulfasalazine induced a moderate delay in the onset of behavioural indicators of pain in mouse models, and treatment with (S)-4-carboxyphenylglycine had no apparent results. This data suggests that the limitation of extracellular glutamate released from cancers in bone with sulfasalazine may provide some alleviation of the often severe and intractable pain associated with bone metastases.</p> / Master of Science (MSc)

Telomerase is a ribonucleoprotein that is reactivated in cancer cells to allow for continuous cellular division and indefinite growth. With telomerase being expressed in more than 85% of all cancer, it is imperative that we understand how to selectively inactivate and degrade this unique DNA polymerase. In doing so, we can specifically target tumor cells to erode their telomeres so that they will undergo apoptosis or senescence. Through this research, we have learned that telomerase can be degraded in the nucleoplasm by Hsp90 chaperone inhibition and in the cytoplasm by the dominant negative mutant, D712A V713I. These findings should guide future drug design to target sites on telomerase that interact with Hsp90 and catalytic divalent metal ions. Previous studies have shown that chaperones function to stabilize the RNP and that their inhibition results in ubiquitin-mediated degradation. However, a detailed understanding of how telomerase is signaled for degradation is not well defined. We showed that Hsp90 inhibition causes telomerase to be degraded by a nuclear ubiquitin/proteasome pathway such that exportation to the cytoplasm is not required. Using confocal fluorescence microscopy and immunoprecipitation /Western analysis, we showed that nucleoplasmic GFP-hTERT is ubiquinated and degraded within 2 hrs of exposure to the Hsp90 inhibitor, Radicicol. Upon combined treatment with the proteasome inhibitor, MG132, degradation is inhibited as shown by Western analysis and fluorescent intensity. Additionally, fluorescent pattern with inhibition of degradation shows telomerase aggregation and co-localization with the nuclear proteasome and not with nucleoli. However, the combined treatment with the exportin inhibitor, Leptomycin B, resulted in complete loss of fluorescence. Taken together, these data suggest that Hsp90 inhibition causes telomerase to immediately undergo nuclear degradation, which may function in the nuclear quality-control of telomerase. The dominant negative expression of telomerase has been shown by many investigators to cause shortening of telomeres. However, the mechanism of how it functions and its fate inside the cell are still unknown. After stably expressing the wild-type and dominant negative mutants GFPhTERT in cells, we show that the D712A V713I mutation causes the ubiquination and degradation of the mutant and wild-type hTERT which eventually leads to the shortening of telomeres. Degradation appears to be cytoplasmic since the additional mutation for the nuclear export signal (nes) and treatment with the exportation inhibitor are able to prevent the reduction in protein levels and fluorescence. Based on this cytoplasmic degradation and the additional co-localization of the GFPDNhTERT to the nucleoli, we propose two new mechanisms of dominant negative hTERT utilizing the theory of interactive dimerization. First, the heterodimer of DNhTERT : wt hTERT may be degraded at a faster rate than the wt hTERT homodimer. Second, the heterodimer may be sequestered in the nucleoli thus diminishing the wild-type hTERT access to the telomere in the nucleoplasm. Overall, we have shown that telomerase can be degraded in the nucleoplasm or cytoplasm depending on the mechanism of inhibition. The significance of this is a better understanding of how Hsp90 inhibition and dominant negative hTERT expression cause the degradation of wild-type hTERT. We have also suggested potential mechanisms of dominant-negative hTERT effect and resistance. With this knowledge, future drug therapies can be designed based on these inhibitors to not only inactivate but also to cause the degradation of an enzyme that is crucially important for the immortalization of cancer cells.

The primary study aim was to evaluate the latent biological effect of childhood sexual abuse (CSA) on adults by quantifying acquired cytogenetic changes and cortisol levels in identical twins who were discordant (N=22) or concordant (N=2) for a history of CSA. Although the difference scores for cortisol values between discordant identical co-twins were not significantly different from zero, a trend was observed for the twins exposed to intercourse, the most severe form of CSA, to have a blunted cortisol awakening response. Acquired cytogenetic changes were assessed by scoring telomere lengths and somatic cell abnormality frequencies via a cytokinesis-block micronucleus (MN) assay. No significant difference in overall telomere intensity values was observed between co-twins, but chromosome-specific telomere differences were observed in the individuals exposed to intercourse compared to their unabused co-twins ([χ2(45)= 62.88; p= 0.040 and χ2(45)= 73.72; p= 0.004). Specifically, shortened telomeres were observed on the short arms of chromosomes 3, 5, & 6, and long arms of chromosomes 11 & 13. A significant increase in MN frequencies was observed in the abused twins compared to unabused twins (t=2.65; df=16; p=0.009). A significant interaction between micronuclei frequencies and age was also observed, suggesting that the biological effects of stress are cumulative (coefficient [SE] = 0.030 [0.009]; p=0.0006). However, the pattern of chromatin present in MN, which was assessed using spectral karyotyping methodologies, was not limited to the subset of chromosomes with telomeric attrition. In summary, this is the first assessment of acquired chromosomal abnormalities, chromosome-specific telomere lengths and cortisol levels in identical adult twins discordant for exposure to CSA. Given that a portion of biological changes were most pronounced in the intercourse discordant twins, these findings support a possible dose-response relationship with CSA severity. Our data also suggest that the MN assay is a superior tool in assessing the latent effects of stress compared to either cortisol profiling or the measurement of telomere lengths. Collectively, application of the information gained from these studies may allow for novel screening techniques to identify individuals who are most at risk for developing stress-associated disease states.

Normal somatic cells have a limited proliferative capacity in vivo and in vitro, termed senescence and later, thought to contribute to molecular and cellular organismal aging. There are several studies that demonstrate the importance of the GH/IGF axis in longevity, aging and cellular senescence. One primary component of the IGF signaling involves IGFBP-3. It is well documented that IGFBP-3 levels are significantly increased in senescent human diploid fibroblasts however IGFBP-3 function is not known in this system. Interestingly, Werner syndrome fibroblasts, commonly used as a model of cellular aging, have upregulated IGFBP-3 levels in young and late passage cells compared to age matched normal fibroblasts. It is known that suppression of p38 MAPK activity in WS fibroblasts can reverse the senescence and promotes cell proliferation. As increased IGFBP-3 expression is associated with cellular senescence, and suppression of p38 MAPK can reverse senescence in WS fibroblasts, it is hypothesized that “IGFBP-3 can induce senescence, by activating the p38 MAPK signaling pathway.” Our studies demonstrate IGFBP-3 and novel IGFBP-3R can induce senescence in young fibroblasts, while suppression of IGFBP-3 in pre-senescent fibroblasts, can delay the onset of replicative senescence. We identified ROS accumulation in IGFBP-3/IGFBP-R-induced senescent cells which we speculated may be signaling p38 MAPK activation. Inhibition of ROS accumulation suppressed p38 signaling and prevented IGFBP-3/IGFBP-3R-induced senescence. To evaluate the sequence of activation we inhibited p38 activity prior to senescence induction. Interestingly, p38 inhibition prevented IGFBP-3/IGFBP-3R-induced senescence, suggesting IGFBP-3 signals ROS induction which activates p38 signaling. We next examined the significance of IGFBP-3R in IGFBP-3-induced senescence. Suppression of endogenous IGFBP-3R inhibits IGFBP-3-induced senescence. We aimed to identify a possible regulatory mechanism for IGFBP-3 upregulation. Using sequence analysis software we identified 3 possible highly conserved miRNA sequences aligned to IGFBP-3. miR-19a appeared to have the most significant downregulated expression in late passage fibroblasts compared to early passage. Furthermore, overexpression miR-19a in late passage cells, significantly decreased IGFBP-3 expression, suggesting miR-19a may silence IGFBP-3 expression in senescence. Making a direct mechanistic connection between senescence and aging is significant and unraveling how IGFBP-3/IGFBP-3R can induce senescence could prove beneficial in understanding the aging process.

To repair, replace, or regenerate damaged or diseased tissue has been a long-standing, albeit elusive, goal in medical research. Here, we characterize patient-derivable mesenchymal stem cell types, termed adipose-derived stem cells (ASCs). These cells, which can be derived from liposuction fat and lipoaspirate saline, are sources for patient-derivable extracellular matrix (ECM), fibrinogen (Fg) and adipose tissue extracellular matrix, and may prove useful for synthesizing new bone tissue analogues in vitro. Traditionally and rapidly isolated ASCs were thoroughly characterized as multipotent, having osteogenic, adipogenic, and chondrogenic differentiation potential, and they exhibited comparable proliferative lifespans. These ASCs also shared an indistinguishable immunophenotype when compared to bone marrow-derived mesenchymal stem cells, suggesting that these cells are an excellent source for bone following tissue engineering experimentation. In order to synthesize bone ex-vivo, electrospun scaffolds of fibrinogen (Fg), polydioxanone (PDO), and Fg:PDO blends were seeded with early passage ASCs, fibroblasts, or osteosarcoma cells and were maintained for 21 days in osteogenic or regular growth media. Constructs were analyzed both histologically and molecularly for evidence of osteoblastogenesis. Using SEM, the appearance of regular, mineralized-appearing structures were found in osteogenic-induced ASC seeded scaffolds beyond 14 days, only in the scaffolds containing Fg. Further, at 21 days of culture, Fg scaffolds with ASCs in osteogenic media became hard and brittle. Robust new collagen synthesis and matrix remodeling were observed on all Fg scaffolds, the levels of which were elevated over time. Pronounced mineralization was found throughout bone-induced ASC scaffolds, while control scaffolds (BJ foreskin fibroblasts) showed no mineral deposition (although they did demonstrate excellent cellularity). Analysis of gene expression (qRT-PCR) indicated that electrospun Fg supported osteoblastogenesis through the upregulation of alkaline phosphatase and osteocalcin gene expression. To confirm our gene expression results, osteogenic-induced ASCs on Fg scaffolds were also shown to secrete osteocalcin in the extracellular matrix, a key marker in osteoblastogenesis. Thus, electrospun Fg is an excellent material for ASC growth, proliferation, and osteogenic differentiation, providing an ideal system for furthering basic bone model-based research and for advancing regenerative medicine. In addition to establishing Fg as a source of scaffolding, we developed and characterized a novel method for isolating and subsequently electrospinning adipose tissue matrix. Because adipose ECM contains many primordial matrix proteins important for embryonic development and regeneration (such as laminin, type IV collagen, and fibronectin), adipose ECM may prove to be an autologous tissue engineering matrix and stem cell culture substrate. We show here that adipose tissue ECM can, in fact, be electrospun into a nanofiberous mesh, histologically shown to contain connective tissue, collagens, elastic fibers/elastin, proteoglycans, and glycoproteins in the newly synthesized matrix. We also show that this novel electrospun adipose tissue scaffold is capable of supporting stem cell growth. Taken together, experiments using ASCs cultured on extracellular matrices of electrospun Fg or adipose ECM present an excellent framework for future advances in regenerative medicine therapeutics and research.

Title of Dissertation: FUNCTION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 7(IGFBP7) IN HEPATOCELLULAR CARCINOMA By Dong Chen. Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring the development of innovative and effective targeted therapies. The oncogene Astrocyte Elevated Gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates Insulin-like Growth Factor Binding Protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarrays by real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at the IGFBP7 locus. Stable IGFBP7- overexpressing clones were established in the background of AEG-1- overexpressing human HCC cells and were analyzed for in vitro proliferation, senescence, in vivo tumorigenesis and angiogenesis. HCC cell lines infected with an adenovirus expressing IGFBP7 (Ad.IGFBP7) were analyzed by using in vitro cell cycle, apoptosis, in vivo tumorigenesis assays. Results: IGFBP7 expression is significantly downregulated in both human HCC patients’ samples and cell lines compared to normal liver and hepatocytes. IGFBP7 expression was also found to inversely correlate with the stages and grade of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence. When injected into nude mice, in vivo growth was profoundly suppressed, potentially as a result of inhibition of both angiogenesis and IGF1R activation by IGFBP7. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing Reactive Oxygen Species (ROS) and activating a DNA damage response. N-acetylcysteine could neutralize ROS and rescue the cells from apoptosis. In early phase after Ad.IGFBP7 infection, activation of cell cycle control proteins like Rb, p53, ATM, ATR, CHK1 and CHK2 were identified and G2/M cell cycle arrest was recorded by FACS. Ad.IGFBP7 infection resulted in the activation of p38 MAPK, and a p38 MAPK inhibitor SB 203580 could block the apoptotic process. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intra-hepatic metastasis. In a nude mouse subcutaneous model, xenografts from human HCC cells were established in both flanks and only left- side tumors received intratumoral injection of Ad.IGFBP7. Ad.IGFBP7 markedly inhibit growth of both left-sided injected tumors and right-sided un- injected tumors by profound suppression of angiogenesis. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Targeted overexpression of IGFBP7 may be a potential novel and effective therapy for HCC.