Research suggests that the cyclic AMP (cAMP) signaling pathway is implicated in the development of alcoholism. Previous work in our laboratory has demonstrated that alcohol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; human type 7 AC (AC7) is most enhanced by ethanol as measured by cAMP accumulation assay in whole cells. We hypothesize that alcohol enhances AC activity by directly interacting with the protein and that alcohol effects on AC can be studied using recombinant AC expressed in bacteria. Our objectives include: 1) design and optimization of the conditions for protein expression and protein purity for recombinant AC proteins; 2) identification of the importance of each cytoplasmic domain to the alcohol effect through chimeric analysis; and 3) investigation of the interaction between alcohol and AC stimulators such as MnCl2, forskolin, and Gsá through concentration-response experiments. To examine these objectives, we expressed in bacteria, and purified recombinant AC proteins carrying the C1a and/or C2 domains of AC2, AC7, and AC9. We present the optimal conditions for the expression and purification of multiple recombinant AC proteins and show that purified recombinant AC proteins retain enzymatic activity and alcohol responsiveness. Through chimeric analysis, we found that the C1a and C2 domains both contribute to the alcohol effect on AC7, however, AC7 C1a may play a stronger role. We also find that recombinant AC responds to alcohol differently under varied conditions of AC activation by MnCl2, forskolin, and Gsá. Through concentration-response experiments we found that there is some interaction between alcohol and forskolin or Gsá, but alcohol does not appear to be competing with forskolin or Gsá at an allosteric site. Overall the results suggest that alcohol interacts with AC proteins directly, independent of stimulants examined, and causes a conformational change, which results in either enhancement or inhibition depending on stimulation conditions.

Mesenchymal stem cells are multipotent cells that can differentiate into many cell types. However, the molecular mechanism controlling this process remains unclear. We utilized rat dental follicle stem cells (rDFSCs) and human adipose derived stem cells (hASCs) to study the mechanisms controlling osteogenesis and adipogenesis. Elevations in the intracellular Ca2+ concentration are a phenomenon commonly observed during stem cell differentiation but cease after the process is complete. The Transient Receptor Potential Melastatin 4 (TRPM4) is an ion channel that controls Ca2+ signals in excitable and non-excitable cells. However, there are no studies on TRPM4 in stem cells. In another study, we investigate the mechanism by which arginine vasopressin (AVP), a neuropeptide hormone secreted mostly from the posterior pituitary gland increased Ca2+ signals and inhibited adipogenesis in hASCs. The overall goal of our studies is to investigate the effect of TRPM4 and AVP on stem cell differentiation and Ca2+ signaling. First, we identified TRPM4 gene expression and its characteristics such as Ca2+-activated, voltage dependent and monovalent conducting properties in rDFSCs. Molecular suppression of TRPM4 transformed the normal agonist-induced first and secondary phases of Ca2+ signals into a gradual and sustained increase which enhanced osteogenesis but inhibited adipogenesis in rDFSCs. Next, we examined TRPM4s impact on Ca2+ signals and adipogenesis in hASCs, which is a more suitable stem cell type for adipogenic studies. Suppression of the TRPM4 diminished the histamine-induced Ca2+ signals mainly via H1 receptors. The increases in intracellular Ca2+ were due to influx via voltage-dependent Ca2+ channels of the L-type (Cav1.2) and release from the endoplasmic reticulum (ER). Lastly, we determined the role of AVP on adipogenesis in hASCs. These cells were responsive to AVP stimulation by increasing intracellular Ca2+ via V1a receptors, Gq-proteins and the PLC-IP3 pathway. Both Ca2+ release from the ER and influx from the extracellular space contribute to the Ca2+ signals. AVP supplementation to the differentiation media decreased the number of adipocytes during adipogenesis. The effect of AVP on adipocyte formation was reversed by the V1a receptor blocker V2255. In conclusion, TRPM4 and AVP control Ca2+ signals which affect stem cell differentiation.

Nucleotide excision repair (NER) is a multi-step cellular process that removes bulky and/or helix-distorting DNA lesions, such as UV induced cyclobutane pyrimidine dimers (CPDs) and bulky chemical adducts. Transcription coupled repair (TCR) is a subpathway of NER dedicated to rapid removal of lesions in the transcribed strand of actively transcribed genes. The TCR mechanism in bacteria has been relatively well elucidated. However, TCR in eukaryotic cells appears to be extremely complicated. The exact nature of the TCR signal and the mechanism of the transcription-repair coupling have been long-standing enigmas. This dissertation focused on how the TCR repressors and facilitators interplay with RNA polymerase II (RNAP II) to carry out TCR in yeast Saccharomyces cerevisiae. By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, we mapped interactions between Spt5 and RNAP II in S. cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR. We also demonstrated that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced CPDs, increases survival of UV irradiated yeast cells but attenuates TCR. In contrast, G730D mutation of Rpb1, which abolishes transcription bypass of CPDs, enhances TCR. Our findings suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. Sen1 is an RNA/DNA helicase that has been shown to mediate termination of noncoding RNAs and some mRNAs. Like deletion of Rad26 or Rpb9, the Sen1 N-terminal deletion (1-975 residues) increases the UV sensitivity of the GGR-deficient cells. Moreover, the Sen1 N-terminal deletion decreases TCR in rad7Δ and rad7Δ rad26Δ cells but not that in rad7Δ rpb9Δ cells. Our findings suggest that the N-terminal domain of Sen1 contributes to Rad26-independent TCR.

Post-traumatic stress disorder (PTSD), a trauma- and stressor-related disorder, is a condition that can develop in response to life-threatening situations. According to the Diagnostic and Statistical Manual of Mental Disorders 5 (DSM-5), a diagnosis of PTSD necessitates exposure to a life-threatening event, intrusive recollections, avoidance of associated stimuli, hyperarousal, and a significant social impairment. All of these symptoms must persist for at least 30 days and not be due to illness, medication, or substance abuse. To date, no definitive diagnostic biomarkers have been identified for PTSD. Recent research, however, points toward physiological abnormalities in the brain, hypothalamic-pituitary-adrenal (HPA) axis, and immune system that may be partially responsible. Many chronic conditions such as hypertension, heart failure, and metabolic syndrome perpetuate in a state of increased inflammation and oxidative stress, exacerbating their pathophysiology. In many psychiatric conditions such as depression and anxiety disorders, neurotransmitter modulation may play a critical role in their pathogenesis. Based upon the literature and work from our laboratory, we hypothesized that similar pathophysiological mechanisms may play a role in PTSD development. We tested our theory by creating a PTSD-like syndrome in rats with the use of a predator exposure/psychosocial stress animal model. We then conducted a series of in vivo and ex vivo experiments in an attempt to discover the roles of inflammation, oxidative stress, and neurotransmitter modulation in PTSD development. First, we evaluated inflammation and oxidative stress in the brain, adrenal glands, and blood in response to the predator exposure model. We then analyzed neurotransmitter modulation in the hippocampus and prefrontal cortex. Next, we investigated the anti-inflammatory and neuromodulating effects of the histone deacetylase inhibitor (HDACi) valproic acid (VA) on inflammation/oxidative stress and neurotransmitters. Finally, we employed the selective-serotonin reuptake inhibitor (SSRI) sertraline to ascertain why SSRIs have historically been ineffective in treating PTSD. Taken together, our findings indicate inflammation, oxidative stress, and aberrant neurotransmitter profiles may play a significant role in PTSD development and progression. In addition, VA may prove to be a legitimate pharmacologic alternative in PTSD treatment, as SSRIs may increase the noradrenergic response and actually exacerbate anxiety in a clinical setting.

Informal and incidental use of peer education is common practice, but it has lacked definition in its implementation. Considerable literature is in existence. However, there is a lack of quality evidence on the application and effectiveness of this educational intervention. This study is an analysis of existing data on the intervention peer teaching and learning in clinical education. In the context of this thesis peer teaching and learning is undergraduate health science students teaching and learning from one another in clinical practice settings. The purpose of this study is to increase the theoretical body of knowledge, which informs this educational practice. / A search was conducted of health science and educational electronic databases using the terms peer, clinical education and undergraduate. The set limitations of the search were publication after 1980, English language and research paper. From this thirteen articles met the inclusion criteria, after rigorous critical and quality appraisal. This appraisal was conducted with a tool developed by the researcher that allowed the synthesis of both quantitative and qualitative studies specific to education. The authors of included studies were contacted regarding knowledge of existing articles that had been missed in the initial search. Hand searching of two key journals, Nurse Education Today and The Journal of Nursing Education, produced no further studies. The selected studies were rated according to the National Health and Medical Research Council, 2000 (NHRMC) levels of evidence for guideline development. The results have been collated into a narrative summary. / According to the NHRMC (2000) guidelines the evidence is low, it was accumulated from both quantitative and qualitative studies and is listed, along with the recommendations, in the final chapter of this thesis. This study demonstrated mostly positive outcomes on the effectiveness of peer teaching and learning; it increases students' confidence in clinical practice, enhances learning in the psychomotor, affective and cognitive domains. And evidence exists that it allows for greater student numbers in a clinical practice area. The negative aspects identified included poor student learning particularly if personalities or learning styles were not compatible and a reduction in time spent singularly with the clinical instructor. / Thesis (MNursing)--University of South Australia, 2004.

Thesis (PH.D.)--Syracuse University, 2003. / "Publication number AAT 3113234."

Thesis (PH.D.)--Syracuse University, 2003. / "Publication number AAT 3081641."

Thesis (Ph. D.)--Syracuse University, 2005. / "Publication number AAT 3194020."

Thesis (Ph. D.)--University of Sydney, 2001. / Title from title screen (viewed Jan. 22, 2009) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Physiotherapy, Faculty of Health Sciences. Includes bibliography. Also available in print form.

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