Thesis (M.S.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

Brain cancers inflict a disproportional burden of mortality upon sufferers. Due to the difficulties of diagnosis and treatment, survival is low with most patients succumbing to the disease within a year of diagnosis. This has been the imperative factor to underpin some of the molecular mechanisms regulating gliomas and to aid with the development of novel disease biomarkers specifically targeting immortalisation (telomerase). 1[mmortalisation not only requires telomerase but also an imbalance/inactivation of DNA repair functions such as 06 methylguanine-DNAmethyltransferase (MGMI) which is a major obstacle with regards to glioma chemotherapy. The primary focus of this study was to evaluate whether there was an association betweenh TERT and its many subunits: TEPI (human telomerase-associated protein 1), TNKS ( tankyrase), DCKI (dyskerin) and PARPI (poly (ADP-ribose) polymerase1 ) in human glioma compared to normal brain tissues and cell lines. Evidence suggests that hTERT was the only gene that was transcribed in all the glioma cell lines and tissues, while absent in the normal. Thus hTERT may represent a simple but reliable biological marker for distinguishing glioma tissues from normal. The hTERT gene is subjected to its own highly coordinated regulation in normal and cancer cells however, whether hTERT is regulated via methylation/demethylation in glioma is unknown. 5azadC treatment reduced hTERT expression, which resulted in the downregulation of telomerase protein and its activity. However, an inverse correlation between hTERT and MGMT expression was observed after treatment. Evidence shows that hTERT inactivation enhances sensitivity towards some chemotherapeutic agents, thus a combination of 5azadC with various chemotherapeutic agents proved to be more effective than chemotherapy administered on its own. Although 5azadC treatment activates MGMT and subsequently hyposensitises glioma cells toward alkylating agents, specifically TMZ (temozolomide), it may compensate by offering telomerase sensitised cells, thus providing an alternative avenue of therapy for the 50% of the glioma patients chosen for this study that have an unmethylated MGMT promoter. However, further clinical developments concerning this approach will be required. 5azadC is known for its toxicity and its effects are diverse, thus it is not favoured as a therapeutic agent. To address these issues three DNMTI (methylation gene) siRNAs were used, downregulating DNMTI directly and hTERT indirectly (possibly via the promoter methylation inhibition mechanism). Combining siRNA and chemotherapeutic agents (TMZ or taxol) enhanced the effects of both the drugs, thus offering an alternative method of treatment using lower concentrations of the drug and hence, reducing side effects, and improving the life expectancy of glioma patients. All glioma tissues used in this study, irrespective of grade or invasiveness, transcribed hTERT at approximately similar copy numbers however, only some of these tissues translated this to protein. This could be due to hTERT mRNA splicing or the involvement of other regulating factors. Several studies have reported that protein translation decreases with age, this would explain why only two of the older patient samples had detectable telomerase activity when they all expressed hTERT mRNA. However, additional studies using a larger cohort of glioma tissues would be needed to further understand the precise mechanism for the discrepancy in hTERT translation in the older glioma patients. Four novel sets of investigations were documented in this thesis resulting in publications: a) 77VKS expression was evaluated in glioma tissues for the first time, b) the effects of the demethylating agent 5-aza-2'-deoxycytidine (5azadC) on the expression of hTERT and MGMTin glioma cells was evaluated, C) combining DNMTI siRNA and chemotherapeutic agents (TMZ or taxol) enhancedth e efficacy of the chemotherapeutiacgents and, finally d) although, h TERT transcription was found in all glioma tissues used in this study irrespective of grade or invasiveness (at similar copy numbers), a discrepancy in translation was documented. Some of the finding from this study may well become the starting point for integrating translational research in to future clinical trial designs for cancer, specifically for glioma patients.

The extent of decomposition of a body can be used, in conjunction with accumulated degree days (ADD), to provide an estimate of the post-mortem interval (PMI). PMI estimations are important in aiding police to narrow down the possible identity of a body, and to include or exclude suspects, and also to establish the order of death for inheritance purposes when two or more potential beneficiaries die at around the same time. Previous studies have shown the decomposition pattern in hanging bodies to be different from that of a body on the ground, but the sample sizes used have been small. This study presents the results of a series of decomposition studies on hanging bodies in a variety of situations; clothed and unclothed, and fully or partially suspended. The study used domestic pigs (Sus scrofa) which enabled large enough sample sizes for statistical robustness. Pigs lying on the ground were used as controls. The pattern of decomposition in hanging pigs was found to differ sufficiently from that of pigs lying on the ground to require the creation of a novel decomposition scoring scale, which was used successfully to score both clothed and unclothed fully suspended bodies, as well as the upper, suspended, part of partially suspended bodies. The presence of loose, lightweight clothing, which did not impede insect access, was found to affect both the pattern and rate of decomposition in hanging pigs, with the clothed bodies decomposing faster than the unclothed bodies (p < 0.05, F2, 477 = 1238). The variations in the start weights of the pigs used for these studies was found to have a statistically significant effect on the rate of decomposition for both the hanging bodies and those on the ground (p < 0.05, F5, 714 = 1962) but the effect was so small as to make no practical difference across the range of start weights encountered. The effect of variation in start weight may be of greater concern, however, in scoring very heavy, obese, bodies and may be exacerbated by the increased fat-to-muscle ratios encountered in such bodies. Finally a set of ADD prediction tables were produced for the hanging and surface pigs. Further work is needed to establish to what extent these tables can be used for humans and, in light of the growing obesity problems in humans, to investigate the effect of weight and increased fat-to-muscle ratios on the pattern and rate of decomposition.

Volume 1 (A/B) of the thesis investigated the innervation pattern, the cellular mechanism(s) of stimulus-secretion coupling (SCC), the effects of age and the in eraction between calcium and magnesium signalling in secretory cells taken from the thyroid gland, the parotid land, the ancreas, the stomach and the lacrimal gland. 1n addition, the study investigated the roles of anti-ulcer and anti-secretory drugs in the stomach and calpains in relation to vision. The study was designed to understand diseased states ( eg diabetes mellitus, pancreatitis, gastric ulcers, cataractogenisis and the dry eye syndrome) compared to healthy conditions. The results have demonstrated that, in addition to parasympathetic nerves, the pancreas, parotid and the lacrimal glands are innervated with adrenergic and non-cholinergic, non-adrenergic (peptidergic) nerves. Stimulation of these intrinsic secretomotor nerves can result in the secretion of proteins and enzymes from the pancreas, the parotid and lacrimal glands. Exogenous application of either acetylcholine (ACh), noradrenaline (NA), or a number of neuropeptides (vasoactive intestinal peptide (VIP), substance P, neuropeptide Y (NPY) and several others), can elicit protein and enzyme secretion. Apart from nervous control, exocrine pancreatic secretion is regulated by both endocrine and paracrine hormones. Cholecystokinin-octapeptide (CCK), secretin and histamine can stimulate protein and enzyme secretion from the pancreas. In addition to innervation pattern, this study also investigated the cellular mechanisms of stimulus-secretion coupling. The results have shown that there are four functionally distinct intracellular signalling pathways via which secretagogues (neurotransmitters, hormones and drugs) can elicit their responses in secretory epithelial cells. These include the utilization of such second messengers as calcium (Ca2+), adenosine 3, 5 cyclic monophosphate ( cyclic AMP), protein kinase C (PKC) and tyrosine kinase (TK).

The high mobility group protein HMGA2 is an architectural transcription factor, which is expressed during embryogenesis. Aberrant expression causes benign and malignant tumor formation. The protein possesses three "AT hook" domains and an acidic Cterminal. HMGA2 is natively unstructured, however it forms a homodimer. In this study site-directed mutagenesis was used to create single methionine mutants, HMGA2Q37M, HMGA2I71M and HMGA2Q85M. These mutants were cross-linked using EDC and then cleaved using CNBr to determine which domains are involved in homodimer formation. Our results indicate that the second "AT hook" domain may interact with the C-terminal. We then labeled a peptide containing the C-terminal (CTP) with tetramethylrhodamine-5- maleimide (TRM). We found that the CTP-TMR binds to HMGA2Α95-108, which lacks the C-terminal. These results suggest that the C-terminal is required for homodimer formation. The techniques used within this study can be applied to forensics and with further research HMGA2 may have a forensic application.

Due to the threat of bioterrorist acts, there is a need to develop techniques that rapidly detect possible bioagents. Amplicon length heterogeneity-polymerase chain reaction (ALH-PCR) presumptively identifies eubacteria in samples by detecting differences between the lengths of the hypervariable regions of the 16S rRNA gene. To study the efficiency, reproducibility, and reliability of the technique, sputum from cystic fibrosis (CF) patients has been chosen as the model system. There is an abundance of microorganisms in the sputum of the CF lung. Using ALH-PCR, the complex microbial diversity and vast community composition in the lungs of the CF patients were studied. Twenty-four out of twenty-six CF samples were presumptively identified to contain Pseudomonas aeurginosa, a known CF pathogen. Sputum profiles were also compared over time and ALH was able to demonstrate that the CF lung flora is a dynamic community and may be affected by antibiotics.

The purpose of this research was to develop a strategy for the DNA typing of Cannabis sativa. DNA typing was accomplished by using the Amplified Fragment Length Polymorphism (AFLP) method. Cannabis (marijuana) was selected because it is an illegal substance grown across the United States and in other countries and the sourcing of this plant is of interest to law enforcement agencies. While some peaks may be shared, the overall peak profile is expected to contain some peak differences between individuals. The AFLP procedure was performed on 25 different cannabis samples using four different primer combinations. In all of the AFLP profiles, there were significant peak differences that allowed for all the samples to be distinguished. The samples that were genetically related displayed the same peak profiles in the electropherograms and the samples that were not related possessed unique profiles. The development and validation of the AFLP method would lead to the creation of a database that could then be used to link cloned samples and to track distribution networks and, ultimately, individual plants could be linked together.

The purpose of this research is to introduce a method for the forensic elemental analysis of cotton fibers for the purpose of increasing the discrimination between otherwise similar cotton evidence using microwave digestion Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and Laser Ablation- Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). A quadrupole ICP-MS and UV laser ablation (266nm) instruments were used for the analysis. A cotton standard reference material (IAEA V-9) was used to validate the developed methods producing good accuracy with typically 10 % bias and good precision (typically 5% RSD) for the element list: 25Mg, 27Al, 55Mn, 57Fe, 88Sr and 137Ba. It was found that the LA-ICP-MS method resulted in improved precision over the solution ICP-MS method. Twenty four (24) raw cotton samples and five white cotton T-Shirts were analyzed with the developed methods. It was also found that all the raw cotton samples from different sources were distinguishable from each other, as were all the cotton T-shirts resulting in zero type I errors and zero type II errors for the pairwise comparisons.

I. Investigations were carried out to establish (a) the regulation of magnesium homeostasis in the exocrine pancreas under agonist stimulation (b) that secretagogueevoked cellular processes in the exocrine pancreas are sensitive to physiological changes in Mg2+ and (c) the intracellular roles of Mg2+ in the secretagogue-evoked exocytotic processes in the exocrine pancreas. H. CCK8 and ACh can induce a dose-dependent transmembrane efflux of Mg2+ which results in a subsequent lowering of [M g2+] . The routes for movement of Mg2+ do not appear to be associated with those sites of Ca2+ mobilisation from either the internal Ca2+ stores or across cell membranes but appears to be associated with those of Na+ and is ATP-dependent. Stimulation with secretin results in an elevation of [Mg2+] compared to resting values. The secretin-evoked elevation of [Mg2+] may be coupled to G protein activation of adenylate cyclase. Digital imaging technology further demonstrates that mobilisation of Mg2+ follows a different route from that of Ca2 . The CCK8-evoked Ca2 signal is initiated in an area towards the luminal pole of the pancreatic acini and rapidly spreads throughout the entire cell. Stimulation of single pancreatic acini with CCK8 results in an initial transient increase in [Mg 2 ] throughout the entire cell which is followed by a sustained decrease in [Mg2+] which is more prominent around the basolateral membrane. III. Pertubations in extracellular and intracellular Mg2+ had marked effects on the CCK5- and secretin-evoked pancreatic juice flow and protein output in (a) the isolated perfused intact pancreas, and (b) amylase release in pancreatic segments and permeabilised pancreatic acini. A nominally Mg2+ deficient saline augments the CCK8-induced responses, whereas elevated Mg2+ attenuated secretion compared to normal (1.0 mM) Mg2 conditions. Parallel effects were found on Ca 2 mobilisation in Fura 2 loaded acini suspensions and on oscillations of Ca2+ in single acini stimulated with CCK8. These effects of Mg 2 appear to be mediated by direct ffil modification of Ca2+ release, propagation and re-uptake into cytoplasmic stores. The effects of Mg2+ on secretin-evoked secretion followed a bell shape characteristics curve for Mg2+dependent enzyme activity, i.e. elevating and reducing the intracellular free magnesium levels attenuate the secretin-induced response. IV. Infusion of CCK8 and secretin evoked time course increases in both pancreatic juice flow and total protein output in anaesthetised rats and in the isolated perfused intact pancreas. Simultaneous application of CCK5 and secretin caused some degree of attenuation in secretory responses. This reduction in secretion may not involve protein kinase C or cyclic AMP but may occur at some point prior to the activation of protein kinase A. Secretin may influence mobilisation of Ca2+ which is required to initiate secretion, possibly by stimulating influx of Mg 2 . V. Electrical stimulation of vagus nerves led to elevated pancreatic juice flow and protein output in anaesthetised rats. Secretin attenuated these secretory parameters. Activation of protein kinase C with TPA, had no significant affect on these nervemediated responses. Electrical field stimulation (EPS, 50 V, 20 Hz, 1 msec) andlor ACh stimulation of pancreatic segments results in marked amylase release; 45Ca2+ influx and elevations in [Ca 2+]i. Secretin had little effect on these parameters but attenuated the EPS and ACh-induced responses. The secretin-evoked inhibition of EFS and ACh -induced secretion was abolished when extracellular Mg2+ was absent. Secretin may control nerve-mediated secretory responses by stimulating an influx of Mg2+ to interact with Ca2+. VI. The present investigations have demonstrated that transport mechanisms exist in the exocrine pancreas to regulate cytosolic free Mg2+ levels and that exocytotic processes exist in the exocrine rat pancreas which are sensitive to Mg2+ within a physiological range. Mg2 may act as an intracellular modulator, primarily by controlling mobilisation of Ca2+.