Ph.D. - Biomedical Sciences (Cell & Molecular Biology)
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Item Biophysical Characterization of Zebrafish Transient Receptor Potential Cation Channel, Subfamily M, Member 7.([Honolulu] : [University of Hawaii at Manoa], [August 2015], 2015-08) Jansen, ChadTRPM7 is a non-selective divalent ion channel that belongs to the transient receptor potential superfamily; it is permeable to divalent ions such as calcium and magnesium as well as trace elements such as manganese and zinc. Although TRPM7 is permeable to many divalent ions, it is regulated by intracellular magnesium and magnesium adenosine triphosphate. Moreover, TRPM7 is thought to be involved in magnesium homeostasis of the cell. Magnesium is important for many processes within an organism. When serum levels of magnesium drop, a state of hypomagnesemia is reached. Hypomagnesemia is associated with many symptoms such as tremors, convulsions and even coma. TRPM7 has been implicated in hypomagnesemia, as well as breast cancer metastasis and delayed neuronal death post brain ischemia. Human TRPM7 has been characterized and studied, but limitations do exist within animal models to study TRPM7. Global knockout of TRPM7 in mice leads to death at embryonic day 7.5. This fact limits the developmental studies that can be performed on a mouse. A nonmammalian model, zebrafish, may be able to bridge the gap in studies of TRPM7. There exists zebrafish with a loss-of-function mutation in TRPM7 that survive; these mutants show skeletogenesis defects and kidney stone formation. Zebrafish TRPM7 has yet to be biophysically characterized. Here, it is investigated whether zebrafish TRPM7 is biophysically similar to human TRPM7. Zebrafish TRPM7 and three truncated mutants of zebrafish TRPM7 were overexpressed in human embryonic kidney cells. The truncated mutants of TRPM7 were utilized as a means to narrow down the magnesium and magnesium adenosine triphosphate binding sites within zebrafish TRPM7. This study shows that: 1- Zebrafish TRPM7 is biophysically similar to human TRPM7 in regards to regulation, divalent ion permeability and pH sensitivity, 2- Zebrafish TRPM7 is less sensitive to osmolarity than human TRPM7, but remains sensitive. 3- Common human TRPM7 inhibitors do not inhibit Zebrafish TRPM7. Additionally, investigation of the three truncated zebrafish TRPM7 mutants suggests that there is a third magnesium-binding site. Experimental designs with zebrafish as an animal model for TRPM7 pathologies and diseases should take into account the differences in zebrafish TRPM7 and human TRPM7.Item Investigations of leptospira in small mammalian host species in the Hawaiian islands([Honolulu] : [University of Hawaii at Manoa], [December 2012], 2012-12) Wong, MayeeConsidered the most widespread and prevalent of zoonoses, the emergent infectious leptospirosis disease is found throughout tropical regions in particular, with annual mean incidence rates in Hawaii consistently the highest in the United States. As a tropical archipelago with relatively low host and leptospiral diversity, Hawaii represents an exceptional opportunity for investigations in the ecology and evolution of this bacterial pathogen. In an effort to gain a better understanding of disease transmission dynamics and environmental drivers in Hawaii, the studies presented in this dissertation each take a distinct approach to examining the associations between three main components underlying the ecology of leptospirosis across the archipelago; namely, the Leptospira pathogen, animal hosts, and climate. First, I employed a longitudinal dataset of animal infection prevalence from a period of 14 consecutive years across five maintenance host species and three main islands to describe the epizootiological distribution of pathogenic leptospires in Hawaii. In a second study, I combined field biology and molecular lab techniques to characterize the 16S rRNA genetic diversity of Leptospira amongst a community of small mammals in a local rainforest. Finally, Hosmer-Lemeshow goodness-of-fit and Wald assessments of multivariate logistic regression models were used to investigate the association between rainfall and leptospiral animal infection prevalence at multiple spatio-temporal scales. The key findings in this dissertation address evolutionary patterns of host specificity, provide a preliminary examination of leptospiral genetic diversity in host vectors, and show that precipitation is an environmental driver of host infection prevalence at specific spatial and temporal scales. These results shed light on leptospiral transmission dynamics in a tropical region enzootic for the bacterial pathogen, and lay the foundation for an integrated eco-evolutionary model of leptospirosis in Hawaii.Item Effect of Sirt1 and telomerase on stem cells(2008) Coussens, Matthew J.Item Item A new model for sperm chromatin structure and its relationship to function(2004) Sotolongo, BarbaraItem Photoaging of skin : a functional genomics approach(2004) Urschitz, Johann G.E.Item Quorum sensing in the Vibrio fischeri-Euprymna scolopes symbiosis(University of Hawaii at Manoa, 2003-12) Lupp, Claudia; Ruby, Edward G; Biomedical Sciences (Cellular & Molecular Biology)Quorum sensing is a cell density-dependent bacterial gene regulatory mechanism used for the expression of colonization-related genes. The symbiotic relationship between the luminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid Euprymna scolopes serves as a model system to study the molecular processes underlying bacterial colonization. This system is especially well-suited for the investigation of the impact of quorum sensing on colonization because (i) it is an easily accessible, natural, two-species colonization model, and (ii) quorum sensing regulates luminescence expression in V. fischeri, which allows the non-invasive detection of quorum-sensing activity both in culture and in symbiosis. While the impact of one of V. fischeri's quorum-sensing systems, lux, on luminescence expression and symbiotic competence has been extensively studied, little was known about other putative systems. The results of this study demonstrate that the V. fischeri ain system is essential for both maximal luminescence expression and symbiotic competence. The ain system predominantly induces luminescence expression at intermediate cell densities, which occur in culture, while the lux system is responsible for luminescence expression at the high cell densities found in symbiosis, suggesting the sequential induction of luminescence gene expression by these two systems. Furthermore, the ain quorum sensing system is important for the processes underlying colonization initiation, while the impact of the lux system is apparent only in later stages of the symbiosis, indicating distinct functions of these two systems during the colonization process. A global transcriptome. analysis of quorum-sensing mutants revealed that ain quorum sensing represses motility gene expression, providing a likely explanation for the initiation defect. Although it has been known that many bacterial species possess multiple quorum-sensing systems, this is the first study demonstrating that two quorum-sensing systems are employed to specifically regulate functions important at distinct cell densities occurring during the colonization process.Item Symbiont-induced changes in host gene expression: The squid-Vibrio symbiosis(University of Hawaii at Manoa, 2003-12) Kimbell, Jennifer Loraine; McFall-Ngai, Margaret; Biomedical Sciences (Cellular & Molecular Biology)All animals exist in lifelong relations with a complement of bacteria. Because of the ubiquity of these symbioses as well as the derived biomedical applications, the study of both beneficial and pathogenic host-microbe associations has long been established. The monospecific light organ association between the Hawaiian sepiolid squid Euprymnascolopes and the marine luminous bacterium Vibrio fischeri has been used as a experimental model for the study of the most common type of animal-bacterial interaction, i.e., the association of coevolved Gram-negative bacteria with the extracellular apical surfaces of polarized epithelia. A fundamental step for understanding the mechanisms of host-symbiont associations lies in defining the genetic components involved; specifically defining changes in host gene expression. The studies presented in this dissertation identify and characterize V. fischeri-induced changes in host gene expression at both the transcript and protein level.