Complex user-end procedures for the execution of computationally expensive processes and tools on high performance computing platforms can hinder the scientific progress of researchers across many domains. In addition, such processes occasionally cannot be executed on user-end platforms either due to insufficient hardware resources or unacceptably long computing times. Such circumstances currently extend to highly sophisticated algorithms and tools utilized for analysis of fluorescent imaging data. Although an extensive collection of cloud-computing solutions exist allowing software developers to resolve these issues, such solutions often abstract both developers and integrators from the executing hardware particulars and can inadvertently incentivize non-ideal software design practices. The discussion herein consists of the theoretical design and real-world realization of an efficient architecture to enable direct multi-user parallel remote utilization of such research tools. Said networked scalable real-time architecture is multi-tier, extensible by design to a vast collection of application archetypes, and is not strictly limited to imaging analysis applications. Transport layer interfaces for packetized binary data transmission, asynchronous command issuance mechanisms, compression and decompression algorithm aggregation, and relational database management systems for inter-tier communication intermediation enable a robust, lightweight, and efficient architecture for networking and remotely interfacing with fluorescent imaging analysis processes. / M.S. / Collaboration amongst researchers within various technical domains who rely on information processing and analysis tools can be strengthened through the deployment of scientific computing infrastructure that enables their usage via a web interface. The architecture of such infrastructure is preferably efficient, lightweight, and simple while retaining potential future integration capabilities with additional research tools. This work presents the theoretical design and realization of an architecture for networking fluorescent imaging analysis processes so as to make them remotely usable within internal computer networks and across the world wide web.

No description available.

Cyclic 3',5'-guanosine monophosphate (cGMP) is a key signaling molecule involved in a myriad of physiological processes, including vascular smooth muscle (VSM) tone, water- and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation and bone formation. Increased occurrence of vascular disorders including erectile dysfunction, hypertension, stroke and coronary artery disease, have made it increasingly important to study the dynamic interplay between cGMP synthesis and hydrolysis in VSM cells. This dissertation examines the spatial distribution of intracellular cGMP, [cGMP]i, in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP]i, we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP]i in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for real-time, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG. We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP]i formation.

The purpose of this thesis paper is to investigate womanist theology and method, along with restoration practices involving spirituality and healing within the context of the visual arts. The thesis exhibition will attempt to create new visual possibilities that inform womanist theological scholarship in terms of promoting contemporary female religious imagery within a metaphorical language. While womanist theology is steeped in interdisciplinary practices, it has yet to consider seriously the studio arts as a means to explore and develop the womanist language. This study will investigate how essential and natural the visual arts assist our understanding of spirituality, especially through a womanist context.

No description available.

A Cytokeratin 8 (K8)/Green Fluorescent Protein (GFP) fusion construct was created to better understand the behavior of K8 within cancer cells. This intermediate filament (IF) protein is a member of the cytoskeletal gene family along with actin and tubulin. IF's are normally expressed in a tissue specific and differentiation dependant manner, in which their role is more supportive than essential to the cell. Such roles include rigidity of cellular shape, protein trafficking, cellular locomotion, and cell signaling platforms. K8 mutation, over expression, and aberrant post translational modifications have been observed in various carcinoma cell lines to be the cause of several phenotypes including apoptosis inhibition, drug resistance, transformation, Mallory-Denk body (MDB) formation, localization at the plasma membrane, and secretion of the protein. In order to study these abnormal phenotypes the K8 gene was generated and inserted into the GFP over expression vector. This allowed for the study of K8 within a well defined cervical cancer cell line named Hela. This study intended to provide answers to K8's localization at the plasma membrane in carcinoma cell models while avoiding criticisms to previous immunohistochemical localization studies. A model which exhibits established phenotypes found in the literature was thus created which has the potential to address several paramount questions related to K8's role in supporting the development and progression of cancer. It could also be utilized as an assay for the discovery of K8 filament formation inhibitors, which may prove useful in combination with current chemotherapeutics. The model could also be used to provide weight to diagnostics, such as the Cancer Recognition test, which utilizes antibodies against K8 as biomarkers for malignancy via an Enzyme-Link ImmunoSorbent Assay (ELISA).

The AKR strain of mice have a genetic trait for spontaneous development of lymphocytic leukemia. In this study, leukemic mice were found to have significantly larger (p<0.01) thymuses and spleens than preleukemic mice. The enlarged leukemic tissues were densely packed with a light staining cell, with a hollow-appearing nucleus. Tissues from preleukemic mice were observed to be infiltrated with a smaller, darker-staining lymphocyte. Fluorescent antibody staining was done on preleukemic and leukemic tissues, using three antisera against murine lymphocyte theta antigen, and an antiserum against murine IgG. Significantly brighter fluorescence, (p <0.05) with theta-specific antisera, was found in leukemic thymuses,spleens, and kidneys than in the same preleukemic tissues. Leukemic tissues had significantly brighter fluorescence (p <0.05) than preleukemic tissues with IgG antiserum.

Published information on the structure and regulation of H. pylori promoters is limited. The work presented in this thesis describes the cloning and characterisation of promoter regions from a clinical isolate of H. pylori, and the development of an alternative, non-radioactive method for verifying the location of transcriptional start sites of bacterial promoters. H. pylori 4187E promoters were randomly cloned into the promoter-trap vector pKK232-8 in Escherichia coli DH5α using two sets of restriction enzymes. Vector pKK232-8 contains a promoterless chloramphenicol acetyltransferase (CAT) gene. Seventy-four promoter-containing clones were isolated from selective media based on their resistance to chloramphenicol. The strength of each promoter was analysed qualitatively, using chloramphenicol minimum inhibitory concentrations, and quantitatively, using CAT assays following exposure of the clones to pH 4 and pH 7. Selected promoter fragments were subcloned into the GFP reporter vector pFPV25, containing a promoterless gfp gene. The subclones were exposed to buffered LB broth at pH 4, 5, 6, 7 and 8, for varying lengths of time, to study acid-induced regulation of gene expression. Subclones were examined qualitatively, using visual examination of GFP fluorescence and fluorescence microscopy, and quantitatively, using flow cytometry following acid shock. DNA sequences were determined for 61 of the 74 H. pylori promoters, and sequence alignments with the published H. pylori strains (26695 and J99) were performed. The transcriptional start site of 27 H. pylori promoter fragments was experimentally mapped using a fluorescence-based primer extension protocol developed by our group. Potential -35 and -10 sequences were identified for each promoter, and a new consensus sequence for H. pylori promoters was proposed based upon these results. This study has considerably expanded knowledge of H. pylori promoter sequences and transcriptional start sites based on those which also function in E. coli. It has also revealed several H. pylori promoters which appear to respond to acid stress

No description available.