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No description available.

Thesis (Ph.D.)--University of Texas at Dallas, 2007. / Includes vita. Includes bibliographical references (leaves 38-40)

Thesis (Ph.D.)--University of Iowa, 2009. / Thesis supervisor: Debashish Bhattacharya. Includes bibliographical references (leaves 34-37).

Almost all bacteria contain genes that may lead to their growth stasis and death.Normally, these toxins are believed to be neutralized with their cognate antitoxinsfrom a toxin-antitoxin (TA) operon. These modules are also abundant in pathogenic bacteria suggesting a role for them both in normal bacterial physiology and pathogenicity. Their functions have been subject to intense debates. Due to the cell killing capability of the toxin and the gene silencing capability of the antitoxin, they have been utilized for basic research, biotechnology and medical applications. However, further advancements of these applications have been impeded by our limited knowledge of the biology of TAs. Among these TA systems is the Ibs/Sib (A-E) family. Here, we discuss our efforts in characterizing these systems, with a focus on the IbsC/SibC member. Studying them has shown to not be straightforward due to the complexity of their underlying mechanisms and the current approaches being laborious and lacking sensitivity to be applied to these low abundant molecules. We have developed fluorescence-based platforms to take advantage of sensitive and high throughput and resolution techniques such as Fluorescence Assisted Cell Sorting (FACS) to study these molecules instead of relying on traditional culturing methods. While developing these platforms, we gained insights about the biology and regulation of these molecules. To expand this knowledge, we actively pursued investigating the regulation of these molecules at the transcriptional and post-transcriptional levels, both in their native context and in artificial systems. The rest of this thesis summarizes our efforts in solving one of the biggest pieces of the Ibs/Sib puzzle, namely their physiological expressions. With the strategies we have optimized for specific detection of these low abundance molecules, and the knowledge of their biology and regulation presented, we are now at an exciting phase to interrupt the long pause in the study of functions by these molecules and advancement of TA-based applications. / Thesis / Doctor of Philosophy (PhD) / Almost all bacteria contain genes that may lead to their growth stasis or death. Normally, these toxins are believed to be neutralized with their cognate antitoxins. In spite of the efforts to understand these toxin-antitoxin (TA) systems, their physiological roles are subject to intense debate. These systems are hard to study mainly because 1) they are only activated under specific conditions and 2) they are low in abundance. Current approaches are not high throughput and sensitive enough. In this thesis, we developed DNA constructs, bacterial strains and methodologies to facilitate the study of these molecules, particularly the Ibs-Sib family. We next employed these tools to gain a fundamental knowledge of their expression under different conditions, which revealed surprising information about the function of these molecules. We believe that future studies can greatly benefit from the tools offered here to tremendously enhance our understanding of these systems and lead to useful applications.

No description available.

Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data.

Diabetes is a complex group of metabolic disorders with genetic, immunological, and environmental etiologies. Decades of diabetes research have elucidated many genetic drivers of normal islet function and dysfunction. Furthermore, genome wide associated studies (GWAS) have discovered that most diabetes susceptibility loci fall outside of coding regions, which suggests a role for noncoding elements in the development of disease. This highlights our incomplete understanding of the islet regulome and suggests the need for detailed functional analyses of noncoding genes to precisely determine their contribution to diabetes susceptibility and disease progression. Transcriptome analyses have revealed that the eukaryotic genome is pervasively transcribed. Strikingly, only a small proportion of the transcriptome is subsequently translated into protein; the majority is made up non-protein coding RNAs (ncRNAs). The most abundant class of these ncRNAs are called long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides that lack protein-coding potential. The establishment of lncRNAs, once dismissed as genomic dark matter, as essential gene regulators in many biological processes has redefined the central role for RNA in cells. While evidence suggests a role for lncRNAs in islets and diabetes, in vivo functional characterization of islet lncRNAs is lacking. For my thesis project, I sought to understand the lncRNA regulatory mechanisms that promote pancreas development and function. We conducted comparative transcriptome analyses between embryonic mouse pancreas and adult mouse islets and identified several pancreatic lncRNAs that lie in close proximity to essential pancreatic transcription factors. One of the candidate lncRNAs, Pax6 Upstream Antisense RNA (Paupar), mapped near Pax6, a gene encoding an essential pancreatic regulatory protein. We demonstrate Paupar is enriched in glucagon-producing alpha cells where it promotes the alternative splicing of Pax6 to an isoform required for activation of essential alpha cell genes. Consistently, deletion of Paupar in mice resulted in dysregulation of Pax6 alpha cell target genes and corresponding alpha cell dysfunction. These findings illustrate a distinct mechanism by which lncRNAs can contribute to cell-specific regulation of broadly expressed transcription factors to coordinate critical functions within a cell.

Wang, Dakui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 208-234). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.