Thesis (M.S.)--University of Toledo, 2005. / Typescript. "A thesis [submitted] as partial fulfillment of the requirements of the Master of Science degree in Chemistry." Bibliography: leaves 171-174.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 99-111). Also available in electronic version.

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology, March 2001. / Includes bibliographical references. Also available on the Internet.

Disk performance for random access fares significantly worse compared to sequential access. Time required to transfer random blocks to or from disk is dominated by seeking and rotational delay. To improve the throughput and reduce the latency, one can apply techniques to increase the sequentiality of disk accesses, such as block rearrangement and replication. We introduce an approach to improve read performance by replicating blocks into file system free space at the block level. This makes the replication module independent of the file system and therefore easier to implement and verify. A solution that requires no changes to the file system is also easier to adopt. Supporting a new file system is a matter of writing a user-space component that understands its free block data structures. We implemented a prototype as a stacked device driver for Linux and evaluated its performance on a number of workloads. / Science, Faculty of / Computer Science, Department of / Graduate

The interaction between Murine Cytomegalovirus (MCMV) and the cell cycle has been investigated in synchronized murine cells. Based on the following evidence it was concluded that MCMV replication depends upon the host S phase: (1) the normal latent period of viral growth in exponentially growing 3T3 cells (12 h). was protracted until the host S phase (ca. 20 to 24 h) in synchronized cells infected in early G—l; (2) G-l arrested 3T3 cells failed to support viral replication; (3) entry of the virus was equally efficient in G-l, S and exponential cells; (4) in exponential 3T3 cells viral DNA synthesis began at 10 h post infection, and in synchronized cells it began approximately 16 to 18 h after infection, or early S phase. Therefore, the replication of viral DNA requires host S phase events. Another herpesvirus, Herpes Simplex Virus type-1 (HSV-1) replicated independently of S phase. However, a mutant of HSV-1, deficient in its ability to induce thymidine kinase, demonstrated a dependency upon S phase similar to MCMV. These data indicate a key role of thymidine kinase in the ability of HSV-1 to replicate outside of S phase. However, MCMV induced neither a cellular nor a viral thymidine kinase, and thymidine kinase was not essential for normal viral replication. When G-l arrested 3T3 cells were infected with MCMV, viral DNA synthesis did not initiate and the lytic cycle was reversibly blocked. The non-replicating viral genome remained viable in G-l cells and could be activated at any time by stimulating the cells to enter S phase. The G-l non-permissive system was studied to help ascertain the cell cycle requirements of MCMV. Specifically, two approaches were pursued. In vitro endogenous MCMV DNA synthesis was first studied in G-l, S phase, and exponential 3T3 cells. Under the appropriate conditions, nuclei from infected cells synthesized viral DNA when they had the capacity to dp so in vivo. Nuclei from G—1 phase cells synthesized cell DNA only and not viral DNA. Infected G-l and S phase cells contained a new DNA polymerase which was distinguished from the host enzyme by the high salt requirement for maximal activity. The putative viral DNA polymerase was inhibited by antiserum prepared against infected cell proteins. Therefore, the novel DNA polymerase present in infected G-l and S phase cells was a viral gene product. The second approach involved a comparison of viral transcription in permissive and non-permissive 3T3 cells. The kinetics of hybridization in solution were analyzed by a computer program which evaluated the number of viral RNA classes and the fraction of the viral genome coding for each class. This study revealed the following: (1) in permissive cells by 6 h post infection (early, i.e. before viral DNA synthesis), two classes of viral transcripts were detected, differing by 7 fold in concentration. The abundant class was transcribed from approximately 7% of the viral DNA and the scarce class from approximately 20%. (2) in permissive cells at 24 h post infection (late), abundant and scarce classes (differing by 6 fold in concentration) were transcribed from approximately 10 and 33% of the viral DNA respectively. (3) in non-permissive cells at 6 h, only one class of RNA was present, representing approximately 15% of the viral DNA. (4) in non-permissive cells at 24 h post infection, a single RNA class was observed which was transcribed from approximately 24% of the viral DNA. Summation hybridization experiments indicated that in non-permissive (G-l) cells, only those regions of the DNA which code for early RNA are transcribed. A model has been proposed to describe cell cycle dependent replication of MCMV. It is concluded that MCMV does not replicate in G—l cells due to the absence of specific S phase 'helper-functions' which are required either for the initiation of viral DNA synthesis directly, or for the transcription of viral DNA sequences. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In this study, DNA methylation was examined during the eukaryotic cell cycle, and shown to occur throughout the S phase as well as during the "early" G₂ phase. However, DNA synthesis and methylation of newly synthesized DNA did not occur simultaneously, but the latter lagged behind DNA synthesis by about two hours. Once added during the S phase, the methyl groups were stably maintained in the DNA. Various compounds which are known to affect DNA synthesis in tissue cultured cells, were tested for their ability to alter the methylation status of DNA. The effects of three DNA synthesis inhibitors, viz. hydroxyurea (HU), 1-S-D-arabinofuranosyl cytosine (ara-C) and aphidicolin were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40 (SV 40) transformed line (SVWI-38) and a y-irradiation transformed cell line (CT-1). HU was shown to enhance hypermethylation of pre-existing DNA strands in the normal cells, while ara-C and aphidicolin caused hypermethylation of newly synthesized DNA strands. The effects of various concentrations of a known inducer of gene expression, sodium butyrate, were examined on these three cell lines as well. During a 16-20 hour treatment period, at butyrate concentrations of between 5 and 20 mM, no adverse effect on cell morphology was observed. Cell growth, in the presence of butyrate for 14 hours, showed that butyrate was more toxic on the transformed cells than on the normal cells. However, at 5 mM butyrate, DNA synthesis was inhibited by 75% in the normal cells, and was unaffected in the transformed lines. RNA synthesis was not affected in the transformed cells, whilst in the normal cell line, RNA synthesis was decreased to 76% of the control value, at sodium butyrate concentrations as low as 5 mM. Protein synthesis also was unaffected in the transformed cells and only slightly (+ 10%) inhibited in the normal cells at 20 mM butyrate. SDS polyacrylamide gel electrophoresis of proteins synthesized in the presence of 10 mM sodium butyrate, showed that most proteins were unaffected. Two high molecular weight proteins in the WI-38 cells appeared to be modified during butyrate. treatment, while one protein was induced by butyrate treatment in the CT-1 cells. More importantly though, butyrate treatment also resulted in hypermethylation of DNA, as shown by MSP 1 and Hpa II restriction endonuclease digestion and high-pressure liquid chromatography analysis. Butyrate appeared to specifically cause hypermethylation of pre-existing DNA strands in the WI-38 cells, while the SVWI-38 and CT-1 cells showed preferential hypermethylation of newly synthesized DNA strands. However, the hyper-methylated state was only heritable if the methylation event occurred in newly synthesized DNA. Hypermethylation on pre-existing DNA was rapidly lost in the subsequent generation. It would therefore appear that methylcytosines are only maintained in the DNA if they are generated on newly synthesized DNA. This study has clearly shown that the heritability of DNA methylation patterns is closely linked to DNA replication.

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Human immunodeficiency virus type 1 (HIV-1) and Mycobacterium tuberculosis (Mtb) coinfection remains a major global health threat. Both pathogens synergistically drive pathogenesis of the other. The risk of developing active tuberculosis (TB) is increased in people living with HIV-1, even in those receiving antiretroviral therapy (ART), whilst TB was responsible for 15 % of HIV-related deaths in 2020. Mtb co-infection increases the likelihood of transcriptionally activating HIV-1 replication potentially due to bioactive Mtb lipids engaging macrophage surface receptors, thus triggering signaling pathways which activate human transcriptional factors (hTF) and production of inflammatory cytokines capable of activating HIV-1 transcription. This work investigated the hypothesis that clinical Mtb strains with single nucleotide polymorphisms (SNP) in lipid-metabolizing genes, required for cell wall lipid biosynthesis, differentially affect HIV-1 replication and human macrophage inflammatory response during Mtb-HIV-1 co-infection in vitro. Monocyte derived macrophages (MDM) were the predominant model used to investigate this phenomenon. Infections, in the presence or absence of HIV-1 co-infection, were performed using either lineage 2 or lineage 4 clinical strains with non-synonymous SNP in polyketide synthase 2 (pks2) required for sulfolipid 1 (SL-1) biosynthesis and compared to control infections using phylogenetically close clinical strains without the SNP of interest and canonical lineage 2 and 4 laboratory strains (H37RvP1939/T605, CDC1551WT and HN878WT). Secreted cytokines and chemokines were measured in supernatant (SN) by Luminex. The effect of Mtb on HIV-1 viral production was assessed by measuring HIV-1 Gag p24 in the SN of co-infected MDM or SN of HIV-1 infected MDM incubated with conditioned media from Mtb-infected MDM. The influence of Mtb on HIV-1 transcriptional activity was measured using a transgenic cell line (TZM-bl) with Luciferase reporter under HIV-1 long terminal repeat (LTR) expression. The impact of incubating TZM-bl cells in Mtb-induced conditioned media before or after HIV-1 infection was assessed. One pair of phylogenetically close clinical strains with and without a pks2 SNP of interest (EX30Q1939/A605 and MRC16P1939/A605) with interesting lipid and inflammatory phenotypes, and H37RvP1939/T605 as a lineage 4 control, were subject to single nucleotide mutagenesis using recombineering to either revert SNP of interest to match the alleles of H37Rv or introduce the SNP of interest into the control strains. The wild-type and mutant strains were used in a trans-well assay to infect MDM in the presence of HIV-1 co-infection in the top chamber, while simultaneously mimicking the bystander effect of cytokine-mediated HIV-1 regulation in the bottom chamber which was only infected with HIV-1. Results demonstrate there was increased cytokine production by MDM infected with MRC16P1939/A605 in both the presence and absence of HIV-1 co-infection compared to its phylogenetically close paired strain EX30 Q1939/A605. The data shows that there was no difference in LTR activity in TZM-bl cells co-incubated with inflammatory environment between the strains of interests, however co-incubation of TZM-bl cells with Mtb-induced inflammatory environment generally increased LTR activity during HIV-1; a proxy for HIV-1 replication. In the trans-well co-infection assay, a significant positive association between production of HIVp24 and secretion of CCL2 was observed, whilst IL-1β secretion showed a significant negative relationship with the production of HIVp24, with donor variability in baseline cytokine production also associated with the extent of HIVp24, CCL2, IL-1β and IL-8 production. Introduction of the pks2 T605A SNP into H37RvP1939/T605 and reversion in EX30Q1939/A605T significantly modified their inflammatory phenotype. Together these results support the hypothesis that Mtb clinical stains with genetic variation in cell wall lipid biosynthesis impacts the inflammatory milieu and, subsequently, HIV-1 replication during co-infection. The outcome of Mtb-HIV co-infection is therefore not homogenous but contingent on the phenotype of infecting Mtb strain and individual.

DNA replication initiates at a precise location on the bacterial chromosome, the origin of replication (oriC). This work has localized the origin of DNA replication on the Sinorhizobium meliloti chromosome to a region spanning the hemE gene. A genetic dissection of the locus revealed that a much larger fragment of DNA (1802 bp) is required for a functional oriC than that of the other characterized alpha-proteobacterial chromosome origin from Caulobacter crescentus. Site-directed mutations of predicted DnaA binding sites has identified several essential elements for replication of the plasmid borne oriC. Mutations in these DnaA boxes also reduce transcription of hemE and thus it is likely that transcription of hemE and replication of the S. meliloti chromosome are coupled. The ColEl plasmid pUCP30T can autonomously replicate when the S. meliloti oriC is cloned into the suicide vector (pTH838) and can be efficiently mobilized out of S. meliloti into E. coli. The pTH838 oriC plasmid when transferred into S. meliloti results in both small and large colonies and both of these transconjugant classes take longer to form than the S. meliloti recA::Tn5 recipient. We attributed this phenotype to the very low copy number of the pTH838 plasmid which was determined to be 0.053 - 0.135 copies per chromosome. The DnaA protein responsible for replication initiation in many bacteria has been purified and used in electrophoretic mobility shift assays. The DnaA protein interacts specifically with sequences in the hemE - Y02793 intergenic region and upstream of the repA2 gene on the pSymA megaplasmid. The DnaA protein has also been implicated as a link between DNA replication and cell division in S. meliloti as overexpression of DnaA in both E. coll and S. meliloti results in filamentation. / Thesis / Master of Science (MSc)