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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2005. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0059. Adviser: Curtis Lively. "Title from dissertation home page (viewed Feb. 21, 2007)."

<p>Glucose metabolism remains an intensely explored topic of cancer biology since the initial discoveries of Otto Warburg nearly 80 years ago. Many solid tumors metabolize glucose primarily to lactate despite the availability of oxygen, revealing a dependence on glycolysis that may serve as a basis for targeted therapy. In particular, a diet devoid of carbohydrate may minimize the growth capabilities of glucose-dependent cancers. As our interests lie in prostate cancer, we examined whether a ketogenic diet devoid of carbohydrate (NCKD) would reduce the growth rate of tumors derived from human prostate cancer cell lines in a murine xenograft model.</p> <p>Our initial experiments utilized the LAPC-4 cell line, a human androgen-sensitive prostate cancer cell line, in a SCID-mouse xenograft model to determine the effects of an NCKD on tumor growth and animal survival relative to two other diets: (1.) a Western-type diet (WD) reflecting consumptions patterns of men diagnosed with prostate-cancer in the Western world and (2.) a low-fat diet (LFD) representing the present standard of care. Following this study, we conducted a second study utilizing a different human prostate cancer cell line (LNCaP) in order to assess whether our initial observations were robust across multiple prostate cancer tumor models and to also further explore the molecular underpinnings of our observations. Both studies revealed the NCKD leads to a reduction in tumor growth rate and greater overall mouse survival relative to the WD. In addition, the NCKD was equivalent in these parameters to the LFD. We also observed key associations between survival and extent of urinary ketosis as well as favorable changes in insulin and insulin-like growth factor-1 (IGF-1) and gene expression that would be predictive of prolonged survival in mice consuming the NCKD.</p> <p>We believe these data provide compelling evidence to consider a potential therapeutic role for dietary carbohydrate restriction in prostate cancer. We hope these results ultimately serve as a basis to conduct future clinical trials assessing whether dietary carbohydrate restriction, either alone or in combination with more conventional therapies, provides clinicians with an additional weapon against prostate cancer.</p> / Dissertation

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Suzanne M. de la Monte. Includes bibliographical references.

Arcanobacterium pyogenes is a gram-positive, fastidious, facultative anaerobe that can persist in a number of animal species both as a commensal and as a pathogen. The economic impact from A. pyogenes -related bovine mastitis and liver abscess infections makes the characterization of possible targets for vaccine development important. One such target, pyolysin (PLO), is a member of the cholesterol-dependent cytolysin (CDC) family and a major virulence factor and host protective antigen in A. pyogenes pathogenesis. Here, sequence analysis of the chromosomal region surrounding plo, which encodes PLO, indicated that the plo gene and an upstream open reading frame, orf121, constitute a genomic islet conserved across geographically diverse isolates. Two genes downstream of plo, ftsY and ffh, encode homologues of the protein components of the signal recognition particle (SRP) pathway which may be involved in plo expression and secretion. However, the role of ftsY and ffh in plo expression could not be defined as they were found essential in A. pyogenes, and ffh was unable to complement a heterologous system. Analysis of plo mRNA and PLO levels across a growth curve indicated that plo is transcriptionally or post-transcriptionally regulated. Sequence analysis of the plo promoter region identified three 11-mer repeats, R1, R2 and R3, and two putative sigma70-like promoter sequences, P1 and P2. Primer extension experiments suggested active transcription from P1 and P2 in vitro. Analysis of transcriptional fusions of successive truncations of the plo promoter region and site-directed mutations in the promoter motifs to a chloramphenicol acetyl transferase (cat) gene, indicated a role for each motif in the regulation of plo expression in vitro, with R2 required for activation and P2 being the regulated promoter. While a null mutation in orf121 did not have a significant effect on plo expression in vitro, gel shift analysis confirmed the ability of some factor within A. pyogenes soluble cellular extracts to bind specifically to the plo promoter region. The identification of cis- and trans-acting factors involved in plo expression provides a basis for characterizing PLO expression in vivo, and may lead to a better understanding of the global regulation of pathogenesis in A. pyogenes.

Acquired immunodeficiency syndrome (AIDS) has been associated with several cardiovascular abnormalities including dilated cardiomyopathy (DCM). DCM is characterized by dilation of the left ventricle and abnormal systolic and diastolic left ventricular function and is often associated with myocarditis and alterations in the extracellular matrix. The prevalence and severity of AIDS-associated DCM necessitates a better understanding of its disease process. To study AIDS DCM, the LP-BM5 murine AIDS (MAIDS) model, which offers many similarities to AIDS and several advantages as a model, was used. The cardiac function of MAIDS mice was compared to control mice using a conductance catheter system and left ventricular dimensions and compliance were significantly altered indicating a dilated cardiomyopathy. Competitive polymerase chain reaction was used to quantify the LP-BM5 retrovirus in splenic and cardiac tissue from MAIDS mice and illustrated active viral replication in spleen as well as heart tissue during the disease process. Immunohistochemistry and Northern blot analysis were used to determine the role of inflammation in this process: No staining was observed for immune cell infiltrates or the inflammatory mediators tumor necrosis factor-alpha and inducible nitric oxide synthase. The content of cardiac collagen, the major determinant of ventricular architecture, was significantly decreased in MAIDS mice compared to controls and LP-BM5 was shown to infect cardiac fibroblasts in vitro. Pharmacological treatment with zidovudine and/or indinavir prevented cardiac dysfunction through decreases in viral load without functional cardiotoxicity.

Previous work in the laboratory of Dr. Richard Friedman identified the eis gene as capable of enhancing the survival of avirulent Mycobacterium smegmatis within U-937 macrophage-like cells. Studies were undertaken to identify the promoter for eis in both M. smegmatis and M. tuberculosis H37Ra. A 412 base pair region upstream of the eis coding sequence, as well as a DinR-like cis element, was necessary for the full expression of eis in both mycobacteria. To study the expression of eis further, a destabilized luciferase-based reporter system was constructed for fusion of the 412 base pair eis promoter. The expression of eis was monitored throughout culture growth of both M. smegmatis and M. tuberculosis H37Ra using this novel reporter and quantitative real-time PCR. In M. smegmatis, eis was induced upon transition from logarithmic to stationary phase as detected by both luciferase activity and real-time PCR. In M. tuberculosis H37Ra, expression from the eis promoter driven luciferase construct could not be detected, although real-time PCR showed that eis was constitutively expressed. Analysis of groEL2 expression by real-time PCR in comparison with phsp60 driven luciferase production showed that this novel luciferase system could also detect expression changes in M. tuberculosis H37Ra. The lack of luciferase expression from the eis promoter suggested that the utility of this system was dependent on the promoter. eis and groEL2 expression were then monitored in both mycobacteria during the infection of U-937 cells. In M. smegmatis, analysis of eis expression using luciferase suggested that eis was not induced during initial infection, but showed that eis is repressed between 3 h and 24 h of infection. Luciferase production from the eis promoter did not change upon the infection of macrophages with M. tuberculosis H37Ra, suggesting that the eis promoter construct was non-functional. Real-time PCR analysis of eis expression during infection of U-937 cells with M. tuberculosis H37Ra showed that eis is constitutively expressed during macrophage infection. A comparison of groEL2 expression and luciferase activity from phsp60 showed high variability in heat-shock expression and suggested that the luciferase system was not suitable for detecting groEL2 expression changes during macrophage infection.