Indiana University-Purdue University Indianapolis (IUPUI) / Chlamydia trachomatis is an obligate intracellular bacterial pathogen that can infect the eyes, genital tract, and disseminate to lymph nodes in humans. Many C. trachomatis infections are clinically asymptomatic and can become chronic if left untreated. When humans are infected with C. trachomatis, a cytokine that is produced is interferon-gamma (IFN-γ). In vitro, IFN-γ stimulates expression of the host enzyme indoleamine 2,3-dioxygenase. This enzyme converts free intracellular tryptophan to N-formylkynurenine. Tryptophan starvation induces C. trachomatis to enter a viable-but-nonculturable state termed persistence, which has been proposed to play a key role in chronic Chlamydial disease. To circumvent host induced tryptophan depletion, urogenital strains of C. trachomatis encode a functional tryptophan synthase (TS). TS synthesizes tryptophan from indole and serine, allowing Chlamydia to reactivate from persistence. Transcriptomic analysis revealed C. trachomatis differentially regulates hundreds of genes in response to tryptophan starvation. However, genes that mediate entry, survival, and reactivation from persistence remain largely unknown. Using a forward genetic screen, we identified six Susceptible to IFN-γ mediated Persistence (Sip) mutants that have diminished capacities to reactivate from persistence with indole. Mapping the deleterious persistence alleles in three of the Sip mutants revealed that only one of the mutants had a mutation in TS. The two other Sip mutants mapped had mutations in CTL0225, a putative integral membrane protein, and CTL0694, a putative oxidoreductase. Neither of these genes plays a known role in tryptophan synthesis. However, amino acid (AA) competitive inhibition assays suggest that CTL0225 may be involved in the transport of leucine, isoleucine, valine, cysteine, alanine, and serine. Additionally, metabolomics analysis indicates that all free amino acids are depleted in response to IFN-γ, making this amino acid transporter essential during persistence. Taken together we have identified two new chlamydial persistence genes that may play a role in chronic chlamydial disease.

<p> The phenomenon of genetic recombination is of fundamental importance to the evolution and adaptation of species, and is a valuable laboratory aid to the biological scientist. Probable mechanisms of control of recombination are largely unknown, due partly to the difficulty of obtaining artificial mutants affecting the process. The studies reported here avoid this difficulty by the use of different factors controlling recombination which occur naturally in the species Neurospora crassa and N. sitophila. Studies of hybrid N. crassa strains carrying factors from N. sitophila are described, and some models for the control of genetic recombination are discussed. </p> / Thesis / Master of Science (MSc)

<p> In order to compare the mode of action of mutagens which cause mass conversion of green Euglena to colorless forms lacking chloroplasts, mutants were selected either for resistance to U.V. light or for resistance to N-methyl-N-nitroso-p-toluenesulfonamide (MNTS). The U.V. resistant mutants selected showed strong resistance to U.V. but the slow growth of these mutants indicated that they probably carried multiple lesions. Some of the problems inherent in selection techniques were explored and discussed. A mutant selected for resistance to MNTS was found to have the ability to convert MNTS to an innocuous compound N-methyl-p-toluenesulfonamide. Experiments using the MNTS resistant strain provide information concerning the mode of action of MNTS and the related mutagen nitrosoguanidine.</p> / Thesis / Doctor of Philosophy (PhD)

atfish production is the largest aquaculture industry in the United States and infectious agents are responsible for 45% of all economic losses. Ictalurid herpesvirus 1 or Channel catfish virus (CCV) has a great economic impact on channel catfish aquaculture; yet it also has the potential for becoming a highly efficient vaccine vector eliciting long-lived immune responses against itself and, as a recombinant, other important catfish pathogens (bacteria, myxosporean, and fungi). However, little is known about CCV’s genome, its gene functions or genetic interactions with its host. Better understanding of CCV biology and pathogenesis could enable more rational vaccine design and other control strategies for CCV. My thesis is that “systems biology” can enable much more rapid understanding of CCV biology and pathogenesis. To test this thesis I needed to first more fully annotate the CCV genome, then construct a rapid system for generating CCV mutants and recombinants for systems biology research and then apply these tools in a systems biology experiment. I experimentally annotated the CCV proteome by proteogenomic mapping followed by real-time PCR and confirmed the expression of 37 of the 76 previously predicted ORFs (25 for the first time) as well as 17 novel ORFs. I next constructed two different infectious clones of CCV: one as three overlapping bacterial artificial chromosomes (BACs) and the other as a full length CCV BAC. These CCV BACs facilitate CCV mutant and recombinant production and I regenerated a genotypically wild-type and an attenuated virus. To further simplify CCV mutant production, I next adapted the CCV infectious clone for lambda phage crossover recombination cloning to enable sequence transfer into a specific CCV locus by a simple one-step in-vitro reaction. Finally, I used the CCV infectious clone, in combination with affinity purification, to identify interacting partners of the CCV zinc RING finger proteins ORF9, ORF11 and ORF12 to provide insight into the topology of one presumptive CCV-channel catfish molecular interaction network module. The work in this dissertation supports my thesis and the CCV BAC tools were patented; together these provide tools to facilitate and accelerate the development and testing of better CCV vaccines.

No description available.

No description available.

<p> Mre11 and Rad50 form a complex with Nbs1 (MRN) in mammals and Xrs2 (MRX) in yeast. The MRN complex plays a role in many cellular processes, such as DNA damage sensing, DNA repair, cell cycle checkpoint and telomere maintenance. Rad50 contains a conserved ATP binding motif and its ATPase activity is essential for ATM activation in vitro. Using a tethering approach, I have shown that Rad50 can be targeted to telomeres through its fusion to hRap1. The fusion of hRap1 to Rad50 did not alter the property of Rad50. The fused wild-type Rad50 promoted telomerase-dependent telomere lengthening. However, the fusion proteins containing loss-of-function mutations in Rad50 (K42E and S1202R) did not. I have also shown that the fused wild-type Rad50 was able to form irradiation-induced foci in a manner similar to unfused Rad50. In contrast, the two defective mutants of Rad50 failed to accumulate irradiation-induced foci. Expression of the fusion proteins containing Rad50 mutants also interfered with the ability of endogenous Mre11 protein to form foci post irradiation. Thus our data suggest that the Rad50 mutants may function as dominant-negative alleles in cells.</p> / Thesis / Master of Science (MSc)

The cell cycle of budding yeast is controlled by a complex chemically reacting network of a large group of species, including mRNAs and proteins. Many mathematical models have been proposed to unravel its molecular mechanism. However, it is hard for people with less training to visually interpret the dynamics from the simulation results of these models. In this thesis, we use the visualization toolkit D3 and jQuery to design a web-based interface and help users to visualize the cell cycle simulation results. It is essentially a website where the proliferation of the wild-type and mutant cells can be visualized as dynamical animation. With the help of this visualization tool, we can easily and intuitively see many key steps in the budding yeast cell cycle procedure, such as bud emergence, DNA synthesis, mitosis, cell division, and the current populations of species. / Master of Science / The cell cycle of budding yeast is controlled by a complex chemically reacting network. Many mathematical models have been proposed to unravel its molecular mechanism. However, it is hard to visually interpret the dynamics from the simulation results of these models. In this thesis, we use the visualization toolkit D3 and jQuery to design a web-based interface and help users to visualize the cell cycle simulation results. It is essentially a webpage where the proliferation of the wild-type and mutant cells can be visualized as dynamical animation.

Vitamin C (ascorbic acid) is one of the most essential organic compounds required by the human body for normal metabolic function. Unfortunately, this valuable nutrient is not produced in the human body but most plants and animal can produce this molecule. Although ascorbic acid was not isolated until the early part of the twentieth century, it was known that eating limes and other citrus fruits could ward off the affects of scurvy as early as the 1500's. Ascorbate serves many critical functions in plants as well as the human body. In both, it works as a cofactor in the production of hydroxyproline-rich compounds and helps protect molecules such as proteins, lipids and fatty acids from oxidation. Although the biochemical pathway in animals has been known since the 1950's (Jackel et al., 1950), the exact process by which ascorbic acid is made in plants has eluded scientists. It was shown in 1963 that the inversion of the hexose carbon chain, which occurs in the animal pathway, is not a possible mode of synthesis in plants (Loewus, 1963). As an alternative, a non-inversion pathway was proposed, which achieves ascorbic acid using D-mannose and L-galactose as intermediates, referred to as the Smirnoff-Wheeler pathway (Wheeler et al., 1998). It was shown that transforming lettuce (cv. Grand Rapids and Black Seeded Simpson) and tobacco (cv. Xanthi) with the terminal enzyme in the animal biosynthetic pathway (GLO; L-gulono-gamma-lactone oxidase) increases the ascorbic acid content between 4 and 7 fold. It was also shown through feeding studies that wild type tobacco plants had elevated ascorbate levels when fed the animal precursor (Jain and Nessler, 2000). These data suggest that at least part of the animal pathway could be present in plants, along with the Smirnoff-Wheeler (1998) pathway. To further investigate this discovery, wild type and ascorbic acid-deficient Arabidopsis thaliana were transformed with the glo. Homozygous lines of these transformants were generated and the ascorbic acid levels were compared to the untransformed wild type and mutant plants. Although the wild type plants containing glo did not show a significant increase in ascorbic acid production, all five of the vtc mutant lines had an increased ascorbic acid content relative to wild type level. These data suggest that an alternative pathway is present in plants that does not require many of the steps in the published Smirnoff-Wheeler (1998) pathway to produce ascorbic acid. / Master of Science

Les flavonoïdes (anthocyanes, flavonols et proanthocyanidines) sont des éléments clés de la qualité organoleptique des baies de raisin. Chez les végétaux, l’expression des gènes de la voie de biosynthèse de ces composés est contrôlée par des complexes protéiques organisés autour des facteurs de transcription de type MYB. Dans le cadre de cette thèse, une première approche s’est intéressée aux mécanismes de régulation de l’expression du gène VvMyb5a et de l’activité biologique de la protéine codée par ce gène. L’analyse du promoteur VvMyb5a a montré que son activité au cours du développement de la baie serait plutôt placée sous contrôle hormonal. Des expériences de double hybride ont révélé que la protéine VvMyb5a pouvait interagir avec une protéine kinase de type GAMYB et une protéine WD40. Une deuxième approche, basée sur l’analyse globale du transcriptome de mutants naturels de vigne affectés dans la biosynthèse des anthocyanes, a permis d’identifier deux nouveaux gènes MYB nommés VvMybPA1 et VvMyb24. L’expression différentielle de ces gènes dans des baies de cépages rouges et blancs a été confirmée et leurs caractérisations fonctionnelles ont été engagées chez Arabidopsis thaliana. / Flavonoids, like anthocyanins, flavonols and condensed tannins, are key elements of he organoleptic quality of grape berries. In plants, expression of genes encoding enzymes of he flavonoid biosynthetic pathway is controlled by small protein complexes organised around MYB transcription factors. In the present work, we first focused on the regulatory mechanisms of VvMyb5a expression and on the biological activity of the corresponding protein. Promoter analysis indicated that VvMyb5a expression is probably mainly controlled by hormones. A yeast two-hybrid screen revealed that VvMyb5a can interact with a GAMYB ype protein kinase and a WD40 protein. In a second time, global transcriptome analysis of grapevine natural mutants deficient in anthocyanin biosynthesis led to the identification of wo new MYB genes, named VvMybPA1 and VvMyb24. Differential expression of these two genes in red and white berry skins was confirmed by RT-PCR and their functional characterizations have been initiated in Arabidopsis thaliana.