Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2009. / Title from PDF t.p. (viewed on Jul 22, 2010). Source: Dissertation Abstracts International, Volume: 70-12, Section: B, page: 7350. Adviser: Carl E. Bauer.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6187. Adviser: Abigail A. Salyers. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4250. Adviser: Carol Gross. Includes bibliographical references. Also available online.

<p> <i>Candida albicans</i> is a yeast-like fungal pathogen that can cause infections ranging from superficial to life-threatening systemic candidiasis. Current treatments for systemic candidiasis are available but often ineffective and toxic. Consequently, it is necessary to develop new therapeutic approaches. The purpose of this study was to construct antibody-based fusion proteins that can bind to <i>C. albicans</i> cells and eliminate them. Two such fusion proteins were constructed. Each one is composed of M1 Fab as the antibody component that binds to <i> C. albicans</i> mannan and the antifungal peptide HPRP-A1. HPRP-A1 was attached via a 15-amino acid linker to either the C-terminus of the constant light chain of M1 Fab (M1 Fab-HPRP-CL) or the N-terminus of the variable light chain of M1 Fab (M1 Fab-HPRP-VL). Binding of the fusion proteins to purified <i> C. albicans</i> mannan was assessed with enzyme-linked immunosorbent assay and the half maximal effective concentration (EC₅₀) for each fusion protein was estimated. EC₅₀ for M1 Fab-HPRP-CL was 273.6 compared to 74.1 for the original M1 Fab (<i>p</i> &lt; 0.05), whereas M1 Fab-HPRP-VL did not show any binding activity, indicating a negative impact on the antibody binding by the linked peptide. Similarly, M1 Fab-HPRP-CL also showed reduced binding for <i>C. albicans</i> cells when compared to M1 Fab as determined with immunofluorescence microscopy and flow cytometry. The effect of M1 Fab-HPRP-CL on the growth of <i>C. albicans</i> cells was analysed using microdilution and absorbance. At 16 &micro;M, the growth of yeast cells treated with M1 Fab-HPRP-CL was 47.1 % of the growth control, compared to 43.5 % for M1 Fab (<i>p</i> > 0.05) and to 1.9 % for HPRP-A1 by itself (<i>p</i> &lt; 0.001). Moreover, HPRP-A1 killed <i>C. albicans</i> at 32 &micro;M and 64 &micro;M, while M1 Fab and M1 Fab-HPRP-CL did not, indicating a loss of the antifungal activity of HPRP-A1 when attached to the antibody. These data together provide valuable insights into the development of novel antibody-based therapeutics as an alternative treatment for candidiasis.</p><p>

Thesis (Ph.D.)--Brown University, 2008. / Vita. Adviser: Wen-Ming Chu. Includes bibliographical references.

<p> Photosystem II (PSII) is recognized as the main site for high light induced damage. One of the core subunits of PSII, D1 protein encoded by the <i> psbA</i> gene, is identified as a high turnover protein that undergoes degradation and replacement as a part of the repair process in PSII. Studies on D1 repair have shown the synchronous nature of D1 degradation and synthesis. FtsH a AAA protease, is known to cause D1 degradation. Therefore, it is widely speculated that damaged D1 is replaced by newly synthesized D1. Although, there is no direct evidence suggesting the removal of damaged subunits only. Alternatively, an induction of the general increase in the turnover rate of D1 could trigger the replacement of all D1 subunits in a random fashion. In this work, I have addressed these two alternate hypotheses by developing a genetic system involving dual D1 expression. Strains were constructed with the parallel expression of a WT <i>psbA2</i> in the ectopic and a damage prone mutant <i>psbA2</i> at the native locus. Firstly, a WT ectopic strain (eWT) was constructed by transforming a <i>psbA</i> deletion strain with a WT <i>psbA2</i>, which was synthesized using synthetic biology and fusion PCR. Secondly, mutant <i>psbA2</i> was introduced into the native locus of the eWT strain. All the dual D1 strains showed high PSII activity. Higher PSII levels represent increased proportion of PSII with WT D1 incorporated, as the mutant does not correspond to high PSII levels. Immunoblot analysis of a dual strain nS345P: eWT against D1 did not show an accumulation of the S345P form, which exists as pre-D1. However, deletion of the FtsH protease in nS345P: eWT showed an accumulation of pre-D1 along with mature D1. This indicates that S345P mutant forms are targeted for repair in the dual strain. Additionally, <i>psbA2</i> gene dosage effect was also studied by expressing two WT <i>psbA2</i>. Similar levels of PSII and D1 were observed in the single and double WT strain. This suggests that level of PSII is being regulated and that D1 is not the regulating factor. Therefore, PSII homeostasis is maintained by some other protein factor. </p>

<p> The ascomycete fungus <i>Magnaporthe oryzae,</i> causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and <i>M. oryzae</i> is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made <i> M. oryzae</i> an ideal model system for understanding general plant-pathogen interactions as well. </p><p> The objective of this dissertation was to characterize the <i>M. oryzae</i> homologs of <i>Histoplasma capsulatum RYP</i> (<i><u> R</u>equired for <u>Y</u>east <u>P</u>hase </i>) genes that are required for transition to the parasitic phase. <i> H. capsulatum</i> is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37&deg;C, the host body temperature. Four<i>H. capsulatum RYP</i> genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. <i> RYP1</i> is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. <i>RYP2</i> and <i> RYP3</i> are homologs of <i>vosA</i> and <i>velB,</i> members of the Velvet family, best characterized in <i>Aspergillus nidulans, </i> where they coordinate morphological differentiation with secondary metabolism. <i>RYP4</i> is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the <i> M. oryzae RYP1</i> homolog, <i>RIG1</i> (<i><u> R</u>equired for <u>I</u>nfectious <u>G</u>rowth </i>), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. <i>&Delta;rig1</i> was blocked in the transition to infectious hyphal growth, similar to <i> H. capsulatum ryp1,</i> which could not transition to the yeast phase. Deletion mutants of <i>M. oryzae RYP2, RYP3,</i> and <i>RYP4 </i> homologs were similar to the wild type in somatic growth and pathogenicity indicating that although <i>RIG1</i> is a pathogenicity factor conserved in plant and animal pathogens, such conservation does not apply to all of the <i>RYP</i> pathogenicity genes identified in <i>H. capsulatum. </i> </p><p> <i>&Delta;rig1</i> is the first <i>M. oryzae</i> mutant known to be blocked in production of primary infection hyphae. Overall, the study suggests limited parallels exist in phase transition of fungal pathogens of plants and animals.</p>

<p>RNA interference (RNAi) acts as an antiviral defense mechanism in fungi, plants, nematodes, insects, and mammals. In antiviral RNAi, virus-specific double-stranded RNA is processed into small interfering RNAs (siRNAs) to guide specific viral RNA degradation by the RNAi machinery. Although antiviral RNAi is non cell-autonomous in plants, it is unknown if antiviral RNAi is also systemic in animals. In this dissertation, I characterized the nematode <i> Caenorhabditis elegans</i> mutants defective in systemic RNAi in their antiviral RNAi response induced by either the replication of a Flock house virus-derived replicon or the infection of Orsay virus. The results from these genetic studies provided evidence for the first time to support an antiviral function of systemic RNAi in animals. Comparison of the population of viral siRNAs by deep sequencing further revealed that <i>C. elegans</i> mutants with strong defects in systemic antiviral RNAi were all partially defective in the biogenesis of the viral secondary siRNAs. A possible role for the viral siRNAs in systemic antiviral RNAi is discussed. </p>

<p> Env7 is a vacuole membrane-localized protein kinase that is orthologous to the human serine/threonine protein kinase, STK16. It is evolutionarily well-conserved throughout Eukarya, and it has one ortholog in Bacteria. Phylogenetic analyses of sequences homologous to Env7 revealed clades that are inconsistent with established eukaryotic phylogeny, suggesting that both horizontal and vertical gene transmission are responsible for their conservation. Conserved amino acid residues and motifs that are potentially important to Env7's catalytic activity, localization, and interactions with other proteins were also identified and assessed. Additionally, one such conserved motif&mdash;the glycine-rich loop&mdash;was mutated in an effort to affect ATP binding in Env7. The phenotype resulting from this mutation was a slightly increased number of mutant cells exhibiting multi-lobed vacuoles under normal conditions.</p>

<p> Fungi are thought to be one of the most diverse groups of organisms in the Arctic. They drive mineral and energy cycles and influence the occurrence of other organisms as mutualists (mycorrhizae, endophytes, lichens), decomposers and pathogens. Nevertheless, information on fungal biodiversity and distribution patterns in relation to environments across the Arctic is still sparse. Molecular methods were used to examine the diversity and community structures of ectomycorrhizal fungi (EMF) associated with two principal arctic host plants, <i> Salix arctica</i> and <i>Dryas integrifolia,</i> as well as total soil fungal communities of adjacent disturbed and undisturbed areas of patterned-ground features across the five bioclimatic subzones (A-E) of the North American Arctic. Key findings include the following: (1) More diverse fungal communities had been observed than previously known. These communities encompass nearly all fungal phyla and included all fungal guilds. However, a few species-rich fungal families dominated these fungal communities. (2) Surprisingly, species richness did not decline with latitude. (3) The most abundant fungal taxa were widely distributed in and beyond the Arctic. Yet root (EMF) and soil fungal communities showed niche preferences in regard to bioclimatic subzones. Furthermore, disturbed and undisturbed patterned ground features harbored different soil fungal communities with the exception of the coldest subzone A. In contrast, EMF community composition was not affected by host plant identity. (4) Fungal communities in the warmest subzone E were distinct from the other arctic subzones and the majority of taxa matched fungi from the boreal forest. (5) Key drivers of fungal community and guild composition along the bioclimatic gradient included regional climate, pH as well as vegetation composition and productivity across the subzones. At the local scale of patterned-ground features, fungal communities were correlated with vegetation composition and microclimate. With a warming climate, I would expect an enhanced colonization of patterned-ground features by vascular plants that would then affect fungal community structure not only at the species level, but also at the level of fungal guilds. In particular I would expect increases in fungi that are symbiotic with plants and a northward shift of both plant and fungal taxa.</p>