<p>Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus <em>Phytophthora </em>are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus <em>Saprolegnia</em>, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen <em>Phytophthora infestans</em> and the fish pathogen <em>Saprolegnia monoica</em>.</p><p> </p><p>A new Oomycete <em>CesA</em> gene family was identified. It contains four homologues designated as <em>CesA1, CesA2, CesA3</em> and <em>CesA4</em>. The gene products of <em>CesA1, 2</em> and <em>4 </em>contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete <em>CesA </em>genes. <em>CesA3</em> is the dominantly expressed <em>CesA </em>homologue in the mycelium of both <em>S. monoica</em> and <em>P. infestans</em>, while <em>CesA1</em> and<em> CesA2</em> are up-regulated in virulent life stages of <em>P. infestans</em>. <em>CesA4</em> was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole <em>CesA</em> gene family in <em>P. infestans</em> lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on <em>SmCesA</em> gene expression, which was accompanied by an increased b-glucan synthase activity <em>in vitro</em>. In addition, these inhibitors slowed down the growth of the mycelium from <em>S. monoica</em>. Zoospores from <em>P. infestans</em> treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the <em>CesA</em> gene family.</p><p>Altogether these results show that at least some of the <em>CesA1-4</em> genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by <em>P. infestans</em>.</p><p> </p>

The therapeutic potential of a novel multi-targeted small-interfering RNA (siRNA) was investigated in human breast cancer cells. Previous studies had identified an siRNA that specifically and potently inhibited expression of thymidylate synthase (TS) by directly targeting human TS mRNA. TS is a folate-dependent enzyme that catalyzes the key reaction involved in synthesizing nucleotide precursors for DNA biosynthesis, and as such, it plays a critical role in maintaining cell growth. The goal of this thesis was to design and develop a novel siRNA molecule that targeted TS mRNA as well as a cellular mRNA that encodes a different cellular protein involved in cancer cell growth and proliferation, such as a member of the ErbB family. Gene sequence analysis was performed and identified an overlapping sequence between TS and ErbB2 mRNAs. An siRNA duplex was then designed to simultaneously target human TS and ErbB2 mRNA. Transfection of the multi-targeted siRNA (TS1M17) revealed that both ErbB2 and TS proteins were significantly suppressed in a time and dose-dependent manner in ErbB2-overexpressing human breast cancer SKBR3 cells. The corresponding mRNA levels, as determined by RT-PCR, were also decreased. Protein levels of other ErbB family members, including ErbB1 and ErbB3, remained unchanged with siRNA treatment. An ErbB2-specific siRNA (B2450) inhibited ErbB2, but had no effect on TS expression demonstrating the specificity of the multi-targeted siRNA against both TS and ErbB2. Mismatched (TS1-Mismatch) and control (GL2) siRNAs had no inhibitory effects on expression of the two target proteins. Suppression of activated ErbB2, as determined by expression of phosphorylated ErbB2 protein, was observed with transfection of TS1M17 siRNA. In addition, the expression of downstream signaling proteins, such as phosphorylated mitogen activated protein kinase (p-MAPK), p27Kip1, p21Cip1, cyclin D1, and survivin were significantly changed. In contrast, control siRNAs did not exert any inhibitory effects on downstream signaling. Taken together, these findings suggest that TS1M17 siRNA inhibits signaling of the ErbB2 pathway. The effect of TS1M17 siRNA on cytotoxicity was analyzed by WST-1 assay. Upon transfection into SKBR3 cells, the TS1M17 siRNA significantly suppressed cell proliferation with an IC50 value of 0.65 nM, which is 154-fold more potent than ErbB2- and TS-specific siRNAs. This study suggests that targeting expression of ErbB2 and TS, two key proteins involved in distinct and critical pathways for cancer growth and proliferation, with a single siRNA molecule may provide a novel approach for cancer chemotherapy.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 96-107).

Reproduction de : Thèse de doctorat : Qualité et sécurité des aliments : Toulouse, INPT : 2004. / Titre provenant de l'écran-titre. Bibliogr. 174 réf.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 92-99).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xii, 152 p. : ill., (some col.). Includes abstract and vita. Advisor: Anthony Young, Molecular, Cellular, and Developmental Biology Program. Includes bibliographical references (p. 137-150).

Modular polyketide synthases (PKSs) are the largest enzymes known to man and are responsible for synthesizing some of the most important human medicines. Their ability to construct stereochemically-rich carbon chains containing diverse substituents has inspired the biosynthetic community to engineer these factories for the in vitro synthesis of a small library of polyketide compounds. New complex polyketides are discovered every year, yet the lack of compound prohibits characterization and testing of these new compounds for medicinal properties. Smaller polyketide compounds generated in vitro could be organically manipulated to generate larger, more complex polyketide natural products and natural product analogs. Chemoenzymatic approaches like this would be extremely beneficial to the scientific community; however, there are still obstacles that must be overcome before the use of PKS for the preparative synthesis of an in vitro generated polyketide library would prove fruitful: purchasing substrates such as methylmalonyl-CoA is cost-prohibitive, PKSs are often difficult to express and purify, and the products generated are typically nonchromophoric. The use of a malonyl-CoA ligase from Streptomyces coelicolor (MatB) was investigated for the enzymatic synthesis of polyketide extender units such as methylmalonyl-CoA (Chapter 2). MatB synthesized a total of 5 CoA-linked extender units in vitro: malonyl-, methylmalonyl-, ethylmalonyl-, hydroxymalonyl- and methoxymalonyl-CoA. Two ternary complex structures of MatB with bound product and leaving group were also solved to sub-2Å resolution. MatB generated extender units were employed in the module-catalyzed synthesis of a triketide pyrone. The selectivity of a PKS module to incorporate a variety of side chains into triketide pyrones was also investigated (Chapter 3). A total of 10 triketide pyrone compounds were synthesized, 5 produced via modular "stuttering" and one possessing a terminal alkyne chemical handle. Lastly, nonchromphoric polyketide products were made visible upon copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) with fluorescent sulforhodamine B azide revealing insights into in vitro reactivites of a PKS module (Chapter 4). The work described in this dissertation has helped advance the scientific community towards procuring an in vitro synthesized polyketide library for future synthetic applications. / text

Septins are a highly conserved family of eukaryotic proteins having significant homology within and among species. In the budding yeast, Saccharomyces cerevisiae, a septin-based hierarchy of proteins is required to localize chitin in the bud neck prior to septum formation. However, this process has not been clarified in a filamentous, conidiogenous fungus capable of yeast growth, such as Wangiella dermatitidis, a polymorphic agent of human phaeohyphomycosis. Prior studies of this melanized mold showed that some chitin synthase mutants (wdchsΔ) have defects in yeast septum formation, suggesting that the septins of W. dermatitidis might functionally associate with some of its chitin synthases (WdChsp). To test this hypothesis, four vegetative septin homologs of S. cerevisiae were cloned from W. dermatitidis and designated WdCDC3, WdCDC10, WdCDC11, and WdCDC12. Of the four, only WdCDC3 functionally complemented completely a strain of S. cerevisiae with a ts mutation in the corresponding gene, although WdCDC12 did so partially. Functional characterizations by mutagenesis of the four W. dermatitidis septin genes revealed that resulting mutants (wdcdc[delta]) each had unique defects in yeast growth and morphology, indicating that each septin carried out a distinct function. Furthermore, when a wdcdc10[delta] mutation was introduced into five different wdchs[delta] strains, weak genetic interactions were detected between WdCDC10 and WdCHS3 and WdCHS4, and a strong interaction between and WdCHS5. Cytological studies showed that WdChs5p was mislocalized in some septin mutants, including wdcdc10[delta]. These results confirmed that in W. dermatitidis septins are important for proper cellular morphogenesis, cytokinesis, and especially septum formation through associations with some chitin synthases. / text

No description available.

The discovery of nitric oxide (NO) as the endothelial-derived relaxing factor has led to significant research on NO and the proteins involved in its function, generation, location and regulation. Synthesis of NO by blood vessel endothelial cells results from the enzymatic oxidation of arginine by endothelial nitric oxide synthase (eNOS) resulting in the formation of equimolar amounts of NO and citrulline. Citrulline is sequentially recycled to arginine by successive reactions involving the enzymes argininosuccinate synthase (AS) and argininosuccinate lyase (AL), respectively. eNOS activity has been shown to be regulated by post-translational modifications including dynamic phosphorylation on multiple serine/threonine and tyrosine residues and dynamic O-linked beta-N-acetylglucosamine (O-GlcNAc) modifications on serine/threonine residues.Previous studies showed that even though intracellular endothelial arginine levels range from 0.1 to 0.8 mM and the Km of eNOS for arginine is 3 uM, the addition of exogenous arginine caused an increase in NO production. To explain this "arginine paradox" we hypothesize that there is a separate and distinct cellular source of arginine substrate directed to NO production and that this source is maintained through the regeneration of arginine via a citrulline-NO cycle. The presented research has provided the following evidence in support of this hypothesis: Citrulline stimulates NO production in an arginine-rich medium, without an increase in intracellular arginine. The enzymes of the citrulline-NO cycle, eNOS, AS and AL, co-fractionate with caveolin-1 in an endothelial cell caveolar membrane fraction. In vitro interaction assays demonstrate protein-protein interactions between fusion tagged AS or AL with eNOS or caveolin-1. Simultaneous monitoring of apparent citrulline and NO production demonstrates an efficient and essential coupling of the reactions of the citrulline-NO cycle. Glucosamine treatment of endothelial cells results in increased NO production in the basal state and decreased NO production in the stimulated state.Our findings demonstrate the enzymes of the citrulline-NO cycle, eNOS, AS and AL, are functionally associated, the reactions are efficiently coupled and enzyme activities are changed by post-translational modifications based on nutrient levels. These alterations ensure a constant and distinct source of arginine which is available for NO production to ensure vascular health.