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<p>Totally 84 differentially expressed mRNA clones from infective L3 larvae of the parasite <em>Haemonchus contortus</em>, a blood sucking nematode,<em> </em>were analyzed with single strand hybridization assay (SSH). Altogether 79 clones were sequenced, edited, and compared with proteins found via BLAST in GeneBank. The aim was to investigate gene expression and potential protein expression following storage at 5 °C for 32 weeks. mRNA was extracted from fresh and stored L3. The SSH derived products were cloned into <em>E. coli,</em> purified and sequenced with Big Dye Terminator v3.1, and then compared with uploaded sequences in GeneBank. BLAST showed 59 (70 %) reliable protein results, where 39 (66 %) were of nematode origin.. Three sequences (4 %) were recognized as <em>H. contortus</em>-related metabolic proteins. Further research is necessary to elucidate the role of these proteins in relation to storage<em>. </em>Many of the sequences (36 %) are also present in other nematodes, such as <em>Caenorhabditis briggsae </em>and <em>C. elegans,</em> where whole-genome projects have been conducted. Bigger and more accurate databases need to be developed. Maybe the most significant future project is to sequence the whole genome of <em>H. contortus</em>.</p>

Totally 84 differentially expressed mRNA clones from infective L3 larvae of the parasite Haemonchus contortus, a blood sucking nematode, were analyzed with single strand hybridization assay (SSH). Altogether 79 clones were sequenced, edited, and compared with proteins found via BLAST in GeneBank. The aim was to investigate gene expression and potential protein expression following storage at 5 °C for 32 weeks. mRNA was extracted from fresh and stored L3. The SSH derived products were cloned into E. coli, purified and sequenced with Big Dye Terminator v3.1, and then compared with uploaded sequences in GeneBank. BLAST showed 59 (70 %) reliable protein results, where 39 (66 %) were of nematode origin.. Three sequences (4 %) were recognized as H. contortus-related metabolic proteins. Further research is necessary to elucidate the role of these proteins in relation to storage. Many of the sequences (36 %) are also present in other nematodes, such as Caenorhabditis briggsae and C. elegans, where whole-genome projects have been conducted. Bigger and more accurate databases need to be developed. Maybe the most significant future project is to sequence the whole genome of H. contortus.

Only two out of more than 200 V. cholerae serogroups, classified on the basis of LPS structure, are associated with epidemic or pandemic cholera. These toxigenic serogroups carry phage-derived pathogenicity islands coding for the main virulence factors for establishment of cholera disease – cholera toxin (CTX) and toxin-coregulated pilus (TCP). The latter also serves as a bacterial surface receptor for the CTXΦ – the filamentous phage which carries the cholera toxin genes into otherwise harmless to human, environmental bacterium V. cholerae. In its natural aquatic habitat V. cholerae is subject to predator grazing, bacteriophage killing, temperature and pH changes, seasonality of plankton blooms and other environmental factors. Therefore understanding V. cholerae pathogenic and virulence potential requires the knowledge of its interaction not only with human host but also members of aquatic environment and environmental factors. V. cholerae is capable of killing the nematode Caenorhabditis elegans. Using a reverse genetics approach, we demonstrated that the quorum sensing regulated protease PrtV is essential for this killing. Other proteases did not seem to contribute to virulence in this model. The data from this study suggest that the PrtV could be important to V. cholerae in its natural niche for its resistance to the grazing predators. The PrtV protease belongs to an M6 family of metallopeptidases which is represented by an Immune Inhibitor A protease from the insect killing bacterium Bacillus thuringiensis. To characterize the protease in more detail, the PrtV was cloned, overexpressed in V. cholerae and purified from the culture supernatant. The enzyme was calcium stabilized and inhibited by metal ion chelators. In tests with in vitro cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell detachment and death. Using human blood plasma as a source of potential substrates, and by tests with purified candidate substrate proteins, we have identified fibrinogen (all α, β and γ chains), fibronectin and plasminogen to be degraded by the protease. Additionally, PrtV was found to alter the stability of V. cholerae cytolysin implicating its role in modulation of the reactogenicity of V. cholerae secreted factors. Pigmentation has been considered to be important in microbial pathogenesis because it has been associated with virulence in many microorganisms. Using transposon mutagenesis we identified the mutated locus of a pigment producing V. cholerae strain to encode a gene of a tyrosine catabolic pathway. The mutation in a putative homogentisate 1,2-dioxigenase gene lead to accumulation of homogentisic acid, its spontaneous oxidation and formation of a dark pigment. The pigment producing strain was altered in its ability to survive UV exposure and H2O2 stress, and was more efficient in colonizing the suckling mouse intestine compared to the wild type strain. Under the in vitro growth conditions the major virulence factor TcpA and CT expression was found to be somewhat enhanced too.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 77 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 71-77).

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Magistrantūros studijų baigiamajame darbe pateikiami baltojo gūžinio kopūsto veislių atsparumo runkeliniam nematodui (Heterodera schachtii Schmidt, 1871) tyrimų in vitro ir in vivo sąlygomis rezultatai. Darbo objektas – in vivo ir in vitro sąlygomis išauginti baltųjų gūžinių kopūstų daigai, užkrėsti runkelinio nematodo (Heterodera schachtii Schmidt, 1871) cistomis. Darbo metodai: įvairių veislių gūžinio kopūsto veislių atsparumas in vitro sąlygomis atliktas įterpiant runkelinio nematodo cistas prie augalo šaknų (Feyaerts, Coosemans, 1992; Jalali, 1998; Soliman et al., 2005). Prieš įterpimą cistos buvo sterilizuotos 0,05 % HgCl2 tirpalu, laikant jas tirpale 3 min, po to tris kartus nuplaunant steriliu distiliuotu vandeniu (Müller, 1978; Feyaerts, Coosemans, 1992; Jalali, 1998). In vivo sąlygomis kopūstų daigai taip pat užkrėsti įterpiant prie augalo šaknų runkelinio nematodo cistas. Iš augalo šaknų nematodai buvo išskirti modifikuotu piltuvėliniu Bermano metodu kambario temperatūroje esant 2 parų ekspozicijai naudojantis binokuliaru MБC – 1 (Zuckerman ir kt., 1985; Šlepetienė, 1981). Augalų atsparumo vertinimas atliekamas nustatant naujos kartos patelių/cistų, rastų ant šaknų sistemos, skaičių (Harrewijn, 1987; Lelivelt, 1995; Voss et al., 1999; Cook, Noel, 2002). Darbo rezultatai. Baltojo gūžinio kopūsto veislės 'Jetma F1', 'Amager Kurzsturnking', 'Zora', 'Brunswick', 'Kamienna Glowa' ir 'Golden Acre' yra tinkamos H. schachtii plitimui, kaip augalai šeimininkai. Tiek in... [toliau žr. visą tekstą] / The master work presents the results of white cabbage variety resistance for sugar beet nematode (Heterodera schachtii Schmidt., 1871) in vivo and in vitro conditions. Object of the work – In vivo and in vitro conditions grown white head cabbage seedlings infected by sugar beet nematode (Heterodera schachtii Schmidt., 1871) cysts. Method of the work – different varieties of cabbages species resistance in vitro is performed by inserting sugar beet cyst nematode to the plant roots (Feyaerts, Coosemans, 1992; Jalali, 1998, Soliman et al., 2005). Prior to insertion of cysts were sterilized 0.05% HgCl2 solution, keeping them in a solution of 3 min, followed by rinsing three times with sterile distilled water (Muller, 1978; Feyaerts, Coosemans, 1992; Jalali, 1998). In vivo cabbage seedlings are also infected with the insertion of the plant's root cyst nematode. The plant's root nematodes were identified by Berman modified funnel method at room temperature for 2 days of exposure using binoculars MБC - 1 (Zuckerman et al., 1985; Slepetiene, 1981). Plant resistance assessment in identifying the next generation of females / cysts found on the root system (Harrewijn, 1987; Lelivelt, 1995, Voss et al., 1999; Cook, Noel, 2002). The results of work. White Cabbages cultivar 'Jetma F1', 'Amager Kurzsturnking', 'Zora', 'Brunswick', 'Kamienna Glowa' and 'Golden Acre' is suitable for (Heterodera schachtii Schmidt, 1871) spread as host plants. Both in vitro and in vivo conditions found that... [to full text]

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