Maize is the staple food for a majority of people in Southern Africa, but plant diseases are responsible for at least 10% of crop production losses. Bacterial leaf streak (BLS) of maize was first reported in South Africa in 1949 and has not been reported elsewhere. Very little is known about the pathogen involved and therefore it is deemed necessary to compile a characteristic profile for the pathogen to prevent the possibility of major crop losses as a result of this disease. This study aimed to use biochemical and phenotypic methods to determine the specific characteristics of the causal agent of BLS. Diseased plant material showing symptoms of BLS were collected during the maize production seasons of 2012 and 2013 within South Africa’s maize production regions namely the North West, Free State, Gauteng and Northern Cape provinces. To prevent contamination, maize leaves were surface sterilised thoroughly before bacterial isolation commenced. Sections of the infected maize leaves were placed on GYC agar plates on which yellow, mucoid bacterial colonies after incubation for 24 to 48 hrs. The isolated bacteria were purified and the molecular identification of the bacteria was conducted in a related study. Although literature indicates that Xanthomonas campestris pv. zeae is the causal agent of BLS, pure cultures obtained from maize leaves showing characteristic symptoms of BLS were identified as species of Xanthomonas, Pantoea, and Enterobacter. To elucidate the pathogenicity of the isolated strains, pathogenicity tests based on Koch’s postulates were performed. Results from the pathogenicity tests confirmed that only the isolate Xanthomonas species was capable of inducing the characteristic BLS symptoms when healthy maize plants were inoculated with the suspected pathogens. It is important to inoculate the maize seedlings at the correct age (four-leaf stage) and the spray method is recommended. Re-isolation was repeated from the same plant material used during the initial isolation process but the isolation method was amended. The optimised isolation method involved the use of a dilution range and spread plate method. Colonies from this isolation technique grew as bright yellow colonies that were identified as Xanthomonas spp. This outcome indicates the importance of surface sterilisation, pulverisation and subsequent dilution of plant materials for isolation of bacterial pathogens from diseases plants. These isolates were used to create protein profiles with SDS-PAGE electrophoresis and carbon utilisation patterns with the Biolog® GN2 system. Protein profiling banding patterns was assessed based on presence/absence criteria. Highly similar protein profiles were observed among the X. campestris pv. zeae isolates but groupings of different protein profiles were determined when minor differences in the protein profiles was taken into account. Xanthomonas campestris pv. zeae was successfully distinguished from the X. axonopodis pv. vasculorum reference strain through unique SDS banding patterns. Banding patterns obtained from cultures grown in a liquid medium (tryptic soy broth) were of a higher quality than the banding patterns obtained from bacteria harvested from solid media (CYG agar plates). Carbon source utilisation data was used to evaluate the average well colour development obtained from each isolate. Statistically significant differences were found among some of the isolates, with some isolates being metabolically more active than other isolates. Substrate utilisation patterns produced by the isolates corresponded to previously published studies on various Xanthomonas species. The cell count of the samples used during carbon utilisation patterns must be standardised in order to obtain reliable results. During this study, the application of Koch’s postulates and two inoculation techniques confirmed that Xanthomonas campestris pv. zeae is the pathogen responsible for bacterial leaf streak of maize. Members of the Pantoea and Enterobacter genera were found on the leaf surface of maize plants infected with BLS but inoculations of healthy maize plants with these bacteria did not result in bacterial leaf streak symptoms on the maize plants. These bacteria were not pathogenic and were considered endophytes. The identified pathogen was characterised through protein and metabolic profiling. The protein profiles of the pathogen obtained through analysis of the major bands of the SDS-PAGE gels were highly similar and distinguishable from the Xanthomonas reference culture. Groupings within the X. campestris pv. zeae group was found when major and minor bands were considered, this may however be altered when the intensities of the bands are used during analysis. Carbon utilisation patterns were assessed using Biolog® GN2 plates. A metabolic fingerprint was created for the pathogen of BLS, it was possible to distinguish between X. campestris pv. zeae and other Xanthomonas strains based on the fingerprint. This fingerprint could be used to identify the pathogen. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Maize is the staple food for a majority of people in Southern Africa, but plant diseases are responsible for at least 10% of crop production losses. Bacterial leaf streak (BLS) of maize was first reported in South Africa in 1949 and has not been reported elsewhere. Very little is known about the pathogen involved and therefore it is deemed necessary to compile a characteristic profile for the pathogen to prevent the possibility of major crop losses as a result of this disease. This study aimed to use biochemical and phenotypic methods to determine the specific characteristics of the causal agent of BLS. Diseased plant material showing symptoms of BLS were collected during the maize production seasons of 2012 and 2013 within South Africa’s maize production regions namely the North West, Free State, Gauteng and Northern Cape provinces. To prevent contamination, maize leaves were surface sterilised thoroughly before bacterial isolation commenced. Sections of the infected maize leaves were placed on GYC agar plates on which yellow, mucoid bacterial colonies after incubation for 24 to 48 hrs. The isolated bacteria were purified and the molecular identification of the bacteria was conducted in a related study. Although literature indicates that Xanthomonas campestris pv. zeae is the causal agent of BLS, pure cultures obtained from maize leaves showing characteristic symptoms of BLS were identified as species of Xanthomonas, Pantoea, and Enterobacter. To elucidate the pathogenicity of the isolated strains, pathogenicity tests based on Koch’s postulates were performed. Results from the pathogenicity tests confirmed that only the isolate Xanthomonas species was capable of inducing the characteristic BLS symptoms when healthy maize plants were inoculated with the suspected pathogens. It is important to inoculate the maize seedlings at the correct age (four-leaf stage) and the spray method is recommended. Re-isolation was repeated from the same plant material used during the initial isolation process but the isolation method was amended. The optimised isolation method involved the use of a dilution range and spread plate method. Colonies from this isolation technique grew as bright yellow colonies that were identified as Xanthomonas spp. This outcome indicates the importance of surface sterilisation, pulverisation and subsequent dilution of plant materials for isolation of bacterial pathogens from diseases plants. These isolates were used to create protein profiles with SDS-PAGE electrophoresis and carbon utilisation patterns with the Biolog® GN2 system. Protein profiling banding patterns was assessed based on presence/absence criteria. Highly similar protein profiles were observed among the X. campestris pv. zeae isolates but groupings of different protein profiles were determined when minor differences in the protein profiles was taken into account. Xanthomonas campestris pv. zeae was successfully distinguished from the X. axonopodis pv. vasculorum reference strain through unique SDS banding patterns. Banding patterns obtained from cultures grown in a liquid medium (tryptic soy broth) were of a higher quality than the banding patterns obtained from bacteria harvested from solid media (CYG agar plates). Carbon source utilisation data was used to evaluate the average well colour development obtained from each isolate. Statistically significant differences were found among some of the isolates, with some isolates being metabolically more active than other isolates. Substrate utilisation patterns produced by the isolates corresponded to previously published studies on various Xanthomonas species. The cell count of the samples used during carbon utilisation patterns must be standardised in order to obtain reliable results. During this study, the application of Koch’s postulates and two inoculation techniques confirmed that Xanthomonas campestris pv. zeae is the pathogen responsible for bacterial leaf streak of maize. Members of the Pantoea and Enterobacter genera were found on the leaf surface of maize plants infected with BLS but inoculations of healthy maize plants with these bacteria did not result in bacterial leaf streak symptoms on the maize plants. These bacteria were not pathogenic and were considered endophytes. The identified pathogen was characterised through protein and metabolic profiling. The protein profiles of the pathogen obtained through analysis of the major bands of the SDS-PAGE gels were highly similar and distinguishable from the Xanthomonas reference culture. Groupings within the X. campestris pv. zeae group was found when major and minor bands were considered, this may however be altered when the intensities of the bands are used during analysis. Carbon utilisation patterns were assessed using Biolog® GN2 plates. A metabolic fingerprint was created for the pathogen of BLS, it was possible to distinguish between X. campestris pv. zeae and other Xanthomonas strains based on the fingerprint. This fingerprint could be used to identify the pathogen. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...

Free-living marine nematodes are the most ubiquitous, abundant and diverse meiofaunal component of benthic communities. These are excellent model organisms, due to its short life span, wide availability and feasibility to cultivate with minimum laboratory facilities. In this study, a population of Diplolaimella dievengatensis Jacobs 1991 (Nematoda, Monhysteridae) from the relatively Pristine estuary of the Guaratuba River in São Paulo, Brazil was isolated and cultivated. The goals were; i- to apply an integrative taxonomic approach in order to compare this population from Brazil with another from the species type-locality in the Belgian Coast, with regard to morphological, life-cycle and the 18S gene of the rDNA molecular data; and ii- to compare, at the light of life history theory, the responses of life history parameters such as; fecundity, growth and survivorship of D. dievengatensis (here as a slower species) and Litoditis marina (Bastian, 1865) (Nematoda, Rhabditidae) (as a faster species) under sublethal exposition to the sodium dodecyl sulfate (SDS) surfactant. The population from Brazil was similar to that from Belgium coast in all parameters. Although morphometric analyses considered the Brazilian D. dievengatensis isometrically larger than the Belgian population regarding some characters, the presence of all diagnostic characters confirmed the similarity of both species. The life-cycle, hatching time, final body length, and biomass parameters were similar in both populations, in which females were larger than males. The population growth, measured as intrinsic rate of natural increase was slightly higher for the D. dievengatensis from Brazil (rm=0.41), than the European population (rm =0.348). Molecular comparison on Genbank showed 99.4% of similarity between both populations, indicating therefore, that D. dievengatensis from Brazil is similar to those from Belgium. In the chapter ii, both species responded differently to SDS exposition. Growth and reproduction rate of D. dievengatensis surprisingly were enhanced at low and intermediate concentrations of SDS (0.001 and 0.003%), while for L. marina these parameters were reduced in all SDS concentrations tested (0.001, 0.003 and 0.006%). The SDS did not affect the survivorship of adults of the slower specie. On the other hand, survivorship of adults of the fast species was significantly affected by SDS and this effect was dependent on adult gender, with reduced rates of males exposed to 0.006% SDS. Although both species are located nearby along the fast-slow continuum, they responded distinctly to of the toxic SDS effect. Effects over L. marina met the trend of faster species in allocating fewer investments in defenses against physiological injuries and on their own somatic maintenance. We propose that the apparent lower resistance of this faster species under stress at the individual level is balanced by their higher reproductive rates, conferring higher resilience to this species at the population level. Finally, it was demonstrated that the marine nematode D. dievengatensis is an important native model organism which can be used in a wide range of studies and experimental tests. Identifying and understanding differential effects of stress in the context of life-history theory is an important aspect which enhanced our understanding about the threats posed by anthropogenic activities on natural communities / Nematodas marinhos de vida livre são o mais onipresentes, abundantes e diversos componentes da meiofauna em comunidades bênticas. São excelentes organismos modelo devido a seu curto ciclo de vida, ampla disponibilidade e viabilidade de cultivo com mínima estrutura laboratorial. Neste estudo, uma população de Diplolaimella dievengatensis Jacobs 1991 (Nematoda, Monhysteridae) do estuário relativamente prístino do rio Guaratuba, São Paulo, Brasil foi isolada e cultivada. Os objetivos foram: ii- aplicar uma abordagem taxonômica integrativa a fim de comparar esta população do Brasil com outra da localidade tipo dessa espécie, da costa da Bélgica, com relação à dados morfológicos, do ciclo de vida e molecular do gene 18S do rDNA; e ii-comparar, à luz da teoria da historia de vida, repostas de parâmetros do ciclo de vida tais como: fecundidade, crescimento e sobrevivência de D. dievengatensis (aqui como espécies mais lenta) e Litoditis marina (Bastian, 1865) (Nematoda, Rhabditidae) (como espécie rápida) sob exposição subletal ao surfactante dodecil sulfato de sódio (DSS) . A população do Brasil foi similar a da costa da Bélgica em todos os parâmetros. Embora analises morfométricas consideraram D. dievengatensis do Brasil isometricamente maior que a população belga em relação em algumas características, a presença de todos os caracteres diagnósticos confirmaram a similaridade de ambas as espécies. Os parâmetros de ciclo de vida, tempo de eclosão, comprimento corporal final e biomassa foram similares em ambas as populações, nas quais fêmeas foram maiores que machos. Crescimento populacional, mensurado como taxa intrínseca de crescimento natural foi ligeiramente mais alta para D. dievengatensisdo Brasil (rm=0.41), que para a população europeia (rm=0.348). Comparações moleculares no Genbank mostraram 99.4% de similaridade entre ambas populações, indicando portanto que a população do Brasil é similar a D. dievengatensis belga. No capítulo ii, ambas as espécies responderam distintamente a exposição ao SDS. Supreendentemente as taxas de crescimento e de reprodução de D. dievengatensis foram incrementadas sob concentrações mais baixas e intermediárias (0.001 e 0.003%), enquanto para L. marina esses parâmetros foram reduzidos em todas as concentrações de DSS testadas (0.001, 0.003 e 0.006%). O DSS não afetou a sobrevivência de adultos da espécie mais lenta. Por outro lado, a sobrevivência de adultos da espécie mais rápida foi significativamente afetada pelo SDS e esse efeito foi dependente do sexo, com taxas reduzidas em machos expostos a 0.006 % de DSS. Embora ambas as espécies estejam proximamente dispostas ao longo do gradiente \"rápido-lento\", elas responderam diferentemente ao efeito tóxico do DSS. Efeitos sobre L marina se enquadram no padrão de espécies mais rápidas ao alocar menores investimentos para as defesas contra danos fisiológicos e para sua própria manutenção somática. Nós propomos que a aparente menor resistência desta espécie rápida sob estresse ao nível individual é balanceada por suas altas taxas reprodutivas, conferindo mais alta resiliência a essa espécie ao nível populacional. Por fim, foi demonstrado que D. dievengatensis é um importante organismo modelo nativo que pode ser usado em uma ampla diversidade estudos e testes experimentais. Identificar e compreender diferentes efeitos do estresse dentro do contexto da teoria da história de vida é um aspecto importante, o qual tem aumentado nosso conhecimento sobre as ameaças de atividades antropogênicas sobre comunidades naturais

Made available in DSpace on 2016-04-28T20:15:32Z (GMT). No. of bitstreams: 1 2009 - Gisele Santos de Meireles.pdf: 1867896 bytes, checksum: 50898c1a65b34d41d9d4352322ce8d1e (MD5) Previous issue date: 2009-02-27 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / This work aimed, to determine the phenotypic analysis from sporulated oocysts of Cystoisospora felis by using a sequential method of cleaning techniques. By using SDS-PAGE at 10 and 12 % were separated 22 protein groups as: 26.65; 29.71; 31.79; 36.41; 44.03; 50.09; 56.08; 62.64; 73.65; 77.55; 85.34; 97.62; 98.66; 101.24; 104.21; 109.23; 110.56; 113.21; 138.48; 180.50; 206.81 and 244.51KDa belonged to estrutucture from C. felis sporulated oocysts. Western Blotting technique was performed after SDS-PAGE and were identified these antigenic proteins: p39.39; p42.18; p44.40; p47.82; p55.46; p58.75; p66.08; p77.41; p92.85; p99.58; p104.10; p112.84; p203.15 and p37.25; p38.80; p67; p69; p77; p93; p99; p103; p111.19; p202.66 KDa from hyperimmune and natural infected rabbits by C. Felis respectively. / Este trabalho teve por objetivo, tra?ar um perfil prot?ico de oocistos esporulados de Cystoisospora felis, recuperados a partir do uso seq?encial de v?rias t?cnicas de purifica??o adaptadas para o uso em oocistos esporulados de C. felis. Com o aux?lio do SDS-PAGE a 10 e 12 % resultou na identifica??o de 22 grupos prot?icos de 26; 29; 31; 36; 44; 50; 56; 62; 73; 77,55; 85,34; 97,62; 98,66; 101,24; 104,21; 109,23; 110,56; 113,21; 138,48; 180,50; 206,81 e 244,51 KDa, pertencentes a estrutura dos oocistos esporulados e esporozoitas de C. felis. Com base nesse resultado e em soro de coelho heter?logo anti-C. felis foi poss?vel desenvolver uma t?cnica de diagn?stico imunoenzimatico com western Blotting para identifica??o de animais infectados de maneira natural ou experimental com C. felis, identificando os seguintes prote?nas antig?nicas: p39.39; p42.18; p44.40; p47.82; p55.46; p58.75; p66.08; p77.41; p92.85; p99.58; p104.10; p112.84; p203.15 e p37.25; p38.80; p67; p69; p77; p93; p99; p103; p111.19; p202.66 KDa, respectivamente.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-10-24T18:46:15Z No. of bitstreams: 1 2013 - Gisele Santos de Meireles.pdf: 16741114 bytes, checksum: 1d4b03cbd2287a0984257f70efb149da (MD5) / Made available in DSpace on 2018-10-24T18:46:15Z (GMT). No. of bitstreams: 1 2013 - Gisele Santos de Meireles.pdf: 16741114 bytes, checksum: 1d4b03cbd2287a0984257f70efb149da (MD5) Previous issue date: 2013-02-27 / CAPES / FAPERJ / This study aimed to determine from the protein profile of oocysts of Cystoisospora felis recovered from the sequential use of various purification techniques adapted for specific use in sporulated oocysts of C. felis. With the aid of SDS-PAGE 12% resulted in identification of 25 groups of protein: 266, 240, 186, 165, 140, 119, 112, 105, 98, 90, 78, 55, 47, 42, 37, 35, 30, 27-28, 25, 22, 19, 18, 16, 14 kDa belonging to the structure of sporulated oocysts and sporozoites of C. felis. Based on this results and heterologous bovine serum anti-C. felis was possible to determine polypeptides dominant relevant to diagnostic immunoassay technique with "Western Blotting", these being immunodominant bands: P208, P138, P113, p106, p62, p56, p51, p48, p44, p38, p36, and p33 p27. In order to avoid misdiagnosis from cross-reactivity a positive control serum anti-C. felis was compared to with positive serum anti-Toxoplasma and Neospora in order to exclude the common protein bands, probably markers of gender and group to identify cattle infected naturally or experimentally with C. felis. As showed, the following specific antigenic protein units: p 206-208, P137-139, p112- 113, p104-107, p27-28 are responsible for determining the animal tested positive or not for Cystoisospora felis. Of the analysis of the variables could be observed that the presence of felines related to the handling, size and type of milking properties facilitates dispersion C. felis. / Este trabalho teve por objetivo, diagnosticar a infec??o por Cystoisospora felis em bovinos atrav?s do Western Blotting, apartir de oocistos obtidos com o uso sequencial de v?rias t?cnicas de purifica??o adaptadas para o uso em oocistos esporulados de C. felis. Com o aux?lio do SDS-PAGE a 12 % resultou na identifica??o de 25 grupos proteicos de: 266; 240; 186; 165; 140; 119, 112, 105, 98, 90, 78, 55, 47, 42, 37, 35, 30, 27-28, 25, 22, 19, 18, 16, 14 KDa, pertencentes a estrutura dos oocistos esporulados e esporozoitas de C. felis. Com base nesse resultado e em soro de bovino heter?logo anti-C. felis foi poss?vel determinar os polipept?deos dominantes relevantes ? t?cnica de diagn?stico imunoenzimatico com ?Western Blotting?, sendo estas, as bandas imunodominantes: p208, p138, p113, p106, p62, p56, p51, p48, p44, p38, p36, p33 e p27. A fim de evitar o diagn?stico equivocado a partir de rea??es cruzadas foi feita a compara??o do soro controle positivo anti-C. felis com o soro positivo anti-Toxoplasma e Neospora com o intuito de excluir as bandas proteicas comuns, prov?veis marcadoras de g?nero e grupo para identifica??o de bovinos infectados de maneira natural ou experimental com C. felis. Sendo evidenciadas, as seguintes unidades proteicas antig?nicas espec?ficas: p 206-208, p137-139, p112-113, p104-107, p27-28 respons?veis por determinar a positividade dos animais testados para C. felis. A partir das an?lises das vari?veis foi poss?vel observar que a presen?a de felinos associados, ao manejo, tipo de ordenha e tamanho das propriedades facilita a dispers?o de C. felis.

In this thesis, coda waves recorded by local seismographic networks will be analyzed to estimate the three-dimensional spatial distribution of scatterers (SDS). This will be done by using the single scattering approximation. This approach leads to a huge system of equations that can not be solved by traditional methods. For the first time, we will use the Simultaneous Iterative Reconstructive Technique (SIRT) to solve this kind of system in seismological applications. SIRT is slow but provides a means to carry out the inversion with greater accuracy. There is also a very fast non-iterative method that allows to carry out the inversion 102 times faster, with a higher resolution and reasonable accuracy: the Filtered Back-Projection (FBP). If one wishes to use this technique it is necessary to adapt it to the geometry of our problem. This will be done for the first time in this thesis. The theory necessary to carry out the adaptation will be developed and a simple expression will be derived to carry out the inversion.FBP and SIRT are then used to determine the SDS in southern India. Results are almost independent of the inversion method used and they are frequency dependent. They show a remarkably uniform distribution of the scattering strength in the crust around GBA. However, a shallow (0-24 km) strong scattering structure, which is only visible at low frequencies, seems to coincide with de Closepet granitic batholith which is the boundary between the eastern and western parts of the Dharwar craton.Also, the SDS is estimated for the Galeras volcano, Colombia. Results reveal a highly non-uniform SDS. Strong scatterers show frequency dependence, which is interpreted in terms if the scale of the heterogeneities producing scattering. Two zones of strong scattering are detected: the shallower one is located at a depth from 4 km to 8 km under the summit whereas the deeper one is imaged at a depth of ~37 km from the Earth's surface. Both zones may be correlated with the magmatic plumbing system beneath Galeras volcano. The second strong scattering zone may be probably related to the deeper magma reservoir that feeds the system.

Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, β-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two β-1,3-endoglucanses while CA whole leaf protein extracts contain at least three β-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 β-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for β-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of β -1,3-endoglucanses.

Gas hydrate formation generally involves gas dissolution, formation of nuclei and growth of new nucleus. On condition of synthesizing experiments without agitation, the formation of hydrate nuclei is comparatively difficult and needs an induction period which is considerably uncertain and random. Some additives such as surfactant sodium dodecyl sulfate (SDS) can increase the formation rate and reduce the induction time. A hydrate formation and mini drilling experimental system was used to carry on methane hydrate formation experiments with small quantity of SDS and SDS- tetrahydrofuran(THF) in deionized water. The reactor is a high pressure cell (40Mpa) made of titanium alloy with 4 transparent windows and an inner volume of about 2.8 liters. The effect of SDS and THF hydrate on the formation rate and amount of methane hydrate was studied by comparative testing and analyzing the collected data of temperature and pressure. According to the results of the tests, the formation rate of methane hydrate in the SDS-THF solution was faster than that in the SDS solution. As a water-soluble hydrate former, THF hydrate nucleation may be benefit of methane hydrate nucleation. A small amount of SDS and THF could dramatically promote the formation of methane hydrate in the pure water, and rapidly increase the amount of methane hydrate too. Therefore, a great deal of time for experiment was saved, which established a good basis for the coming mini drilling and drilling fluid experiments.

Aeromonas hydrophila is an opportunistic pathogen that causes a wide range of symptoms and diseases in fish. Development of a commercial vaccine has been problematic due to the heterogenicity between isolates of A. hydrophila. A new approach using immunoproteomics was used in this study to try to develop a vaccine that would protect against a wide range of A. hydrophila strains. The virulence of 14 isolates of A. hydrophila from different geographical regions was determined in common carp (Cyprinus carpio) indicating that 6 isolates were virulent, while 8 isolates were avirulent. Expression of cellular and extracellular products (ECP) of six of these isolates (4 virulent and 2 avirulent isolates) were examined following culture of the bacterium in vitro, in tryptic soy broth, and in vivo, in dialysis tubing placed within the peritoneal cavity of carp. Two types of molecular weight cut off tubes (25 and 100 kDa) were used for the implants. Whole cell (WC), outer membrane protein (OMP) and ECPs of the bacteria grown in vitro and in vivo were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations of A. hydrophila grown in vitro and in vivo. The production of unique proteins and up and down-regulation of protein expression were observed in all the preparations of bacteria grown in vitro and in vivo. Unique bands were seen in the 1D SDS-PAGE at 58 and 55 kDa for WC and OMP preparations, respectively, for all the isolates cultured in vivo. Bands of increased intensity were observed at 70, 55, 50 and 25 kDa with WC preparations for the virulent isolates cultured in vivo. Analysis of WC preparations by 2D SDS-PAGE indicated differences in the expression of spots between bacteria cultured in vitro and in vivo. A number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0-6.0 were observed in the bacteria grown in vivo. The protein profiles of different preparations (WC, OMP, ECP) of bacteria cultured in vitro and in vivo were screened by 1D Western blot using antibodies from carp artificially infected with different isolates of A. hydrophila to identify potential vaccine candidates. The WC preparations of A. hydrophila (T4 isolate) grown in vitro were also analysed by 2D Western blot. A 50 kDa protein of A. hydrophila was found to be the most immunogenic molecule in both WC and OMP of bacteria grown both in vitro and in vivo. The protection efficacy of this protein was determined in goldfish by vaccinating fish with electro-eluted 50 kDa protein then challenging the fish with A. hydrophila. Fish were also passively immunised with fish sera raised to the 50 kDa protein and then challenged. The relative percentage survival (RPS) was 67 % in the vaccination trial, while the results were inconclusive for the passive immunisation trial. The 50 kDa protein was confirmed to be the S-layer protein of A. hydrophila following identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Recombinant S-layer protein was then produced and the cross-protection efficacy of this protein against six virulent isolates of A. hydrophila was confirmed in a large scale vaccination trial using carp. The RPS value for the 6 isolates of A. hydrophila ranged from between 56 and 87 %. The results of this project suggest that the immunogenic S-layer protein of A. hydrophila could be used as a common antigen to protect fish against infection by different isolates of this pathogenic bacterium.