Submitted by ANNITA MORAIS GIRARDI null (annitamgirardi@gmail.com) on 2016-02-16T19:49:59Z No. of bitstreams: 1 Tese_Annita_Morais_Girardi.pdf: 19183057 bytes, checksum: cf7a9853bcc87aef37b3ca91a2afceea (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-17T13:22:33Z (GMT) No. of bitstreams: 1 girardi_am_dr_jabo.pdf: 19183057 bytes, checksum: cf7a9853bcc87aef37b3ca91a2afceea (MD5) / Made available in DSpace on 2016-02-17T13:22:33Z (GMT). No. of bitstreams: 1 girardi_am_dr_jabo.pdf: 19183057 bytes, checksum: cf7a9853bcc87aef37b3ca91a2afceea (MD5) Previous issue date: 2016-01-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este estudo avaliou o uso da termografia infravermelha e do exame radiológico digital como ferramentas de diagnóstico precoce da laminite em ovinos com acidose ruminal subaguda induzida experimentalmente e os efeitos da ingestão prolongada de alta proporção de alimento concentrado sobre as variáveis clínicas e laboratoriais. Foram utilizadas sete ovelhas adultas, com cânulas ruminais permanentes, que não tiveram prévio contato com alimento concentrado. Para a indução da acidose ruminal, incluiu-se ao volumoso, diariamente, 10% de alimento concentrado até atingir 80%, porcentagem mantida até completarem 19 semanas. A observação dos sinais de diarreia deu-se do quarto ao 22º dia e de laminite do quinto ao 24º dia. O fluido ruminal foi predominantemente líquido, de odor ácido e coloração amarelada. Observou-se elevação inicial nos valores da frequência cardíaca, tempo de sedimentação e flotação no fluido ruminal, plaquetas, neutrófilos segmentados, AST, FA, GGT, cálcio ionizado, magnésio, glicose, ureia, triglicérides e bilirrubinas indireta e total. No início do experimento, notou-se redução da frequência respiratória, linfocitopenia, monocitopenia, hipocalcemia, hipofosfatemia, hiponatremia, hipoproteinemia e hipocolesterolemia. A frequência de movimentos ruminais, o tempo de redução do azul de metileno, os níveis sanguíneos de creatinina, bilirrubina direta, lactato e CK foram reduzidos, enquanto as médias de temperatura, peso e escore corporal, eosinófilos, albumina, cloretos e potássio no sangue aumentaram durante todo o período de observação. Redução do pH ruminal ocorreu nos primeiros dias, a despeito de sua manutenção acima de 5,5 durante as 19 semanas de observação. Mecanismos compensatórios respiratórios e metabólicos, principalmente a acidificação da urina, mantiveram o pH sanguíneo dentro dos limites fisiológicos considerados para a espécie ovina. Dezesseis frações proteicas séricas foram separadas pela eletroforese, dentre elas oito proteínas identificadas pelos seus pesos moleculares, de funções desconhecidas. A maioria das proteínas de fase aguda e as imunoglobulinas identificadas no fracionamento eletroforético variaram durante o período de observação, sugerindo reação inflamatória. Nenhuma alteração macroscópica foi detectada à necropsia. A radiologia digital não permitiu a identificação de alterações podais decorrentes de laminite durante o período de avaliação. A termografia infravermelha detectou aumentos na temperatura da parede do casco mesmo sem a manifestação clínica de dor, indicando que esta técnica pode ser uma ferramenta útil para detectar laminite em ovinos numa fase precoce de evolução da doença. / This study evaluated the use of infrared termography and digital radiologic examination as early diagnostic tools for laminitis in sheep with experimentally induced subacute ruminal acidosis, and the effects of long-term ingestion of a high concentrate diet on clinical and laboratory variables. Seven adult ewes with permanent rumen cannula were used, which did not have any previous ingestion of concentrate feed. For the induction of ruminal acidosis, it was added to the roughage, 10% of concentrate feed until reach 80%, percentage held to the end of the 19-week experiment. Diarrhea signs were observed from fourth to 22nd day and laminitis from fifth to 24th day. Ruminal fluid was predominantly liquid, sour-smelling and yellowish. Initial increases of heart rate, sedimentation and flotation time, platelets, segmented neutrophils, AST, ALP, GGT, ionized calcium, magnesium, glucose, urea, triglycerides, indirect and total bilirubin were noted. At the beginning of the experiment, there was reduction of respiratory rate, lymphocytopenia, monocytopenia, hypocalcemia, hypophosphatemia, hyponatremia, hypoproteinemia, and hypocholesterolemia. Ruminal movement rate, methylene blue reduction time, creatinine, direct bilirubin, lactate and CK blood levels decreased, while body temperature, weight and score, eosinophils, albumin, chloride and potassium blood levels increase throughout the period. Ruminal pH reduction occurred within the first days, despite its maintenance above 5.5 during the 19 weeks. Respiratory and metabolic mechanisms, mainly urine acidification, remained the blood pH between physiologic limits for sheep. Sixteen protein fractions were separated by electrophoresis, among them eight proteins of unknown functions, identified by their molecular weights. Most acute phase proteins and the immunoglobulins identified in the electrophoretic fractionation varied throughout the observation period, suggesting inflammation. No macroscopic alteration was detected at necropsy. The digital radiology did not allow the identification of podal changes resulting from laminitis during the trial period. Infrared thermography has detected increases in the maximum temperature of the hoof wall, even without the clinical manifestation of pain, indicating that this technique can be an useful tool to detect laminitis in sheep at an early stage of the disease. / FAPESP: 2012/09111-7

Orientador: Wilma Aparecida Starke Buzetti / Banca: Caris Maroni Nunes / Banca: Maria Conceição Zocoller Seno / Resumo: Toxocara vitulorum é um parasita nematódeo de alta freqüência no trato intestinal de búfalos, particularmente em bezerros búfalos de um a três meses de idade. Devido à sua alta morbidade e mortalidade, causa consideráveis prejuízos a bubalinocultura. A pesquisa objetivou a obtenção de antígenos de extrato larval solúvel bruto (Ex), do material excretor-secretor (ES) de larvas infectantes e do líquido perientérico (Pe) de adultos de T. vitulorum, bem como a separação das frações protéicas na mistura pelo SDS-PAGE, seguida da análise imunológica por "Western blot" (WB), utilizando-se soros imunes e colostros de búfalos naturalmente infectados com T. vitulorum além de camundongos imunes. O acompanhamento do quadro parasitário dos bezerros búfalos também foi realizado. Pôde-se verificar que os três antígenos, Pe, Ex e ES, apresentaram mobilidades eletroforéticas pelo SDS-PAGE revelando nove (11,5, 14,2, 31, 38, 58, 76, 88, 112 e 165 KDa), onze (11,2, 13,3, 16,5, 22, 25, 32, 43, 53, 68, 82 e 96 KDa) e oito (19, 48, 56, 64, 90, 110, 150 e 190 KDa) bandas protéicas, respectivamente. A maioria dessas frações separadas pela eletroforese, foi reconhecida por todos as amostras de soros e pelo colostro, quando analisada pelo WB. No entanto, somente as bandas de alto peso molecular (68 - 190 KDa) persistiram nos grupos de bezerros búfalos que se encontravam no pico, declínio ou expulsão e na ausência ou autocura, à exceção do antígeno ES, que desapareceu durante o processo de autocura. Já os soros de bezerros búfalos com um de vida, que mamaram o colostro e os daqueles que se encontravam em fase de aparecimento ou ascensão, revelaram com as mesmas frações detectadas no soro e no colostro das búfalas. Os três antígenos reagiram de forma cruzada entre si, quando foram testados com soros homólogos e heterólogos de camundongos imunizados experimentalmente com estes antígenos de T. vitulorum / Abstract: Toxocara vitulorum is a nematode parasite of small intestine of cattle and water buffaloes particularly buffalo calves with one to three months of age, causing high morbidity and mortality. The purpose of this research was the antigen obtaintion and characterization of crude soluble larval extract (Ex), excretory-secretory (ES) of infective larvae, and perienteric fluid (Pe) from adults of T. vitulorum, as well as the separation of protein fractions from the antigenic mixture by SDS-PAGE and analysis of each band by Western blot (WB), using immune sera and colostrum of buffaloes naturally infected by T. vitulorum, and mice experimentally immunized. The parasitological status of the buffalo calves was also evaluated using sequentially coprological examinations. The results showed that three antigens, Pe, Ex and ES, revealed nine (11,5, 14,2, 31, 38, 58, 76, 88, 112, and 165 KDa), eleven (11,2, 13,3, 16,5, 22, 25, 32, 43, 53, 68, 82, and 96 KDa) and eight (19, 48, 56, 64, 90, 110, 150, and 190 KDa) protein bands by SDS-PAGE, respectively. The majority of these isolated bands were recognized by sera and colostrum of all groups of infected animals (buffalo cows one day post parturition and buffalo calves in five different periods of T. vitulorum infection) analyzed by WB. However, only the fractions of high molecular weight (68 - 190 KDa) persisted in the groups of buffalo calves at maximum peak of infection, expulsion and post-expulsion of the parasite or self-cure process, excepting ES antigen, that was not detected during the self-cure process. Sera of buffalo calves at one day of age, after suckling the colostrum and at the beginning of infection reacted with the same bands detected by serum and colostrum of the buffalo cows. The three antigens showed crossed reaction among themselves, when they were tested with homologous and heterologous sera of mice experimentally immunized with them / Mestre

Made available in DSpace on 2014-06-11T19:23:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T20:51:05Z : No. of bitstreams: 1 rocha_tg_me_jabo.pdf: 837046 bytes, checksum: f72346486eb135d1df17c2bfd41fd6ee (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do estudo foi avaliar a transferência de imunidade passiva (TIP) de vacas Canchim aos seus bezerros. Um grupo experimental foi constituído por 13 vacas primíparas e seus bezerros, e o outro por 13 vacas pluríparas e seus bezerros. Foram coletadas amostras de sangue venoso dos bezerros e secreções lácteas das vacas até 1 hora e 1, 2, 7, 15 e 30 dias após o nascimento/parto. No hemograma, notaram-se alterações características do período neonatal, como redução na contagem de hemácias, teor de hemoglobina e volume globular após a ingestão do colostro, além de aumento na contagem de linfócitos e redução da contagem de neutrófilos segmentados. Os exames bioquímicos séricos revelaram aumento nas atividades de GGT (até 3.746 U/L) e ALP (até 1.030 U/L) e nos teores de proteína total (até 7,77 g/dL), globulinas (até 6,01 g/dL), IgA (até 322 mg/dL) e IgG (até 2.918 mg/dL) após a ingestão do colostro, seguidos por redução gradual nestes parâmetros até os 30 dias de idade. As avaliações bioquímicas das secreções lácteas revelaram alta concentração de todos os componentes colostrais analisados, com redução gradual nos seus teores no decorrer do período experimental, à exceção dos minerais, cujas concentrações oscilaram pouco entre os momentos. A TIP foi eficiente em ambos os grupos de bezerros, e a qualidade das secreções lácteas, embora diferentes entre vacas primíparas e pluríparas, não interferiu na passagem de imunoglobulinas da vaca para o bezerro. / The aim of this study was to evaluate the passive immunity transfer from Canchim cows to its calves. One experimental group comprised 13 primiparous cows and its calves and the other group, 13 multiparous cows and its calves. Samples of calves’ venous blood and cow`s lacteal secretions were collected until 1 hour and 1, 2, 7, 15 and 30 days after birth/parturition. In the hemogram, characteristic alterations of this period, such as reduction in the erytocyte count, hemoglobin concentration and corpuscular volume after colostrum intake and rise in the lymphocyte count and reduction in the neutrophil count were noticed. The serum biochemistry revealed augmentation in the GGT (up to 3,746 U/L) and ALP (up to 1,030 U/L) activities and in the total protein (up to 7.77 g/dL), globulin (up to 6.01 g/dL), IgA (up to 322 mg/dL) and IgG (up to 2,918 mg/dL) concentrations after colostrum intake, followed by a gradual reduction in these parameters until 30 days of age. The biochemical evaluation of the lacteal secretions revealed high concentrations of all the colostral components analysed, with gradual reduction in their concentrations along the experimental period, except for the minerals, which showed little oscillation between the moments. The passive immunity transfer was efficient in both groups of calves, and the quality of the lacteal secretions, although different between primiparous and multiparous cows, did not interfere in the transference of immunoglobulins from the cows to the calves.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-23T18:50:16Z No. of bitstreams: 1 GracianaCleciaDantas_TESE.pdf: 5111296 bytes, checksum: b36fdda3c626c34a46d304ad7884c2d2 (MD5) / Approved for entry into archive by Monica Paiva (monicalpaiva@hotmail.com) on 2017-03-23T18:58:12Z (GMT) No. of bitstreams: 1 GracianaCleciaDantas_TESE.pdf: 5111296 bytes, checksum: b36fdda3c626c34a46d304ad7884c2d2 (MD5) / Made available in DSpace on 2017-03-23T18:58:12Z (GMT). No. of bitstreams: 1 GracianaCleciaDantas_TESE.pdf: 5111296 bytes, checksum: b36fdda3c626c34a46d304ad7884c2d2 (MD5) Previous issue date: 2015-05-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O uso de agrot?xicos em cultivo agr?cola ? cada vez maior. Os inseticidas qu?micos usados na agroind?stria causam grandes preocupa??es como a contamina??o ambiental e a falta de seletividade aos organismos. Uma alternativa para esse problema ? a utiliza??o de bioinseticidas. Os bioinseticidas s?o mais seguros que os agrot?xicos comuns e apresentam vantagens como menor toxicidade e alta especificidade. Ademais, podem ser utilizados em menor quantidade, decomp?em-se mais r?pido e sua utiliza??o associada com os inseticidas sint?ticos faz com que o uso de agrot?xicos seja menor, causando baixo impacto ambiental. Em particular, bioinseticidas do tipo baculov?rus t?m sido apontados como uma op??o de substitui??o a estes inseticidas qu?micos na agricultura. Neste trabalho, foi realizada a produ??o in vitro do baculov?rus Spodoptera frugiperda nucleopolyhedrovirus e o estudo do seu perfil eletrofor?tico com base nas prote?nas virais presentes nos v?rus extracelulares deste baculov?rus. As part?culas de v?rus extracelulares obtidas durante a passagem seriada do baculov?rus foram analisadas por eletroforese em gel desnaturante e, como esperado, a poliedrina - prote?na respons?vel pela forma??o do corpo de oclus?o - com aproximadamente 30 kDa foi destaque em todas as passagens analisadas. Prote?nas na regi?o entre 14,4 e 97 kDa tamb?m foram detectadas. A an?lise do efeito da adi??o do horm?nio ?-ecdisona extra?do do Ginseng brasileiro e do horm?nio ecdisona sint?tico na produ??o do baculov?rus Spodoptera foi realizada na terceira e quarta passagens em c?lulas Sf21, momento em que a forma??o dos corpos de oclus?o foi reduzida. Os resultados mostraram que o horm?nio ecdisona n?o influenciou na produ??o dos corpos de oclus?o na terceira passagem. Por?m, na quarta passagem ao se adicionar o horm?nio ecdisona, tanto o sint?tico como o extra?do do Ginseng brasileiro, houve um aumento aproximado de duas vezes na forma??o dos corpos de oclus?o quando comparados ? produ??o sem adi??o de ecdisona. Utilizando este processo de infec??o, foi realizada uma modelagem e simula??o matem?tica que representasse o mecanismo de produ??o do bioinseticida. / O uso de agrot?xicos em cultivo agr?cola ? cada vez maior. Os inseticidas qu?micos usados na agroind?stria causam grandes preocupa??es como a contamina??o ambiental e a falta de seletividade aos organismos. Uma alternativa para esse problema ? a utiliza??o de bioinseticidas. Os bioinseticidas s?o mais seguros que os agrot?xicos comuns e apresentam vantagens como menor toxicidade e alta especificidade. Ademais, podem ser utilizados em menor quantidade, decomp?em-se mais r?pido e sua utiliza??o associada com os inseticidas sint?ticos faz com que o uso de agrot?xicos seja menor, causando baixo impacto ambiental. Em particular, bioinseticidas do tipo baculov?rus t?m sido apontados como uma op??o de substitui??o a estes inseticidas qu?micos na agricultura. Neste trabalho, foi realizada a produ??o in vitro do baculov?rus Spodoptera frugiperda nucleopolyhedrovirus e o estudo do seu perfil eletrofor?tico com base nas prote?nas virais presentes nos v?rus extracelulares deste baculov?rus. As part?culas de v?rus extracelulares obtidas durante a passagem seriada do baculov?rus foram analisadas por eletroforese em gel desnaturante e, como esperado, a poliedrina - prote?na respons?vel pela forma??o do corpo de oclus?o - com aproximadamente 30 kDa foi destaque em todas as passagens analisadas. Prote?nas na regi?o entre 14,4 e 97 kDa tamb?m foram detectadas. A an?lise do efeito da adi??o do horm?nio ?-ecdisona extra?do do Ginseng brasileiro e do horm?nio ecdisona sint?tico na produ??o do baculov?rus Spodoptera foi realizada na terceira e quarta passagens em c?lulas Sf21, momento em que a forma??o dos corpos de oclus?o foi reduzida. Os resultados mostraram que o horm?nio ecdisona n?o influenciou na produ??o dos corpos de oclus?o na terceira passagem. Por?m, na quarta passagem ao se adicionar o horm?nio ecdisona, tanto o sint?tico como o extra?do do Ginseng brasileiro, houve um aumento aproximado de duas vezes na forma??o dos corpos de oclus?o quando comparados ? produ??o sem adi??o de ecdisona. Utilizando este processo de infec??o, foi realizada uma modelagem e simula??o matem?tica que representasse o mecanismo de produ??o do bioinseticida.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-04-06T21:03:10Z No. of bitstreams: 2 Tese - Leonardo Guimarães de Oliveira - 2016.pdf: 2541166 bytes, checksum: 5b0501be9f076d03111ca00fcf4980a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-07T11:18:45Z (GMT) No. of bitstreams: 2 Tese - Leonardo Guimarães de Oliveira - 2016.pdf: 2541166 bytes, checksum: 5b0501be9f076d03111ca00fcf4980a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-07T11:18:45Z (GMT). No. of bitstreams: 2 Tese - Leonardo Guimarães de Oliveira - 2016.pdf: 2541166 bytes, checksum: 5b0501be9f076d03111ca00fcf4980a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The protein profile of hornless Nellore cattle from a segregating population for meat tenderness of extremes shear force, low extreme group (M) and extreme high (D) group, showed differentially expressed proteins. They had greater relative abundance of proteins of the glycolytic process in group M and proteins of the oxidative metabolism evidenced more expressed in group D, fact that probably is correlated to the final tenderness of the meat. Only identified in group D, the cytochrome c protein indicates induction of the apoptotic process in this group of animals. Structural proteins were identified in group M, indicating a possible greater proteolysis. Calpastatin was only identified in group D, this protein is highly related to the final meat tenderness because it is a natural inhibitor of the calpain. Separation of calpastatin by ion exchange column chromatography of two different muscles described two peaks of calpain inhibitory activity: peak 1 (CAST1) and peak 2 (CAST2). CAST 1 activity increased during the post-mortem period in Triceps brachii and showed no difference between days in Longissimus dorsi and on the other hand, total activity of calpastatin and CAST 2 decreased during post-mortem aging. The 115 kDa band of calpastatin decreased its intensity during post-mortem aging in both muscles with more than 70% of the change occurring on the first day. The mitochondrial ATP synthase beta subunit proteins increased and Succinyl CoA ligase decreased after aging and Adenylate kinase isoenzyme decreased on day 7. Calpastatin peaks had weak phosphorylated bands and had different IP and MW patches than 2D-SDS -PAGE. During the purification process of the calpastatin peaks the activity per mg of protein increased but lost half of the total activity presented during the first purification step. For peak 2 of calpastatin the specific activity increased 139.8 times and at the end of this process 36% of the total activity remained. Purified calpastatin was identified on the second-size gel having a molecular weight similar to the western blot. Spots from calpastatin peak 1 and two from calpastatin peak 2 were identified as peptides belonging to the calpastatin molecule. Sequence of peptides identified in Spot from purified peak 1 as part of the inhibitory domain III and IV and of the C-terminus and from the purified peak 2 a sequence of peptides identified as part of the inhibitory domain I, II and III. These results lead us to believe that both peaks, in this case, are degradation products of the intact molecule, and probably the small peptides are broken down during the process. The results of the present study show that purification of distinct forms of active calpastatin is possible, however, the intact form of calpastatin was not present in this purification. The presence of peptides was not conclusive to determine the origin and composition of each active peak. / O perfil protéico de animais da raça nelore mocho de uma população segregante para a maciez da carne de extremos valores de força de cisalhamento, grupo extremo baixo (M) e grupo extremo alto (D), apresentou proteínas diferentemente expressas as quais houve maior abundância relativa de proteínas do processo glicolítico no grupo M e proteínas do metabolismo oxidativo evidenciadas mais expressas no grupo D, fato que provavelmente está correlacionado à maciez final da carne. Apenas identificada no grupo D, a proteína citocromo c indica indução do processo apoptótico neste grupo de animais. Proteínas estruturais foram identificadas no grupo M, indicando uma possível maior proteólise. A calpastatina foi somente identificada no grupo D, esta proteína está altamente relacionada com a maciez final da carne por ser inibidora natural das calpaínas. A separação de calpastatina por cromatografia em coluna de troca iônica de dois músculos diferentes descreveu dois picos de actividade inibitória de calpaínas: pico 1 (CAST1) e pico 2 (CAST2). Atividade de CAST 1 aumentada durante o período post mortem no Triceps brachii e não apresentou diferença entre os dias em Longissimus dorsi e por outro lado, a atividade total de calpastatina e CAST 2 diminuiu durante o envelhecimento post mortem. A banda de 115 kDa da calpastatina diminuiu sua intensidade durante o envelhecimento post mortem em ambos os músculos com mais de 70% da alteração ocorrendo no primeiro dia. As proteínas mitocondriais da subunidade ATP sintase beta aumentaram e a Succinil-CoA ligase diminuiu após o envelhecimento e a Adenilato quinase isoenzima diminuiu no dia 7. Os picos de calpastatina apresentavam faixas fosforiladas fracas e apresentavam manchas em IP e MW diferentes do que 2D-SDS-PAGE. Durante o processo de purificação dos picos de calpastatina a actividade por mg de proteína aumentou mas perdeu metade da atividade total apresentada durante a primeira etapa de purificação. Para o pico 2 de calpastatina a actividade específica aumentou 139,8 vezes e no final deste processo permaneceu 36% da atividade total. A calpastatina purificada foi identificada no gel de segunda dimensão com um peso molecular semelhante ao western blot. Spots do pico 1 da calpastatina e dois do pico 2 da calpastatina foram identificados como peptídeos pertencentes à molécula da calpastatina. Sequêcia de peptídeos identificados em Spot a partir do pico 1 purificado como parte do domínio inibidor III e IV e do terminal C e do pico 2 purificado uma sequêcia de peptideos identificados como parte do domínio inibidor I, II e III. Estes resultados levam-nos a crer que ambos os picos, neste caso, são produtos de degradação da molécula intacta e, provavelmente, os pequenos peptideos são quebrados durante o processo. Os resultados do presente estudo mostram que é possível a purificação de formas distintas de calpastatina activa, contudo a forma intacta de calpastatina não estava presente nesta purificação. A presença de peptídeos não foi conclusiva para determinar a origem ea composição de cada pico ativo.

This diploma thesis deals with an influence of high temperature and water shortage on the protein content of gliadin and glutenin fractions in four varieties of winter wheat: Bohemia, Tobak, Pannonia and var. Syria with designation S46 (IG142780). The crop was grown at 26, 29, 32, 35, 38 and 41 °C during anthesis under control irrigation treatment (with soil moisture higher than 70 %) or under drought stress (with soil moisture lower than 30 %). To separate gliadins, the A-PAGE method was used, and glutenins were separated by SDS-PAGE method. Proteins were quantified by computer densitometry. Significant influence of genotype on the gluten proteins was found. Variety Pannonia has high content of -, 5-gliadins, LMW and HMW glutenins, but low content of other gliadin fractions. It was the opposite in the other varieties. Due to temperature, as well as drought, there was an increase in the content of all gluten fractions, especially of HMW glutenins, 1,2-gliadins and total gliadins. The largest increase in the gluten fractions due to drought was observed in Syria variety. In other varieties simultaneous exposure to drought and heat caused decrease in gliadin content, but increase in glutenin content. Drought at high temperatures reduced gliadin-to-glutenin ratio, mostly in Bohemia variety. This ratio has increased due to the temperature, especially in Tobak variety. For Syria variety, no effect of stress conditions was found on gliadin-to-glutenin ratio.

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During 20th century main concern was to have sanitary water flowing through the tap. In 21st century constant supply of safe drinking water is common at any home in USA. Hence consumers pay attention to aesthetic quality of tap water. Odorous algal metabolites in source water and metals introduced to drinking water due to corrosion of pipes in the distribution system cause taste, odor and color problems, and result in complaints and perception of tap water as unhealthy. Millions of dollars are spent each year by water industry to address and prevent these issues. This research focused on some of the taste-and-odor issues associated with drinking water. First aim was to understand when geosmin, 2-MIB, and nonadienal become detectable, employing two-resistance mass transfer theory to determine the concentration of odorants in bathroom air. Results showed that water temperature and odorant concentration in water play an important role. Next focus was to develop an international odor standard to be used for training of sensory analysis panelists. There are many sensory methods to monitor drinking water to detect the off-flavors however an odor standard has been missing. Hexanal was studied with trained flavor profile analysis panels and was proposed as an ideal odor reference standard to be used for training and sensory assessment of water samples. Main focus was to understand metallic flavor of drinking water caused by iron and copper. It was shown that metallic sensation has taste and retronasal components creating the flavor and humans are very sensitive to it. Occurrence of lipid oxidation in the oral cavity was shown when metals were ingested, that produces carbonyls which are responsible for the metallic flavor. Antioxidants and chelators were investigated to study prevention of lipid oxidation and, chelators were determined to be more effective. Oral epithelial cell cultures were developed as a model for oral cavity to further investigate lipid oxidation and effectiveness of the antioxidants and chelators. This dissertation is a result of inter-disciplinary work and possibly a good example for how problems may be solved by incorporating different methods and point of views from several disciplines. / Ph. D.

The main focus of the PhD study was to develop new gel electrophoresis and ICP-MS based methods to analyze a wide variety of the bio-molecules such as proteins, phosphoproteins and metalloproteins etc. The tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) method is commonly used to resolve low molecular mass proteins, however, it requires a high percentage gel and a very complicated procedure to achieve this separation. This study describes a modification to tricine-SDS-PAGE to make it more effective for the separation of smaller proteins and for coupling to ICP-MS. The modified method employs low percentage PAGE gels and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. This modified method was applied to analyze phosphopeptides. Phosphopeptides are very small in size and difficult to separate using the other techniques such as Laemmli SDS-PAGE, original tricine-SDS-PAGE, immobilized metal affinity chromatography (IMAC), size exclusion chromatography (SEC) etc. In this study a simplified procedure is described based on modifying the original tricine-SDS-PAGE method. A comparative study showed that this modified method successfully resolved a digest mixture of very low to high molecular mass phosphopeptides/peptides. In off-line coupling of this method with ICP-MS, much better recoveries of the peptides from the gel were obtained as compared to traditional methods which indicate the compatibility of this modified method for quantitative studies. An on-line coupling of the modified system with ICP-MS was also demonstrated and it was applied for the separation, detection and quantification of phosphopeptides. Another application of this modified system was the separation of serum proteins. Blood serum contains five major protein groups i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The separation of these five major proteins in a single gel is difficult to achieve using traditional methods. The modified system was shown to be superior for the separation of these serum proteins in a 7% (m/v) native-PAGE gel and a cellulose acetate membrane. A further study was carried out into controlling the factors that cause metal loss and protein fragmentation in SDS-PAGE. Using a reducing sample buffer, and heating to high temperatures (90-100ºC) in alkaline or acidic conditions may cause protein fragmentation and decrease the metal binding affinity. 70ºC was found suitable to prepare the sample at neutral, alkaline or acidic pH as no fragmentation observed. To prevent metal loss, the binding constant (log K) values of metal-amino acids, play the major role. Those metals which have high binding affinities with the amino acids in proteins can also be affected by the variation of the pH so prior information about pH to maintain the binding constant values is essential to minimize metal loss. This was observed in the loss of zinc, and to a lesser extent copper from human serum albumin (HSA) as measured by inductively coupled plasma mass spectrometry (ICP-MS). The method described above was applied for the separation and quantification of the serum proteins obtained from age-related macular degeneration (AMD) patients (where the AMD patients were from Moorfields Eye Hospital, London). Zn and Cu were quantified employing external calibration. Zn concentration showed variation whilst Cu did not show any significant variations in samples from AMD patients. A brief study of the interaction of cisplatin and oxaliplatin with HSA and transferrin was also performed. Cisplatin bound much faster than oxaliplatin with HSA. After 24 hours incubation, cisplatin showed a decrease in signal intensity which indicates that cisplatin binding decreases with time. Cisplatin binding with transferrin as compared to HSA was not significant, which could be the result of unstable Pt-transferrin complex formation. Oxaliplatin did not show high binding to either protein, perhaps due to the presence of the bulky, non polar DACH ligand.

<p>This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.</p>