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Orientador: Maria da Gloria Buzinaro / Resumo: A diarreia neonatal em bovinos é muito comum e resulta em importantes perdas econômicas. O rotavírus constitui um dos principais agentes relacionado à síndrome diarreica, que pode acometer também seres humanos. A ocorrência de rotavírus em bovinos leiteiros e de corte dos Estados de Minas Gerais, Goiás e Mato Grosso do Sul foi analisada por meio de dados epidemiológicos obtidos no período de 2006 a 2010, do Laboratório de Rotaviroses, da Faculdade de Ciências Agrárias e Veterinárias (FCAV/Unesp) de Jaboticabal, São Paulo. No período estudado, foram obtidas 851 amostras de fezes de bezerros, na faixa etária de um a 90 dias de idade, independentemente da manifestação de sinal clínico de diarreia. As amostras foram provenientes de 47 rebanhos leiteiros e 30 rebanhos de corte. A análise foi feita pela técnica de eletroforese em gel de poliacrilamida (PAGE) que indicou animais positivos em 29,9% (23/77) dos rebanhos e 7,1% (60/851) das amostras. Maior prevalência de animais positivos foi encontrada em amostras de bezerros de corte, com 12% (45/370). Dos casos de diarreia, 22,6% (45/199) dos bezerros foram positivos, enquanto 2,3% (15/642) foram detectados em bezerros sem diarreia. O Estado de Goiás apresentou maior prevalência de animais positivos (11,7%, 33/282), seguido por Mato Grosso do Sul (6,5%, 17/260) e Minas Gerais (3,2%, 10/309). A frequência da infecção por rotavírus foi maior em bezerros com idade inferior a 30 dias e nos meses mais chuvosos do ano. De acordo com a mig... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

The aim of this thesis is to evaluate the expression of both TSG101 tumor susceptibility gene and ret oncogene in human thyroid carcinoma specimens. Functional inactivation of TSG101 in mouse fibroblast leads to cellular transformation and the ability to form metastatic tumors in nude mice. No genomic deletion of TSG101 gene has been reported in human cancer, casting a doubt on the role of TSG101 as a classical tumor suppressor. Subsequent studies reveal that TSG101 is a frequent target of spilicing defects, which is correlated with cellular stress and p53 status, and might reflect the cellular environment during the cancer development. Furthermore, recent reports demonstrate TSG101 as a part of the MDM2/p53 regulatory circuitry, a well recognized circuitry that upon deregulation results in tumorigenesis. In this study we have analyzed TSG101 gene expression in 85 specimens of thyroid carcinomas. The results indicated that 100% of papillary carcinomas (48/48), 85% of follicular carcinomas (18/21), 91% of medullary carcinomas (10/11) and 60% of undifferentiated carcinomas (3/5) showed strong to moderate cytoplasmic staining, whereas the staining was completely negative, or cytoplasmic dot-staining in the adjacent non-neoplastic follicular cells. Occasionally, the staining could be found in the nucleus. Subsequently, sequence analysis of 17 papillary carcinoma specimens revealed no mutation in steadiness box region, indicating that it might not be the cause of TSG101 protein overexpression. In summary, our results indicate strong correlation of TSG101 overexpression and thyroid carcinomas. Further experiments are urged to clarify the relationship of TSG101 overexpression and thyroid tumorigenesis. Rearrangement of ret proto-oncogene is unique to papillary thyroid carcinoma (PTC). These rearrangements consist of the fusion of ret tyrosine kinase domain to a variety of heterologous genes, thus generating chimeric transforming oncogenes termed, ret/PTC. The frequency of ret/PTC activation in non-radiation exposured adult populations has been reported to vary from 0-55% depending on the geographic distribution. To detect ret rearrangement and to identify candidate of novel ret/PTC in 62 specimens of PTC collected from southern Taiwan, a RT-multiplex PCR method was used to reveal the possible specimens that harbor ret rearrangements. Type specific-PCR amplification and subsequent sequence analysis of PCR product were performed to identify the known types of ret/PTC. We have identified two cases of ret/PTC1, two cases of ret/PTC3 and one case of ELKS-RET. Excitingly, four cases of unknown ret/PTC type were identified. Hence, 5¡¦-RACE strategy will be employed to identify novel ret/PTC in these four specimens.

Submitted by KARLA ALVARENGA NASCIMENTO null (karlanascimentovet@yahoo.com.br) on 2017-03-21T13:16:57Z No. of bitstreams: 1 Dissertação Karla.pdf: 1655729 bytes, checksum: 6cdf4bfec3aad9e315d583cdf42d5d1a (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-21T21:32:38Z (GMT) No. of bitstreams: 1 nascimento_ka_me_jabo.pdf: 1655729 bytes, checksum: 6cdf4bfec3aad9e315d583cdf42d5d1a (MD5) / Made available in DSpace on 2017-03-21T21:32:38Z (GMT). No. of bitstreams: 1 nascimento_ka_me_jabo.pdf: 1655729 bytes, checksum: 6cdf4bfec3aad9e315d583cdf42d5d1a (MD5) Previous issue date: 2017-02-09 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os pestivírus possuem importância significativa na suinocultura, sendo que os suínos podem ser infectados pelo Vírus da Diarreia Viral Bovina (BVDV) em condições naturais. O presente estudo teve como objetivo induzir a infecção experimental do BVDV-1 em leitões desmamados e avaliar a excreção e possível transmissão do vírus pelas vias lâmina d’água e naso-nasal. Foram conduzidos dois experimentos e uma repetição para cada via de transmissão avaliada. No total foram selecionados 24 leitões, sendo utilizados seis por experimento, divididos em grupos controle (n=2), sentinela (n=2) e infectado (n=2), utilizando isoladores, projetados para estabelecer o contato entre os animais apenas pela via de transmissão em estudo. O grupo infectado recebeu um inóculo contendo BVDV-1, estirpe Singer, enquanto que os grupos controle e sentinela foram inoculados com meio E-MEM. Os animais permaneceram nos isoladores por 25 dias, período em que foram coletadas amostras de suabe nasal diariamente e sangue a cada sete dias. Ao final, os animais foram eutanasiados e necropsiados, sendo realizada coleta de fragmentos de órgãos para histopatologia, imunohistoquímica e RT-PCR. Quando avaliada a via de transmissão lâmina d’água, apenas um animal infectado apresentou excreção de material genômico a partir do 6o dia pós-infecção (dpi), e um animal sentinela apresentou excreção no 20o dpi. Nestes animais, a soroconversão ocorreu no 25o dia apresentando título de anticorpos igual a 20. Um animal infectado não apresentou soroconversão durante o período amostrado, porém houve excreção a partir do 5o dpi. Na repetição do experimento, apenas um animal infectado soroconverteu ao 25o dia, com título de anticorpos de 20, cuja excreção do material genômico foi detectada ao 21o dpi. Nos animais sentinelas, apenas um apresentou excreção no 13o dpi. Em relação ao experimento naso-nasal, os animais infectados apresentaram excreção de material genético a partir do 17o dpi, porém a soroconversão não foi observada em todos os animais. Na repetição do experimento naso-nasal a soroconversão foi detectada em apenas um dos leitões infectados no último dia de experimento, com título de anticorpos de 20. A excreção do material genômico ocorreu nos dois animais infectados, sendo que um animal apresentou excreção a partir do dia 10 e o outro a partir do 18o dpi. Em relação à macroscopia e microscopia, os animais de todos os experimentos não apresentaram lesões significativas. O presente estudo demonstra que os suínos são susceptíveis ao BVDV, possibilitando a excreção e servir como fonte de infecção. Evidenciou-se que a lâmina d’agua pode veicular o BVDV de um suíno infectado para outro, ressaltando a importância e o risco de transmissão. A possibilidade de transmissão do BVDV pela via naso-nasal em leitões não foi provada dentro do período avaliado. / Pestiviruses have significant importance in swine breeding, and pigs can be infected by Bovine Viral Diarrhea Virus (BVDV) under natural conditions. The present study aimed to induce the experimental infection of BVDV-1 in weaned piglets and to evaluate the excretion and possible transmission of the virus through the water and naso-nasal routes. Two experiments and one replicate were conducted for each evaluated route of transmission. In total, 24 piglets were selected, and six were used per experiment, divided into control (n = 2), sentinel (n = 2) and infected (n = 2) groups using isolators, designed to establish contact between animals only by Route of study. The infected group received an inoculum containing BVDV-1, Singer strain, while the control and sentinel groups were inoculated with E-MEM medium. The animals remained in the isolators for 25 days, during which samples of nasal swab and blood were collected every seven days. At the end, the animals were euthanized and necropsied, and organ fragments were collected for histopathology, immunohistochemistry and RT-PCR. When the water-borne transmission pathway was evaluated, only one infected animal showed excretion of genomic material from day 6 post-infection (dpi), and one sentinel animal showed excretion at the 20th dpi. In these animals, seroconversion occurred on day 25 with an antibody titer equal to 20. An infected animal did not present seroconversion during the sampled period, but there was excretion from the 5th dpi. In the repeat of the experiment, only one infected animal was seroconverted at day 25, with antibody titer of 20, whose excretion of the genomic material was detected at 21o dpi. In sentinel animals, only one had excretion at the 13th dpi. In relation to the naso-nasal experiment, the infected animals presented excretion of genetic material from the 17th dpi, but seroconversion was not observed in all the animals. In the repetition of the nasonasal experiment, seroconversion was detected in only one of the infected piglets on the last day of the experiment, with an antibody titre of 20. Excretion of the genomic material occurred in the two infected animals, and one animal had excretion from the Day 10 and the other from the 18th dpi. Regarding macroscopy and microscopy, the animals of all experiments did not present significant lesions. The present study demonstrates that pigs are susceptible to BVDV, allowing excretion and serve as a source of infection. It has been shown that the water table can transport BVDV from one infected pig to another, emphasizing the importance and risk of transmission. The possibility of BVDV transmission by the nasal nasal route in piglets has not been proven within the evaluated period. / FAPESP: 2014/13590-3 / FAPESP: 2015/07098-1

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.

The North American elk or wapiti is an indigenous species to Canada. Understanding of the reproductive physiology of elk is limited, as little research has been conducted in this field as compared to domestic farmed species. In order to make available the tools to study reproductive physiology of the elk this thesis describes the cloning and expression of elk pituitary glycoprotein hormone cDNAs. The common gonadotropin a-subunit, and FSH, LH and TSH b-subunit elk cDNAs were amplified by reverse transcription and polymerase chain reaction (RT-PCR). There was a high degree of nucleotide similarity between the elk a and b subunits when compared with reported sequences from other species. The cDNAs for the pituitary glycoprotein hormone genes were used as probes to investigate seasonal expression in the female elk pituitary gland. Steady state levels of the common a-subunit mRNA was observed regardless of the reproductive season, but a significant increase in expression occurred during the breeding season. The FSH and LH b-subunit genes were expressed at low levels in pituitary glands of animals during presumed anestrous and pregnancy, but levels considerably increased during estrus. In contrast, levels of TSH b-subunit mRNA were similar regardless of the reproductive status. The FSH cDNAs were also transfected into a Chinese hamster ovary (CHO) mammalian expression system, aimed at the production of recombinant elk FSH. Transfected CHO cell lines were screened for expression of a- and FSH b-subunit mRNA by Northern blot. Activity of FSH was equivalent to ~100 mIU/ml of recombinant human FSH (Gonal-FTM), identified by FSH receptor signaling in an in vitro cell based assay. In conclusion, this work represents an advance towards understanding the molecular basis of seasonal reproduction in elk. This information and the availability of elk recombinant FSH will be useful for the application of advanced reproductive technologies required for the rapid expansion of healthy, disease resistant, and genetically superior animals, which are important for domestic production and wildlife management.

The North American elk or wapiti is an indigenous species to Canada. Understanding of the reproductive physiology of elk is limited, as little research has been conducted in this field as compared to domestic farmed species. In order to make available the tools to study reproductive physiology of the elk this thesis describes the cloning and expression of elk pituitary glycoprotein hormone cDNAs. The common gonadotropin a-subunit, and FSH, LH and TSH b-subunit elk cDNAs were amplified by reverse transcription and polymerase chain reaction (RT-PCR). There was a high degree of nucleotide similarity between the elk a and b subunits when compared with reported sequences from other species. The cDNAs for the pituitary glycoprotein hormone genes were used as probes to investigate seasonal expression in the female elk pituitary gland. Steady state levels of the common a-subunit mRNA was observed regardless of the reproductive season, but a significant increase in expression occurred during the breeding season. The FSH and LH b-subunit genes were expressed at low levels in pituitary glands of animals during presumed anestrous and pregnancy, but levels considerably increased during estrus. In contrast, levels of TSH b-subunit mRNA were similar regardless of the reproductive status. The FSH cDNAs were also transfected into a Chinese hamster ovary (CHO) mammalian expression system, aimed at the production of recombinant elk FSH. Transfected CHO cell lines were screened for expression of a- and FSH b-subunit mRNA by Northern blot. Activity of FSH was equivalent to ~100 mIU/ml of recombinant human FSH (Gonal-FTM), identified by FSH receptor signaling in an in vitro cell based assay. In conclusion, this work represents an advance towards understanding the molecular basis of seasonal reproduction in elk. This information and the availability of elk recombinant FSH will be useful for the application of advanced reproductive technologies required for the rapid expansion of healthy, disease resistant, and genetically superior animals, which are important for domestic production and wildlife management.

This research investigated the existence of <10 £gm nitrogen-fixing unicellular cyanobacteria in the South China Sea. The surveys covered the period from February 2004 to January 2005 and a total of seven cruises. The unicellular cyanobacteria that express orange-yellow from cellular phycoerythrin were observed under a fluorescence microscope. Their expressions in nitrogen-fixation were confirmed by the results from reverse transcription polymerase chain reaction (RT-PCR) and whole cell fluorescence immunolocalization of nitrogenase. The nifH gene sequences of the unicellular cyanobacteria collected from the South China Sea was with >90% identities of their nucleotides similar to the nifH gene sequences of unicellular diazotrophs from ALOHA (Hawaii) as well as Synechocystis sp. WH 8501, Cyanothece sp. ATCC 51142, Cyanothece sp. WH 8902, Cyanothece sp. WH 8904 and Synechococcus sp. RF-1. Positive reactions of fluorescence immunolocalization of nitrogenase were only observed in some, not all, of <10 £gm unicellular cyanobacteria, suggesting that cell counting alone can not be used to estimate nitrogen fixation rate. There was great seasonal and spatial variation in the unicellular cyanobacteria cell density. There was, however, no significant relationship between cell density and the investigated environmental factors. Cell density was high when temperature was high or where stratification index of water column was high, such as in summer or in basin in contrast to other seasons or the shelf-slope regions.

The effect of heat shock was observed on the expression of hilA in Salmonella Typhimurium by creating a fluorescence-based reporter strain of Salmonella and by realtime reverse transcriptase polymerase chain reaction (RT-PCR). The hilA gene in Salmonella is known to play an important role in its pathogenesis. hilA is known to be activated when the bacteria encounter stress-inducing conditions. A number of factors have been identified that affect hilA expression, such as, pH, osmolarity, oxygen tension. When Salmonella enter their warm-blooded hosts, they encounter an increase in temperature. Therefore, heat is another stressor that is encountered by Salmonella during infection of their hosts. A fluorescence-based strain of Salmonella was created to study the effect of heat shock. The gene for green fluorescent protein (gfp) was placed under the control of the promoter of hilA on a plasmid. This plasmid was used to transform Salmonella cells to create a fluorescent strain. In this strain, when the hilA promoter is activated, gfp is transcribed, which encodes the green fluorescent protein. This protein can be measured by a fluorescence assay. The results of this study indicated that at 45ºC, hilA is activated. RT-PCR was used to look at hilA expression at different temperature. The results of this study indicated that, compared to 37ºC, higher temperatures like 45ºC and 55ºC significantly activate hilA.

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.