Thesis (Ph.D.)--Mississippi State University. College of Veterinary Medicine. / Title from title screen. Includes bibliographical references.

Vascular stiffening is defined as the reduced ability of the blood vessels to expand in response to an increase in blood pressure. Vascular stiffening is often not appreciated as a disease in and of itself but is important to recognize because it is considered a predictor of many cardiovascular disease states. Mechanisms of vascular stiffening remain largely unknown; however the immune system has been found to play major roles in cardiovascular disease and arterial remodeling. This dissertation therefore seeks to illustrate the role of the adaptive immune system in vascular stiffening. This has been done by modeling vascular stiffness in transgenic mice lacking an adaptive immune system as well as immunosuppression in normal mice using a novel method to stimulate regulatory T cells with a cytokine immune complex. We have found that inhibition of the immune system by the use of a genetic knockout (RAG 1 ⁻/⁻ mice) or suppression of an existing immune system with an IL-2/anti-IL-2 complex reduces the development of angiotensin II-induced vascular stiffening. This dissertation supports the role of the adaptive immune system, and particularly CD4⁺T cells, in the development of vascular stiffening as well as the protective roles of Tregs in the disease. It also highlights the use of the IL-2/anti-IL-2 complex as a new potential therapy for vascular stiffness. Therapeutics that suppress adaptive immune function may be beneficial in the treatment of vascular stiffening.

Apoptosis (programmed cell death) and exercise immunology have been the focus of research for the past five years. Trained athletes are particularly susceptible to a wide variety of viral and bacterial infections and this has been related to oxidative damage which is a mediator of apoptosis. Apoptosis, a normal physiological mechanism has also been implicated in the pathogenesis of a wide-variety of diseases. To date, the link between apoptosis and exercise has not been shown by established methods or ultrastructurally. The objective of the study was t.o determine the effects of a single bout of high intensity exercise on lymphocyte DNA and antioxidant status in trained athletes. The study was carried out in two phases. In the first phase, 11 trained athletes were subjected to a treadmill run to exhaustion using a ramp protocol to determine their maximum oxygen uptake (V02 max). Fifteen millimetres of blood was collected before exercise, immediately after exercise, 24 hours and 48 hours after exercise. Whole blood (4 ul) was used in the determination of DNA damage in lymphocytes using the single cell gel electrophoresis (SCGE) assay. The remaining blood was centrifuged and used for the following: Vitamin C concentration was determined by the 2,4 dinitrophenylhydrazine method, vitamin E concentration was determined by the High Pressure Liquid Chromatography (HPLC) method and lipid peroxides were determined by the measurement ofhydroperoxides. In the second phase, 3 trained athletes who had participated in phase 1, were subjected to a V02 max. test. Blood samples (10 ml) were collected before and immediately after exercise, 24 hours and 48 hours later. Lymphocytes were isolated using Histopaque 1077. An in situ cell death detection kit, Fluorescein was used for the detection and quantification of apoptosis in lymphocytes at a single cell level, based on labelling of DNA strand breaks. Analysis was carried out using flow cytometry. Lymphocytes were also prepared for Transmission Electron Microscopy (TEM) using conventional techniques. The results showed that immediately after exercise there was a non-significant decrease in vitamin C concentrations (p=o, 16), and a non-significant increase in vitamin E (p=0,82) and lipid peroxide concentrations (p=0,21). There was no significant difference in all 3 levels over the 48 hour period, when compared to the pre-exercise values. The SCGE assay revealed that the immediate post exercise samples showed DNA damage in lymphocytes of all subjects as evidenced by fluorescent strands of DNA outside the cell while DNA damage was observed in only one subsequent sample. In the pre-exercise samples, DNA was visualised as a central core, whereas in all samples taken after exercise, DNA was located at the periphery or confined to one pole of the cell. The pattern of DNA distribution seen in the SCGE assay over the 48 hour period were characteristic features of apoptosis. Flow cytometric analysis showed an increase in apoptosis in lymphocytes immediately after exercise with a further increase after 24 hours. After 48 hours the numbers decreased to control levels. TEM showed that majority of cells were normal before exercise while other lymphocytes were smaller with indented nuclei. Immediately after exercise the lymphocytes displayed features of indented nuclei and microsegregation, cell shrinkage, swelling of the endoplasmic reticulum, mitochondria and Golgi. These changes persisted after 24 hours but were not observed after 48 hours when most of the cells showed normal morphology. The ultrastructural changes observed were also characteristic features of apoptosis. These results suggest that high intensity exercise may cause an increase in apoptosis as evidenced by DNA damage in the SCGE assay and fully supported by the results achieved during flow cytometry and by the ultrastructural changes observed. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution. To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution. Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an in vitro purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 166-195).

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-16Bitstream added on 2014-06-13T20:30:22Z : No. of bitstreams: 1 dourado_gkzs_me_arafcf.pdf: 2109934 bytes, checksum: 79ddf2b14ea1fc3f4779c4b22b8e7f81 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Tem sido mostrado que a hesperidina, um flavonóide encontrado nas frutas cítricas e especialmente no suco de laranja, apresenta atividade antiinflamatória e imunomodulatória, entre outras. O presente estudo avaliou os efeitos da ingestão do suco de laranja (SL) como fonte natural da hesperidina, da glicosil hesperidina isolada (GH) e da mistura de ambos (SL-GH), sobre o sistema imune inato de camundongos. Para tanto, foram medidos os parâmetros de produção e inibição da produção de óxido nítrico (NO), e de liberação das citocinas IL-10, IL-12 e TNF- em culturas de macrófagos peritoneais ex vivo. Os macrófagos foram obtidos de grupos de animais previamente tratados com GH, SL, SL-GH ou solução salina (SS). Em seguida as células foram tratadas com GH e lipopolissacarídeo bacteriano (LPS) in vitro. Os resultados mostraram que o tratamento com suco de laranja estimulou a produção de NO de forma moderada (60μmol de NO- 2) pelos macrófagos, aumentou a liberação da citocina IL-10 e diminuiu significativamente a produção de TNF- . O tratamento com G-hesperidina inibiu completamente a produção de NO em macrófagos mesmo na presença de LPS, aumentou a produção da citocina IL-12 e diminuiu a produção de IL-10. Estes resultados sugerem que a ingestão de suco de laranja é benéfica ao sistema imune, podendo apresentar ação contra possíveis agentes infecciosos. A flavanona Ghesperidina isolada pode ser considerada um composto anti-inflamatório nos casos onde ocorre a intensa liberação de óxido nítrico, tais como as doenças inflamatórias / It has been shown that hesperidin, a flavonoid found in citrus fruits and especially in the orange juice, has anti-inflammatory and immunomodulatory, among others. This study evaluated the effects of ingestion of orange juice (SL) as a natural source of hesperidin, isolated glucosyl hesperidin (GH) and a mixture of both (SL-GH) on the innate immune system of mice. To this end, it was measured the parameters of production and inhibiting the production of nitric oxide (NO), and release of cytokines IL-10, IL-12 and TNF-α in cultures of peritoneal macrophages ex vivo. Macrophages were obtained from groups of animals previously treated with GH, SL, SL-GH or saline solution. Then the cells were treated with GH and bacterial lipopolysaccharide (LPS) in vitro. The results showed that treatment with orange juice stimulated the production of NO in a moderate (60µmol NO- 2) by macrophages, increased the release of cytokine IL-10 and significantly decreased the production of TNF-α. Treatment with G-hesperidin completely inhibited NO production in macrophages, even in the presence of LPS, increased the production of cytokine IL-12 and decreased production of IL-10. These results suggest that ingestion of orange juice is beneficial to the immune system, may bring suit against possible infectious agents. The isolated G-flavanone hesperidin can be considered an anti- inflammatory compound in cases where it occurs intense release of nitric oxide, such as inflammatory diseases

CAPES / Esta tese propõe um novo algoritmo baseado em Sistemas Imunológicos Artificiais (SIA) para classificação hierárquica e multi-label, onde os classificadores gerados são representados na forma de regras SE-ENTÃO. A classificação hierárquica e multi-label é considerada desafiadora uma vez que um exemplo está associado a uma ou mais classes organizadas hierarquicamente, sendo que esta organização estrutural de classes deve ser considerada na construção dos classificadores. A técnica proposta aborda a construção de classificadores hierárquicos locais (onde cada classificador processa apenas exemplos de classes em uma região local da hierarquia) e globais (onde um único classificador processa exemplos de todas as classes ao mesmo tempo). A área de aplicação utilizada para validação desta tese foi a predição de função biológica de proteínas usando termos da ontologia gênica como classes a serem preditas pelo SIA. O desempenho do algoritmo é avaliado experimentalmente para 10 bases de proteínas. Os critérios de avaliação do algoritmo nos experimentos computacionais são a precisão preditiva (taxa de acerto e área da curva precision-recall) e a simplicidade do conhecimento descoberto (medida pelo número de regras e número total de condições nas regras descobertas). Os experimentos computacionais permitem identificar parâmetros e procedimentos que influenciam no desempenho da técnica proposta. Os testes comparativos com outras abordagens mostram que sobre alguns conjuntos de experimentos a abordagem proposta se mostrou superior, enquanto em outros conjuntos não foi possível superar a técnica da literatura usada para comparação. / This thesis proposes a new approach based on Artificial Immune System (AIS) for hierarchical multi-label classification, where the classifiers produced by the system are represented in the form of IF-THEN classification rules. Hierarchical multi-label classification is a challenging problem, because an example is associated with one or more classes organized into a hierarchy and the class hierarchy must be considered in the construction of the classifiers. The proposed method addresses the construction of local hierarchical classifiers (where each classifier processes only examples of classes in a local region of the hierarchy) and global hierarchical classifiers (where a single classifier processes examples of all classes at the same time). The application domain used to validate the proposed methods was the prediction of the biological function of proteins, using terms of the Gene Ontology as classes to be predicted by the AIS. The performance of the algorithm was evaluated in computational experiments with 10 datasets of proteins. The evaluation criteria in these experiments were the predictive accuracy (accuracy rate and the area under the precision-recall curve) and the simplicity of the discovered knowledge (measured by the number of rules and total number of conditions in the discovered rules). The computational experiments allowed the identification of parameter settings and procedures that significantly influence the performance of the proposed method. The experiments comparing the proposed method with other methods have shown that in some datasets the proposed method outperformed other methods, whilst in other datasets it was not possible to outperform other methods proposed in the literature.

A hemoglobina possuí um grande potencial antimicrobiano, sendo os peptídeos antimicrobianos (AMPs) derivados de proteínas contêm o grupamento heme denominados hemocidinas. O carrapato bovino Rhipicephalus (Boophilus) microplus é capaz de adquirir e processar grandes quantidades de sangue para seu metabolismo. Neste trabalho, purificamos hemocidinas produzidas no tubo digestório do carrapato. Identificamos 10 fragmentos da hemoglobina bovina que possuem atividade contra Candida albicans (Hb 1-94, 3-94, 1-87, 93-141, 102-141, 103-141, 107-141, 104-141 e 98-114 e Hb\"beta\" 127-145). Determinamos que todos os peptídeos apresentam um alto conteúdo de aminoácidos básicos, além de uma estrutura secundária em \"alfa\"-hélice, características encontradas em outros AMPs. Verificamos também que as duas principais proteases que atuam na produção das hemocidinas são uma cisteíno- e uma aspártico-proteinase. A identificação de diversas hemocidinas, no tubo digestório de R. (Boophilus) microplus, indica uma possível participação das hemocidinas na proteção contra microorganismos / The hemoglobin posses a great potential as an antimicrobial, being denominated hemocidins the antimicrobial peptides (AMPs) derived from heme containing proteins. The cattle tick Rhipicephalus (Boophilus) microplus is capable of processing large amounts of blood to its own metabolism. In this work we purified the hemocidins that are produced in the tick gut. We identified 10 fragments from bovine hemoglobin that presents activity towards Candida albicans (Hb 1-94, 3-94, 1-87, 93-141, 102-141, 103-141, 107-141, 104-141 e 98-114 e Hb\"beta\" 127-145). We determined that all the peptides presents a high content of basic amino acids, besides a secondary structure in \"alfa\"-helix conformation, characteristics found in other AMPs. We also verified that a cistein- and an aspartic-protease are the two main proteases involved on the production of these hemocids. The identification of several hemocidins in the gut of R. (Boophilus) microplus, suggests that these AMPs may play a role in the defense of the tick against microorganisms

Submitted by LUANA CAMARGO SOUSA null (luanacamargo.bio@hotmail.com) on 2016-03-15T18:50:34Z No. of bitstreams: 1 005-2016 - Luana Camargo Sousa - DISSERTAÇÃO MESTRADO.pdf: 1321578 bytes, checksum: 0580f4bb0e26158d5c269f87473f2dad (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-03-15T19:35:29Z (GMT) No. of bitstreams: 1 sousa_lc_me_ilha.pdf: 1321578 bytes, checksum: 0580f4bb0e26158d5c269f87473f2dad (MD5) / Made available in DSpace on 2016-03-15T19:35:29Z (GMT). No. of bitstreams: 1 sousa_lc_me_ilha.pdf: 1321578 bytes, checksum: 0580f4bb0e26158d5c269f87473f2dad (MD5) Previous issue date: 2016-01-20 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente trabalho tem como objetivo avaliar a influência de dietas com diferentes níveis de proteína (P) e carboidratos (CH) na resposta imunológica e nos índices zootécnicos de juvenis de surubim (Pseudoplatystomasp.). O experimento foi conduzido no Laboratório de Aquicultura da Unesp – Campus de Dracena, distribuído em um delineamento inteiramente casualizado com seis tratamentos e quatro repetições (38P/22CH; 38P/26CH; 38P/30CH; 42P/22CH; 42P/26CH; 42P/30CH). Os resultados obtidos foram submetidos à análise de variância (ANOVA) e as médias comparadas pelo Teste de Duncan (5%), através do programa estatístico SAS, versão 9.0. Não foram observadas diferenças significativas entre as dietas para os parâmetros de desempenho: ganho em biomassa, taxa de crescimento específico, conversão alimentar e taxa de eficiência protéica. Os grupos alimentados com a dieta 38P/22CH obtiveram melhor desempenho para comprimento padrão, peso final e ganho de peso. Não foram observadas diferenças significativas em albumina, atividade respiratória dos leucócitos e atividade hemolítica complemento. A dieta 38P/30CH obteve valores médios significativamente maiores para as concentrações de proteína, globulina e lisozima. Para os parâmetros hematológicos, entre as dietas, não houve diferença significativa para as concentrações de eritroblasto, linfócito, eosinófilo, volume corpuscular médio e hemoglobina corpuscular média. Após serem desafiados, os peixes apresentaram uma resposta significativa na concentração de hemoglobina, e uma redução no hematócrito, em resposta ao estresse causado pelo desafio. A melhor resposta produtiva foi observada nos peixes que receberam a dieta 38P/22CH. As condições fisiológicas não foram comprometidas mostrando a capacidade dos peixes em responder ao desafio com LPS. / This study aimed to evaluate the influence of diets with different protein levels (P) and carbohydrates (CH) in the immune response and growth performance of juvenile surubim (Pseudoplatystoma sp.). The experiment was conducted in the Aquaculture Laboratory - Unesp Dracena, distributed in a completely randomized design with six treatments and four replications (38P/22CH; 38P/26CH; 38P/30CH; 42P/22CH; 42P/26CH; 42P/30CH). The results were submitted to analysis of variance (ANOVA) and the means were compared by Duncan test (5%), through the SAS statistical software, version 9.0. No significant differences were observed among diets regarding the growth performance parameters: weight gain, specific growth rate, feed conversion and protein efficiency ratio. Fish fed diets with 38P/22CH diet performed better for standard length, body weight and weight gain. There were no significant differences in albumin, respiratory activity of leukocytes and complement hemolytic activity. The 38P/30CH diet achieved significantly higher average values for the concentrations of protein, globulin and lysozyme. For hematological parameters, there were no significant difference in the concentrations of erythroblast, lymphocytes, eosinophils, mean corpuscular volume, mean corpuscular hemoglobin. After being challenged fish showed a significant response in hemoglobin concentration, and a decrease in hematocrit in response to stress caused by challenge. The best productive response was observed in fish fed diet 38P / 22CH. Physiological conditions were not compromised showing the ability of fish to respond to LPS challenge. / FAPESP: 2014/26723-1