A proteína recombinante inibidora de serinoprotease denominada de Amblyomin-X foi obtida a partir de uma biblioteca de cDNA das glândulas salivares do carrapato Amblyomma cajannense, construída e utilizada para identificar um gene que codifica um inibidor de serinoprotease do tipo Kunitz. O Amblyomin-X inibe a formação da massa tumoral in vivo, no entanto o mecanismo envolvido neste efeito não está totalmente esclarecido. Visto que um dos mecanismos anti-carcinogênicos dos inibidores de serinoproteases é a inibição do processo de angiogênese, este trabalho foi delineado para avaliar as ações do Amblyomin-X sobre a angiogênese in vivo e sobre funções da célula endotelial envolvidas neste processo. A angiogênese in vivo foi estudada em modelo de câmara dorsal por microscopia intravital. Quarenta e oito horas após a implantação da câmara dorsal, os animais receberam tratamento tópico de salina ou de Amblyomin-X por 8 dias, com intervalos de 48 horas a cada dose (10, 100 ou 1000ng/mL). Os efeitos foram avaliados em condições basais e na vigência do crescimento tumoral (injeção de 1x105 células B16-F10 de melanoma murino no tecido subcutâneo). Adicionalmente, os efeitos do Amblyomin-X sobre a permeabilidade vascular foram avaliados pela mensuração espectrofotométrica da quantidade de corante extravasado no tecido dos animais após injeção intradérmica do fator de crescimento do endotélio vascular (VEGF) ou do Amblyomin-X. Uma série de estudos in vitro foram realizados em células endoteliais de linhagem de microcirculação (t-End) para avaliar os efeitos do Amblyomin-X (10, 100 e 1000ng/mL) sobre: 1) a migração destas células, usando modelos bidimensional (2D) de cicatrização in vitro e tridimensional (3D) em câmara de Boyden modificada, na ausência e frente ao fator de crescimento do endotélio vascular (VEGF; 100 ng/mL); 2) sobre a aderência em Matrigel® e 3) sobre a secreção de prostaglandina E2 (PGE2) e a produção de óxido nítrico (NO) por ensaio imunoenzimático e reação de Griess, respectivamente. Ademais, foram avaliados os efeitos do Amblyomin-X sobre a viabilidade das células B16-F10 (1x105) por citometria de fluxo. Os resultados obtidos mostram que a aplicação tópica de Amblyomin-X reduziu o número de vasos no tecido subcutâneo dorsal (10ng/mL = 21,7%; 100ng/mL= 35,7%; 1000ng/mL= 36,8% vs 1° dia de tratamento). O mesmo efeito foi observado na presença de células B16-F10 (1000ng/mL= 44,3% vs 1° dia de tratamento), além de uma redução no desenvolvimento da massa tumoral (1000ng/mL= 88% vs controle). O tratamento com Amblyomin-X reduziu a migração basal das células t-End no modelo 2D (10ng/mL=16,4%; 1OOng/mL=23, 1%; 1000ng/mL=26,8% vs controle) e 3D (10ng/mL=39,2%; 100ng/mL=49,4%; 1000ng/mL=50,4% vs controle); inibiu a adesão destas células endoteliais em Matrigel® (100ng/mL=46,4%; 1000ng/mL=48,4% vs controle); não alterou produção os mediadores químicos NO e PGE2 pelas células endoteliais; não modificou a permeabilidade vascular e não alterou a viabilidade das células de melanoma murino B16-F10. Em conjunto, os dados obtidos mostram que o Amblyomin-X inibe a formação de novos vasos em condições basais e na vigência de crescimento tumoral in vivo que este efeito pode estar relacionado à redução do desenvolvimento tumoral, uma vez que a concentração de Amblyomin-X que inibe a angiogênese não causou citotoxicidade às células tumorais in vitro. Além disso, os mecanismos envolvidos no processo de angiogênese podem ser decorrentes, pelo menos em parte, de prejuízos na migração e adesão das células endoteliais. / The recombinant serine protease inhibitor protein called Amblyomin-X was obtained from a cDNA library of the Amblyomma cajennense salivary glands constructed and used to identify a gene encoding a kunitz type serine protease inhibitor. Amblyomin-X presents inhibitory effect on tumoral mass formation in vivo. Nevertheless, the mechanisms involved in the effects have not been clarified. Considering that interference on angiogenesis process is one of the mechanisms responsible for the antitumor activity displayed by serine protease inhibitors, this project was undertaken to study the Amblyomin-X actions on this process and on related endothelial cell functions. In vivo angiogenesis was studied using dorsal chamber model associated to intravital microscopy. Forty eight hours after dorsal chamber implantation, the animals were topically treated with saline or Amblyomin-X during 8 days, with intervals at each 48hs (10, 100 ou 1000ng/mL). The effects were evaluated at basal conditions or during tumoral development (1x105 B16-F10 murine melanoma cells injected into subcutaneous tissue). In addition, the effects of Amblyomin-X on vascular permeability were evaluated by measuring the dye leakage into dorsal intradermic tissue after local injection of vascular endothelial growth factor (VEGF) or Amblyomin-X, or both. In vitro assays were also performed using endothelial cells from microcirculation (t-End) and the effects of Amblyomin-X (10, 100 e 1000ng/mL) were studied on: 1) cell migration, using bidimensional (2D) and tridimensional (3D) models in modified Boyden chamber using chemotatic factor (VEGF100 ng/mL); 2) Matrigel® adherence and, 3) prostaglandin E2 (PGE2) and nitric oxide (NO) secretion by enzymatic assay and Griess reaction, respectively. In addition, the Amblyomin-X toxicity was evaluated on the B16-F10 cells (1x105), using flow citometry. Results obtained show that topic application of Amblyomin-X reduced the number of vessels in the subcutaneous dorsal tissue (10ng/mL = 21,7%; 100ng/mL= 35,7%; 1000ng/mL= 36,8% vs 1st day of treatment). The same effect was observed in the presence of B16-F10 cells (1000ng/mL= 44,3% vs 1st day of treatment), simultanesouly to a significant reduction on tumoral mass development (1000ng/mL= 88% vs control). Amblyomin-X treatment impaired basal migration of tEnd in the 2D (10ng/mL=16,4%; 100ng/mL=23, 1%; 1000ng/mL=26,8% vs control) and 3D model (10ng/mL=39,2%; 100ng/mL=49,4%; 1000ng/mL=50,4% vs controle); inhibited the adhesion of t-End in Matrigel® (100ng/mL=46,4%; 1000ng/mL=48,4% vs control); did not alter the production of chemical mediators (PGE2 and NO); did not modify the vascular permeability and did not affect the B16-F10 cells viability. Taken together, data here obtained show that Amblyomin-X inhibited the new vessels formation under basal conditions, and during tumoral development. The effect could be related to the reduction of tumoral progress also detected in vivo, asthe schedule of treatment employed did not induce cancer cell toxicity. The mechanisms involved in the reduced angiogenesis may be related, at least in part, to the impaired endothelial cell migration and adhesion.

Bacteroides melaninogenicus strains 2D and K110 were characterized with regard to their pathogenic, collagenolytic, proteolytic, hemagglutinating and metabolic activities. Both strains were members of the subspecies 13. melaninogenicus ss. asaccharolyticus. They possessed a cell-bound oxygen-sensitive collagenase, a cell-bound and a soluble oxygen-sensitive hemagglutinin (HA), and a protease. Both strains produced butyric and phenylacetic acids and were infective in guinea pigs as characterized by their ability to produce necrotic lesions and to be transferred from one animal to another. Strain 2D required hemin for growth and its growth rate was influenced by the addition of free amino acids to the medium. The hemagglutinating and proteolytic activities of strain 2D were investigated further to determine their relationship to infection. The soluble HA was reversibly inhibited by Hg and activity was restored in the presence of reducing agents. Iodoacetic acid caused irreversible inhibition. The HA was sensitive to heat and pronase treatment. Treatment of the red blood cells (RBC) with neuraminidase enhanced HA activity while the presence of galactose in the reaction mixture inhibited it, suggesting the involvement of galactose residues on the RBCs in the reaction.. Adsorption of the HA to RBC followed by elution and gel filtration resulted in the recovery of 50% of the HA activity and a 52-fold purification. Protease production by _B. melaninogenicus strain 2D was dependent on the growth rate of the organism. The protease was reversibly inhibited by HgCl₂ and irreversibly inhibited by iodoacetamide and iodoacetic acid. The enzyme was insensitive to serine protease inhibitors and EDTA. The pH optimum for proteolytic activity was 7.0, which correlates with the pH of its natural environment, the gingival crevice. It is thus classified as a neutral sulfhydryl enzyme. A 774-fold purification of the cellular protease of 2D, with a 160% recovery of activity, was accomplished by precipitation with 60% ethanol, ultracentrifugation and gel filtration through Sephadex G-100 and Sepharose 2B in the presence of urea. Electrophoretic analysis of the protease on SDS-polyacrylamide gels revealed four distinct bands, each of which was shown to be associated with carbohydrate. In the absence of SDS only one band, which did not migrate into the gel, was obtained. Any attempts to further dissociate the protease resulted in the loss of activity. The protease was active against azocoll, azocasein, casein and N,N-dimethylcasein. No glycosidase, lipase, collagenase or HA activities were detected. Protein, carbohydrate and lipid were detected in the preparation. The soluble protease which amounted to 20% of the cellular protease of strain 2D was subjected to gel filtration on Sephadex G-100 and eluted in a single peak at the void volume. The properties of the soluble protease were identical to those of the cell associated enzyme, suggesting the presence of a single proteolytic enzyme which was released into the culture medium with cell lysis or due to shedding of outer membrane fragments. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (M.Sc. (Microbiology))--Mahidol University, 1983.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains viii, 173 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Master of Biological Chemistry) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 37-42.

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T18:53:11Z (GMT). No. of bitstreams: 1 Nascimento_CatiaMariaGeraldadosSantos_M.pdf: 1717590 bytes, checksum: 6c341cf3cd058cf34bc633755af508d8 (MD5) Previous issue date: 2012 / Resumo: Neste estudo foram analisadas oito isolados de Plesiomonas shigelloides, obtidos da bacterioteca do Laboratório de Fatores de Virulência em Bactérias, Instituto de Biologia -UNICAMP - Campinas/SP: sete isolados são provenientes de água doce do Rio Cambé na região de Londrina/PR e, um isolado de ATCC 14029. Os sobrenadantes de culturas de P. shigelloides foram capazes de induzir efeitos anticarcinogênicos in vitro (em células He-La e HEp-2), apesar de apresentarem efeitos citotóxicos em linhagens celulares não cancerígenas (em células CHO e Vero). A atividade hemolítica dos cultivos de P. shigelloides foi detectada em placas com ágar sangue (meio sólido) e em microplaca (meio líquido) contendo hemácias de carneiro. A atividade citotóxica e hemolítica de Plesiomonas shigelloides apresenta característica de termolabilidade. Os isolados de P. shigelloides aderiram em células HEp-2 e em superfícies inertes como o plástico (microplaca de poliestireno) e vidro (lamínula) e, ainda apresentaram a capacidade de formar biofilme in vitro, sendo possível a colonização e formação de biofilmes em células epiteliais e em dispositivos médicos implantáveis como cateteres, próteses e/ou sondas. A produção de exoenzimas hidrolíticas como lípase e proteases (caseinase e elastase) foi detectada nos isolados de P. shigelloides. No teste de sensibilidade antimicrobiana pelo método de difusão de discos, os isolados de P. shigelloides se mostraram sensíveis à maioria dos antibióticos utilizados. Entretanto, outros testes de sensibilidade antimicrobiana se tornam necessários de modo a assegurar uma terapia antibiótica eficiente em casos de confirmação de diagnóstico clínico microbiológico. Neste estudo, os isolados de P. shigelloides expressaram fatores de virulência in vitro como citotoxicidade, adesão, exoenzimas e resistência antimicrobiana que podem potencialmente estar envolvidos na sua patogenicidade, de modo especial às gastroenterites ou complicações extra-intestinais (septicemia) em indivíduos imunocomprometidos (HIV-soropositivo, câncer ou ainda doenças hepatobiliares) / Abstract: We analyzed eight isolates Plesiomonas shigelloides, obtained from the bacterial collection Laboratory of Virulence Factors in Bacteria, Biology Institute - UNICAMP - Campinas/SP: seven isolates are from freshwater in Cambé River, region of Londrina/PR and a isolate ATCC 14029. The culture supernatants P. shigelloides were able to induce anticarcinogenic effects in vitro (He-La and HEp-2 cells), although having cytotoxic effects on non-cancer cell lines (CHO and Vero cells). The hemolytic activity of cultures P. shigelloides was detected on blood agar plates (solid medium) and microplates (liquid medium) containing sheep erythrocytes. The hemolytic and cytotoxic activity P. shigelloides features characteristics of thermo ability. Isolates P. shigelloides joined in HEp-2 cells and inert surfaces such as plastic (polystyrene microplate) and glass (coverslips) and also had the ability to form biofilm in vitro, it being possible colonization and biofilm formation in epithelial cells and devices implantable medical devices such as catheters, implants and/or probes. The production of hydrolytic exoenzymes such as lipase and protease (caseinase and elastase) was detected in isolates P. shigelloides. In antimicrobial susceptibility testing by the disc diffusion method, isolates P. shigelloides were sensitive to most antibiotics. However, other antimicrobial susceptibility testing has become necessary to ensure efficient antibiotic therapy in cases of clinical diagnostic microbiological confirmation. In this study, isolates P. shigelloides expressed virulence factors in vitro as cytotoxicity, adhesion, exoenzymes and antimicrobial resistance that may potentially be involved in this pathogenicity, specially to gastroenteritis or extraintestinal complications (septicemia) in immunocompromised individuals (HIV-seropositive, cancer or diseases hepatobiliary) / Mestrado / Clinica Medica / Mestra em Clínica Médica

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

The overall goal of my thesis research was to understand better the mechanisms that control antigen processing and presentation by class II MHC molecules. Towards this goal I investigated ways in which the physical structure and post-translational modifications of the class II MHC alpha and beta chains and associated molecules might serve to regulate antigen processing and presentation. Specifically, I investigated (1) a hypothesis that Ii might aid binding of foreign antigenic peptides to the class II MHC foreign antigen binding site (desetope), and the application of this hypothesis to the prediction of class II-presented peptides; (2) the proteolytic cleavage of Ii to p25; (3) the proteolytic cleavage of the class II MHC alpha and beta chains, and (4) the phosphorylation of Iiand the alpha and beta chains. In exploring the hypothesis that amphipathic alpha helical peptides digested from foreign antigen, bind to the class II MHC desetope, to be presented to T cell receptors, we found such an extended, amphipathic helix in Ii (Phe146-Val164). A hypothesis was developed that this amphipathic alpha helix of Ii bound to the desetope of class II MHC molecules, and remained there from time of synthesis until catalyzing the charging of the desetope with a foreign peptide. This region of Iicould then be considered to be the prototypic T cell-presented peptide and the "strip-of-helix" algorithm was developed to search the sequences of proteins for similar amphipathic alpha helices. Such peptides might bind to the class II MHC desetope and have a high probability to be presented to the T cell. The strip-of-helix algorithm calculated the mean hydrophobicity (from Kyte-Doolittle values; Kyte and Doolittle, 1982) of sets of amino acids in axial strips down sides of helices for 3 to 6 turns, at positions n, n+4, n+7, n+11, n+14, and n+18. Peptides correlating well with T cell responsiveness had: (1) 12 to 19 amino acids (4-6 turns of an alpha helix), (2) a strip with highly hydrophobic residues, (3) adjacent, moderately hydrophilic strips, and (4) no prolines to break the helix. This algorithm predicted 10 of 12 T cell-presented peptides in 7 well-studied proteins. In a study of the post-translational modifications of Ii, an early proteolytic pathway of the destruction of Ii, resulting in the generation of p25, was described. This 25,000 dalton protein, seen in immunoprecipitates with antibodies to class II MHC molecules or to Ii, was shown to be a C-termina1 fragment of a high mannose form of Ii. The evidence for this conclusion includes the following results. [35S]methionine-1abe1ed Ii and associated molecules were immunoprecipitated, denatured, resolubi1ized and subjected to a second immunoprecipitation with various antibodies. Two antisera to C-termina1 peptides of Ii (183-193 and 192-211), but not an antiserum to an N-termina1 peptide (12-28), immunoprecipitated p25. A monoclonal antibody (mAb) to Ii immunoprecipitated [35S]methionine-1abe1ed p25 but not [35S]cysteine-1abe1ed p25, consistent with the loss of a portion of Ii containing the only cysteine in Ii, Cys28. [35S]methionine pulse-chase labeling demonstrated the maximal appearance of p25 at 20-40 min chase times. p25 molecules were reduced to about 10.5 kD by treatment with endoglycosidases F and H. p25 was, therefore, generated from a high mannose form of Ii in the ER or cis-Golgi. This finding could either implicate that site for class II MHC desetope charging with foreign peptides or reflect a mechanism for degradation of "excess" Ii molecules in the ER. Digestion of class II MHC antigen-Ii complexes with various proteases yielded fragments, migrating at and near p25 in 2-D electrophoretic gels, which were relatively resistant to further digestion. This observation was consistent with the presence of relatively protease-resistant secondary structures (domains) and a relatively protease-sensitive (IgG hinge-like) region in Iinear its insertion into the membrane. In a study of the post-translational modifications of the class II MHC alpha and beta chains, well conserved pairs of basic amino acids in the sequences of these molecules were observed. It was hypothesized these could be sites for proteolytic cleavage, as precedented in other systems (i.e.proinsulin processing). These potential cleavage sites fall in significant locations with respect to the deduced structure of the class II MHC desetope, supporting the hypothesis that these cleavages might either aid or destroy antigen presenting functions. To test this hypothesis we looked for remnant polypeptides of the alpha and beta chains. Polypeptides were observed in gels of immunoprecipitated class II MHC complexes. To identify if such polypeptides were derived from the alpha and beta chains, immunoblotting to electrotransferred polypeptides was attempted, with antisera made to synthesized peptides that mimicked eight regions of the alpha and beta chains. These antisera were produced and characterized by dot blotting, ELISA, western blotting, and immunoprecipitation of native and denatured material. One antiserum, to an alpha chain peptide (77-88), blotted to a polypeptide immunoprecipitated by anti-class II MHC antiserum. This observation supported the hypothesis that the alpha and beta chains undergo proteolytic cleavages, possibly in the control of antigen presentation. It was also demonstrated that Ii and the alpha and beta chains can be phosphorylated under varying culture conditions, but this project was not pursued.

Licentiatavhandling (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 122-136.