Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pathology. Includes bibliographical references.

Made available in DSpace on 2014-10-09T12:23:39Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:58Z (GMT). No. of bitstreams: 1 00203.pdf: 1432783 bytes, checksum: d2a399f27f88b4a93cdad210e33ea6a6 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Erika R. Geisbrecht / Maintenance of muscle tissue during development is greatly dependent upon the extracellular matrix (ECM) to stabilize, sense, and compensate for changes in the local environment. Muscle has a particularly high demand for a dynamic ECM to allow for contraction and to transmit forces necessary for generating movement. Inefficient contraction and/or detachment can lead to muscle tissue damage and the release of damage-associated molecular patterns (DAMPs), which overactivate immune responses and drive the progression of muscle diseases. Our lab uses the Drosophila muscle attachment site (MAS) as a model to characterize novel genes and mechanisms involved in muscle maintenance. Initially, we were focused on characterizing a novel ECM protein, Fondue (Fon), which had previously been shown as a critical mediator of ECM stability in the hemolymph clot. Mutations in fon and the knockdown of fon through RNAi causes body wall muscles to detach and also creates large gaps between muscle hemisegments. TEM analysis of fon mutant MASs revealed a loss of ECM integrity and important support features including disruption of cuticle and tendon architectures, a lack of muscle-tendon interdigitation, and a loss of electron-dense matrix accumulation. More interestingly, a sensitized background screen revealed a subset of coagulation proteins, fon, Tiggrin, and Lsp1γ, that were necessary for stabilizing muscle attachment sites. Further investigation into gene expression profiles of mutants experiencing hypercontraction-induced muscle tissue stress indicated a clear trend of innate immune activation, suggesting a broader connection between muscle development and innate immunity. In fon mutants with muscle detachment, we also observe abnormal melanin accumulation as melanotic tumors or along the larval MASs, activation of Toll signaling in the fat body, and constitutive expression of the antimicrobial peptide (AMP), drosomycin. In a fon-sensitized background assay, we identified genetic interactions between fon and Toll pathway members, including the NFκB inhibitor/IκB, cactus. At the local level, fon-mediated muscle detachment and muscle hypercontraction mutants, Mhc[superscript]S1 and Brkd[superscript]J29, cause JAK/STAT activation within muscle tissue. We propose a model where muscle tissue stress caused by disruptions to muscle homeostasis progresses muscle disease through overactivation of the innate immune system. Understanding the mechanisms by which these two biological processes are intertwined will advance our knowledge of how tissue stresses can be sensed and elicit multi-tissue responses.

Point mutations found in human breast cancer samples introduced into the Drosophila dco gene affect the Drosophila haematopoiesis. I described different haematopoietic phenotypes in such mutants and tested their connection with the Toll and the JAK/STAT signalling pathways.

Although it is sympatric with tsetse flies, Stomoxys calcitrans is not a biological vector of trypanosomes. It is known that haemolymph (HL) and midgut reservoir zone (RH) lectins regulate parasitic infections in some dipteran insects. Agglutinins (lectins) were detected in HL and RH from unfed stable flies (maximum titre 2-6). Increased haemagglutination activity resulted post-feeding (maximum titre 2- 16 - 2- 18). Optimum titres varied according to agglutinogen type and mammalian blood source. Rabbit erythrocytes produced the highest haemagglutination titres followed by human group B, human group 0, horse, human group A, human group AB and sheep. Stomoxys haemagglutination activity was found to be 1.5 - 2.5 times stronger than that of Glossina. Whole blood-fed flies produced the highest titre (2-18), compared to glucose-fed insects, against rabbit erythrocytes. Anti-Trypanosoma brucei brucei titres ranged from 2-6 - 2-7 in both tissues. Similar results were obtained with Leishmania hertigi and Crithidia!asciculata. Purification of the samples was performed in order to draw conclusions with confidence regarding the physico-chemical properties of the agglutinins (lectins) and in order to determine the molecular weight of the agglutinins. Protein contents ofHL and RH samples of flies aged < 12 hours to 3 days were determined. They were 25 - 28 mg/ml and 6.4 mg/ml respectively. Protein contents increased with age reaching 32 mg/ml for HL and 7.2 mg/ml for RH at day 14 post-emergence (p.e.). The contents then started to decrease reaching 22 mg/ml for HL and 5.6 mg/ml for RIi at day 28 p.e. Purified lectins constitute 4.3% of the total protein contents in RH samples (having molecular weights of 26,302 Da, 16,218 Da and 14,028 Da) and, approximately twice, 9.47% of the total protein contents, in HL samples (having similar molecular weights of 28,300 Da, 16,218 Da and 14,600 Da). HL and RH anti-parasite and anti-erythrocyte agglutinins (lectins) were basic glycoproteins in nature, calcium ion dependent for activity, heat labile, freeze-thaw sensitive and required slightly acid to alkaline pH conditions for optimum agglutination. Lectins were specific for galactosyl and glucosyl moieties. In vivo sugar inhibition of RH lectin activity resulted in three-fold increased S. calcitrans mortalities post- T.b. brucei infection, compared to the controls, suggesting a lectin parasite-killing function. However, sugar inhibition of lectins did not lead to transformation of trypanosomes to procyclic forms or to infection of the fly.

The overall aim of this project was to determine the molecular mechanisms by which the flagellin gene from Salmonella enterica serovar Typhimurium (S. Typhimurium) activates the NAIP/NLRC4 inflammasome and its contribution to the host protective immune response against salmonellosis. Inflammasomes are multi-protein complexes formed in response to the activation of pattern recognition receptors (PRRs). The NOD-like receptor (NLR)-family of inflammasome complexes are formed from the cytosolic NLR receptors, ASC adaptor and caspase-1 in response to pathogen- associated molecules or danger-associated signals. The NAIP/NLRC4 inflammasome is activated by the S. enterica flagellar filament protein (FliC), the SPI-1 type III secretion system needle (PrgI) and inner rod proteins (PrgJ). Recognition of these bacterial ligands by the NAIP receptors allows oligomerisation with NLRC4 and subsequent recruitment of caspase-1. Caspase-1 mediates pyroptosis, while recruitment of ASC is also required for cleavage of pro-IL-1β and pro-IL-18 to their active forms by caspase-1. Differential recognition of the flagellar filament proteins (flagellin) by the NAIP/NLRC4 inflammasome forms an important part of my thesis. Here, I have looked at the molecular mechanisms and immunological consequences of the differential recognition of flagellin by the NAIP/NLRC4 inflammasome using S. Typhimurium SL1344 and the non-pathogenic E. coli strain K12-MG1655. An important part of my work was to try and determine which regions of fliC are required for NAIP/NLRC4 inflammasome activation and whether they can be mutated while preserving motility. To do this a panel of ten strains expressing chimeric fliC genes were created and characterised in macrophage infection experiments and bacterial motility assays. My results confirm the C-terminus of FliC is critical for both inflammasome activation and motility in agreement with published reports. To further investigate this differential recognition by the NAIP/NLRC4 inflammasome I modified S. Typhimurium strain of moderate virulence (M525P) to express flagellin from E. coli K12-MG1655. This strain (M525PΔfliC::fliCK12-MG1655CmR) retained motility and both in vitro and in vivo characterisation was carried out in macrophages and using a murine model of sublethal salmonellosis respectively. Activation of the NAIP/NLRC4 inflammasome was impaired in murine macrophages infected with M525PΔfliC::fliCK12-MG1655CmR when compared to those infected with M525P. Mice infected with M525PΔfliC::fliCK12-MG1655CmR had increased liver and spleen bacterial burdens compared to those infected with M525P, indicating that optimal NAIP/NLRC4 inflammasome activation is key for efficient control of microbial spread in vivo. The role of NAIP receptors in inflammasome formation was further investigated with the use of CRISPR/Cas9 to generate mutant murine macrophage cell lines. To investigate the consequence of gene deletions cell lines were designed to lack NAIP 1, 2, 5 and 6, while others were designed to express tagged NAIP proteins to elucidate the cellular localisation of the NAIP proteins during inflammasome formation by microscopy. Characterisation of these cell lines is ongoing, with extensive optimisation of the CRISPR/Cas9 technique undertaken during this study.

Made available in DSpace on 2014-10-09T12:23:51Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:51Z (GMT). No. of bitstreams: 1 01011.pdf: 1513856 bytes, checksum: 5aef640dc2adb97f1d5a80607ef8ffc3 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Orientador: Rosemeri de Oliveira Vasconcelos / Coorientador: Geórgia Modé Magalhães / Banca: Marcela Marcondes Pinto Rodrigues / Banca: Pamela Rodrigues Reina Moreira / Banca: Hélio José Montassier / Banca: Barbara Cristina Mazzucatto / Resumo: Os tumores mamários malignos possuem várias formas de evadir o sistema imune e, dentre elas está a modulação das células dendríticas (DCs), por interferência na sua maturação, resultando em apresentação de antígenos ineficiente aos linfócitos T e consequente indução da tolerância imunológica. As DCs moduladas pelo tumor recrutam muitos linfócitos T regulatórios (Tregs) para o microambiente tumoral, o que amplia os efeitos imunossupressores locais. A forte relação entre as DCs e os linfócitos Tregs no microambiente tumoral ainda foi pouco estudada, principalmente em cães. Por isso, este estudo teve o objetivo de avaliar a relação existente entre as DCs e os linfócitos Tregs em carcinomas mamários caninos do tipo simples de cadelas, por meio da técnica de imunohistoquímica. Foram utilizadas 10 amostras de glândula mamária sem tumor (G1) e 40 amostras de neoplasias mamárias (G2: adenomas; G3: carcinomas papilares; G4: carcinomas tubulares e G5: carcinomas sólidos), que foram submetidas a imunodetecção de linfócitos Treg (FOXP3+), linfócitos T CD4 e T CD8, MHC-II, DCs mieloides (imaturas e maduras), as citocinas TGF-β, IL-10, a enzima Indoleamine 2,3-dioxigenase (IDO) e dos receptores de quimiocina (CCR6 e CCR7). A maioria das células, citocinas e receptores imunológicos mostraram correlação positiva dentro da população tumoral e controles avaliados, principalmente sobre o efeito fixo "idade", que teve alta correlação positiva com o tamanho tumoral e com CCR6. Quanto às correlaçõ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The malignant mammary tumors have several ways of evading the immune system, including the modulation of dendritic cells (DCs), by interfering with their maturation, resulting in inefficient presentation of antigens to T cells and consequent induction of immunological tolerance. Tumor-modulated DCs recruit many regulatory T cells (Tregs) into the tumor microenvironment, which amplifies local immunosuppressive effects. The strong relationship between DCs and Tregs cells into the tumor microenvironment was poorly studied, especially in dogs. Therefore, this study aimed to evaluate the relationship between DCs and Tregs cells in the simple type canine mammary carcinomas, using the immunohistochemical technique. Ten samples of mammary gland without tumor (G1) and 40 samples of mammary neoplasms (G2: adenomas, G3: papillary carcinomas, G4: tubular carcinomas and G5: solid carcinomas) were submitted to immunodetection of Treg cells (FOXP3 +), CD4 and CD8 T cells, MHC-II, myeloid DCs (immature and mature), cytokines TGF-β, IL10, Indoleamine 2,3-dioxygenase (IDO) and chemokine receptors (CCR6 and CCR7). Most of the cells, cytokines and immunological receptors showed positive correlation within the tumor population and controls evaluated, mainly on the fixed effect "age" that had high positive correlation with the tumor size and with CCR6. As for the negative correlations, the CD83 antibody was the only one that had no significant positive correlation with any other variable. It was o... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-10-09T12:23:39Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:58Z (GMT). No. of bitstreams: 1 00203.pdf: 1432783 bytes, checksum: d2a399f27f88b4a93cdad210e33ea6a6 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Made available in DSpace on 2014-10-09T12:23:51Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:51Z (GMT). No. of bitstreams: 1 01011.pdf: 1513856 bytes, checksum: 5aef640dc2adb97f1d5a80607ef8ffc3 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP