Thesis (Ph. D.)--University of Sydney, 2005. / Title from title screen (viewed 22 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Experimental Medicine, Faculty of Medicine. Degree awarded 2005; thesis submitted 2004. Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--Ohio State University, 2004. / Document formatted into pages; contains xv, 178 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 May 20.

Thesis (PhD)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: David Pascual. Includes bibliographical references (leaves 278-316).

Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on Apr. 21, 2009). Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-20Bitstream added on 2014-06-13T21:03:39Z : No. of bitstreams: 1 silvares_mrc_dr_botfm.pdf: 1230751 bytes, checksum: a7b798cd6de13023c34b6f92ec176d41 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A conexão pele-tireoide tornou-se uma fronteira de interesse da dermatoendocrinologia. Neste contexto, a relação entre urticária crônica idiopática (UCI) e autoimunidade tireóidea tem sido estudada nos seus diferentes aspectos. Além disso, de 30% a 40% dos casos de UCI apresentam etiologia autoimune. A tireoidite de Hashimoto (TH) representa a mais prevalente doença autoimune da tireoide (DAT), acarretando, frequentemente, hipotireoidismo. Os indícios que o quadro de UCI é mais grave e menos responsivo aos tratamentos convencionais quando associado à DAT, sugerem um papel da tireotrofina (TSH) no desenvolvimento das lesões urticariformes. O teste do soro autólogo da pele (TSSA) é um teste intradérmico in vivo utilizado para avaliar a autorreatividade, a presença de autoanticorpos funcionais ou de urticária de etiologia autoimune. Apesar dos indícios de associação entre DAT e UCI, os relatos são controversos. Os objetivos do presente estudo foram avaliar: a associação entre UCI e autoimunidade tireóidea; a frequência de alterações tireóideas em pacientes com UCI, com ênfase nas de origem autoimune; a frequência de angioedema em relação às alterações tireóideas; a associação entre TH, em pacientes com UCI, e positividade do TSSA; a expressão gênica do receptor de TSH (RTSH) na pele sã e na pápula do TSSA, de pacientes com UCI, com e sem TH. O estudo foi dividido em dois capítulos. No Capítulo 1, foi realizada uma análise retrospectiva dos prontuários médicos de 223 pacientes com urticária crônica (UC), acompanhados no ambulatório de alergia dermatológica do Hospital das Clínicas da Faculdade de Medicina de Botucatu. Foram selecionados 115 casos com UCI que dispunham dos resultados das dosagens plasmáticas de TSH, tiroxina livre (T4-L) e anticorpos antitireoperoxidase (antiTPO) e antitireoglobulina (antiTG). Tratava-se de 91 mulheres (79,1%) e... / The thyroid-skin connection has become a hot frontier in dermatoendocrinology. In that context, the relation between chronic idiopathic urticaria (CIU) and thyroid autoimmunity has been studied in its different aspects. Besides, from 30% to 40% of CIU cases present autoimmune etiology. Hashimoto’s thyroiditis (HT) represents the most prevalent autoimmune thyroid disease (ATD), frequently causing hypothyroidism. The signs that the CIU picture is more severe and less responsive to conventional treatments when associated with ATD suggest a role of thyrotropin (TSH) in developing urticariform lesions. The autologous serum skin test (ASST) is an in vivo intradermal test to evaluate autoreactivity, presence of functional antibodies or urticaria from autoimmune etiology. Despite the signs of association between ATD and CIU, reports are controversial. The present study aimed to evaluate: the association between CIU and thyroid autoimmunity; the frequency of thyroid alterations in patients with CIU emphasizing on the autoimmune originated ones; the frequency of angioedema in relation to thyroid alterations; the association between HT, in patients with CIU, and the ASST positivity; the TSH receptor (RTSH) gene expression in healthy skin and in the papules of ASST, of patients with CIU, with or without HT. The study was divided into two chapters. In Chapter 1, a retrospective analysis was performed in the medical report of 223 patients with chronic urticaria (CU), assisted at the outpatient of dermatologic allergy of the Clinic Hospital of the Botucatu Medical School. One hundred fifteen cases with CIU and available results of plasmatic dosage of TSH, free thyroxine (FT4), and antithyroperoxidase (antiTPO) and antithyroglobulin (antiTG) antibodies were selected. There were 91 women (79.1%) and 24 men (20.9%), mean age 36.5 years, who started the follow-up between 1984 and 2006. These case ... (Complete abstract click electronic access below)

Asthma is a common chronic respiratory disease where patients suffer from restricted airways and airway inflammation (mainly eosinophilic type 2 (T2) inflammation). Inhaled (ICS)/oral corticosteroids (OCS) and, more recently T2-targeting biologics, are prescribed as mainstay therapies for asthma. Despite these therapies, a subset of asthma patients continues to have symptoms and airway inflammation, suggesting an underlying additional asthma pathology. We have observed multiple airway autoantibodies in 55% of moderate-to-severe asthma patients associated with inadequate response to corticosteroids and anti-T2 biologics. Increased airway degranulation (eosinophilic and/or infective) together with lymphopenia (low lymphocyte counts) underlie these self-reactive/autoimmune-like events. In healthy individuals, regulatory lymphocytes limit the development and activity of self-reactive cells, including those capable of producing autoantibodies. Lymphopenia can lead to skewed non-regulatory to regulatory lymphocyte subsets that support a microenvironment with reduced ability to limit self-reactivity. In this study, wanted to measure B cell subsets by flow cytometry and non-regulatory to regulatory B cell ratios in asthma patients and healthy controls. To understand B cell compartmentalization, we analyzed peripheral and sputum B cells. We identified decreased regulatory B cells (Bregs), in particular CD5+ Bregs, and skewed non-regulatory to regulatory B cell ratios in both circulation and airways of asthma patients. Compared to healthy controls, only patients requiring daily OCS had significantly lower CD5+ Bregs, suggesting a reduced regulatory component in more severe patients. Further, CD5+ Bregs, capable of producing immunomodulatory interleukin-10, were significantly lower in patients with a history of multiple lymphopenic events (70% requiring daily OCS). Together, this supports the need to investigate lymphopenia-induced dysregulation of Bregs, increase in autoreactive B cells and airway autoreactivities, and subsequent progression into a more-severe therapy refractory autoimmune pathology. This opens a new avenue for asthma treatment, particularly for the severe population with airway autoimmune responses often not targeted/controlled by current anti-inflammatory therapies. / Thesis / Master of Science (MSc) / Asthma is a common respiratory disease where patients have difficulty breathing and airway inflammation. Corticosteroids and/or targeting biologic therapies are prescribed to reduce inflammation. Despite these treatments, some patients still have inflammation. In patients that don’t respond well to treatment, we have found evidence of self-attacking antibodies that can further lead to disease severity. These antibodies are produced by self-attacking B cells normally suppressed by regulatory B cells (Bregs). We wanted to measure if asthma patients have reduced ability to limit self-attacking B cells because of reduced Bregs. We found that asthma patients have lower Bregs and an imbalance of non-regulatory to regulatory B cells in the blood and airways. Together, we show B cell subsets that suggest a self-attacking asthma component, not targeted by current asthma treatments. Understanding the involvement of Bregs in asthma opens a new therapeutic option in those that don’t respond well to current treatment.

The first observations of biofilms were made by the “father of microbiology” Antonie van Leeuwenhoek in the 17th century. The number of publications on biofilms has grown exponentially in the last 20 years, highlighting the medical relevance of the field. The complexity of the bacterial biofilm as well as its variability across species provides a continual channel for discovery. While all biofilms differ, there are some components that remain standard such as proteins, polysaccharides and DNA allowing for linkages between seemingly distinct biofilms. Biofilm-associated infections account for more than 65% of all infections implicating the need for understanding the stages of biofilm formation and development. Our lab focuses on the amyloid component of the biofilm and has identified that curli and extracellular DNA (eDNA) complex irreversibly within the biofilms of Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we investigate the formation and pathogenicity of cytotoxic curli intermediates previously unidentified in the in vitro biofilm. The identification of multiple curli conformations within the biofilm biogenesis aides in the understanding of amyloid kinetics in the enteric biofilm. Together, these studies provide a link between biofilm-associated infections and autoimmune responses in the host. In these studies, we planned to isolate curli from different stages in biofilm development to observe their differences both structurally and through their interactions. We identified turbulence has a significant impact on the formation of mature biofilm. We were able to isolate an intermediate form of curli through increased turbulence during biofilm growth. There has never been an intermediate form of curli isolated before our studies due to the high efficiency of the nucleation-precipitation process of curli fiber formation. From this isolation, we characterized these intermediates in comparison to the mature curli complexes. We observed that higher turbulence leads to lesser biofilm formation by sedimentation assay and crystal violet staining. Additionally, we investigated the expression of the curli forming genes csgBA via flow cytometry analysis which indicated that csgBA was preferentially expressed under low turbulence conditions. When investigating the curli conformations isolated from the biofilm, we found that intermediate complexes incorporated less thioflavin T (ThT) indicating lesser amyloid content. We also differentiated the mature and intermediate curli aggregate populations using multiple microscopy techniques. Under confocal microscopy, intermediate fibers seldom measured larger than 100 µm, while mature curli did. Electron dense regions were observed under transmission electron microscopy in the mature curli indicating high fibrillization and compact structure of these aggregates, not seen in the intermediates. Due to known interactions of curli with eDNA in the biofilm, next we investigated the DNA content of the complexes. We hypothesized that the mature structured complexes would have greater DNA content supporting the maturation of the fibrils and the structural compaction. Indeed, we found more DNA could be extracted from the mature curli fibers. Interestingly, we increased the fibrillization of intermediates upon addition of exogenous genomic DNA suggesting DNA incorporation was necessary for the formation of the mature fibrillar aggregates. Intermediates of amyloid β are found to be more cytotoxic than the mature form of the amyloid. For this reason, we hypothesized that curli intermediates could also be cytotoxic. After treating bone marrow-derived macrophages with mature and intermediate curli complexes, we observed that the intermediate aggregates were significantly more cytotoxic to immune cells than mature aggregates. Together, this data implicates a role for the cytotoxic intermediate form of curli in the pathogenesis of Salmonella as well as other enteric bacteria. Curli complexes have been previously described as a novel pathogen associated molecular pattern (PAMP) by their ability to activate numerous receptors in immune cells. The host immune response to curli complexes has been elucidated in our lab. First, binding to Toll-like receptor-2 (TLR2) begins with recognition of the conserved cross-β sheet secondary structure. Mutations disrupting this structure are shown to abrogate immune cell recognition and signaling. For this reason, we next investigated the pathogenicity of the intermediates discovered and characterized above. As cytotoxic oligomers exist for human amyloids, we aimed isolate an earlier form of the intermediate curli and investigate the ability of these conformations to activate host immune responses. As mature curli has been reported to induce anti-dsDNA antibodies in murine models of systemic lupus erythematosus (SLE), we anticipated differences in the autoimmune response to these different curli aggregates as well. First, we isolated and characterized an early form of intermediates isolated at 24 hours which were smaller in size and incorporated less DNA within their complexes than the aforementioned intermediates. Further investigations into the structure of these early intermediates described altered secondary structure by circular dichroism. The lack of fully formed secondary structure in the early intermediates we hypothesized to decrease the ability of these complexes to interact with immune receptors as mature curli. Indeed, we saw decreased response in pro-inflammatory cytokine production as well as type I IFN production. This lack of type I IFN production, lead us to investigate the autoantibody response to the early intermediates. When treating both wild-type and autoimmune-prone mice with curli complexes, early autoantibodies responses were dependent on the DNA content of the complex. However, after continual treatment for 10 weeks, intermediate complexes produced levels of anti-dsDNA similar to that of the mature curli treatment. In addition to anti-dsDNA antibodies, for the first time, other anti-nuclear antibodies, such as anti-C1q and anti-nucleosome, were produced in response to these treatments as well. Finally, chronic exposure to curli complexes led to significant histopathological changes including synovial proliferation and periosteal resorption, in the joints of mice autoimmune-prone mice. Together, this data identified that chronic exposure to curli induced autoimmune sequelae which is thought to be transient in genetically healthy individuals, but leads to joint inflammation in individuals whom are genetically predisposed to autoimmunity. In summary, these data significantly broaden the knowledge of curli amyloid formation during biofilm biogenesis in vitro through the identification of previously unidentified cytotoxic intermediate conformations of curli. Additionally, our work forwards the fields of both autoimmunity and biofilm-associated infection research by providing evidence of the direct impact that chronic exposure to the biofilm has on the host, both transient and long-lasting in those whom are pre-disposed to autoimmunity. / Biomedical Sciences

published_or_final_version / abstract / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy