Systematic characterization of microbes in several tumors including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) has revealed the presence of multiple species of intracellular bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in co-culture of host epithelial cells and bacteria, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging oncomicrobe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a Gram-negative, anaerobic bacterium that is normally found within the oral cavity. However, its selective enrichment in CRC and PDAC tumors is correlated with poor clinical outcomes. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum infection of host cancer cells induced robust secretion of select cytokines that increased cancer cell migration, impacted cell seeding, and enhanced immune cell recruitment. In PDAC, we uncovered additional cytokines that were secreted from both normal and cancerous pancreatic cell lines upon infection with F. nucleatum that increased cancer cell proliferation and migration via paracrine and autocrine signaling, notably in the absence of immune cell participation. In order to examine the contribution of a hypoxic TME on infection dynamics, we used a multi-omics approach that combined RNA-seq and ChIP-seq of H3K27ac to determine epigenomic and transcriptomic alterations sustained within hypoxic CRC cells upon infection with F. nucleatum. Our findings revealed that F. nucleatum can subvert host cell recognition in hypoxia and can modulate the expression of multiple cancer-related genes to drive malignant transformation. Insights gained from this research will pave the way for future studies on the impact of the tumor microbiome in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer. / Doctor of Philosophy / Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States, respectively. Recent systematic characterization of various tumor types revealed the presence of distinct bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in developing host cell-microbe co-culture models, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging cancer-associated microbe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a microbe commonly found within the oral cavity. However, clinical studies revealed that selective enrichment of F. nucleatum in CRC and PDAC tumors significantly correlated with poor prognosis. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum invasion of host cancer cells induced the secretion of select proteins called cytokines that cells use to signal and communicate with each other. These cytokines directly stimulated the cell migration of host cancer cells which is usually associated with increased cancer aggressiveness. In PDAC, F. nucleatum infection induced the secretion of additional cytokines from both cancer cells and normal cells that, in addition to cell migration, impacted the proliferation of cancer cells, another feature of aggressive cancers. F. nucleatum usually thrives in a low oxygen environment that is prevalent in cancer tissue and hence, we examined how a low oxygen environment can influence infection dynamics using sequencing technologies that probe the genomic constitution within cells. Our findings revealed that F. nucleatum can escape recognition in low oxygen environments and can modulate the expression of multiple cancer-related programs within the cell to drive cancer progression. Insights gained from this research will pave the way for future studies on the impact of the tumor-associated microbes in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer.

No description available.

AIDS-related human cytomegalovirus (HCMV) retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. Understanding the pathogenesis of this disease is essential for developing new, safe, and effective treatments for its prevention or management, yet much remains unknown about the virologic and immunologic mechanisms contributing to its pathology. To study such mechanisms, we use a well-established, reproducible, and clinically relevant animal model with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) that mimics in mice the symptoms and progression of AIDS in humans. Over 8 to 12 weeks, MAIDS mice become susceptible to experimental murine cytomegalovirus (MCMV) retinitis. We have found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling (SOCS)1 and SOCS3, host proteins which dampen immune-related signaling by cytokines, including antiviral interferons. Herein we investigated virologic and/or immunologic mechanisms involved in this stimulation and how virally-modulated SOCS1 and/or SOCS3 proteins may contribute to MCMV infection or experimental MAIDS-related MCMV retinitis. Through pursuit of two specific aims, we tested the central hypothesis that MCMV stimulates and employs SOCS1 and/or SOCS3 to induce the onset and development of MCMV retinal disease. MCMV-related SOCS1 and SOCS3 stimulation in vivo occurred with intraocular infection, was dependent on method and stage of immune suppression and severity of ocular pathology, was associated with stimulation of SOCS-inducing cytokines, and SOCS1 and SOCS3 were differentially sensitive to antiviral treatment. In vitro studies further demonstrated that SOCS1 and SOCS3 stimulation during MCMV infection occurred with expected immediate early kinetics, required viral gene expression in cell-type-dependent and virus origin-dependent patterns of expression, and displayed differential sensitivity to antiviral treatment. These data suggest that SOCS1 and SOCS3 are stimulated by divergent virologic, immunologic, and/or pathologic mechanisms during MCMV infection, and that they contribute to the pathogenesis of retinal disease, revealing new insights into the pathophysiology of AIDS-related HCMV retinitis.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-09-18T13:07:03Z No. of bitstreams: 1 Gabriela da Silva Cruz. Avaliação... 2014.pdf: 1237079 bytes, checksum: 7beefa52e21bd18ac4325a1c493249ba (MD5) / Made available in DSpace on 2014-09-18T13:07:04Z (GMT). No. of bitstreams: 1 Gabriela da Silva Cruz. Avaliação... 2014.pdf: 1237079 bytes, checksum: 7beefa52e21bd18ac4325a1c493249ba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / O Trypanosoma cruzi é um parasita intracelular e agente causador da doença de Chagas, que afeta milhões de pessoas em todo o mundo. Sabe-se que durante os processos de inflamação, regeneração e fibrose desencadeados pelo T. cruzi no hospedeiro há a participação de diversos mediadores e fatores. O objetivo deste trabalho foi avaliar a associação entre polimorfismos de nucleotídeos únicos com as formas clínicas e o grau de fibrose em pacientes com doença de Chagas. Os polimorfismos foram analisados por PCR em tempo real. Foram incluídos no estudo 55 pacientes com diagnóstico de doença de Chagas e classificados de acordo com a forma clínica da doença, sendo que 17 apresentavam a forma indeterminada, 15 a forma cardíaca sem disfunção ventricular e 23 a forma cardíaca com disfunção ventricular. Os genótipos CA dos polimorfismos do gene LGALS3 (rs4644 e rs4652); AG e GG do SOCS3 (rs4969170); CT e TT do IL-28B (rs12979860 e 8099917, respectivamente); AG, AG, CC, AG e AG do CLDN-1 (rs10212165, rs3909582, rs9865082, rs9880018 e rs9848283, respectivamente); e CC do CCL5 (rs2280789) foram estatisticamente mais frequentes em pacientes com a forma cardíaca do que com a forma indeterminada da doença. Com relação ao grau de fibrose, os genótipos CC dos polimorfismos do gene LGALS3 (rs4644 e rs4652); AA do SOCS3 (rs4969170); CC do rs12979860 e TT do rs8099917 do IL-28B; AA do rs10212165, AA, AG e GG do rs3909582, CC e CT do rs9865082, AG e GG do rs9880018 e AA do rs9848283 do gene CLDN1; e CC do CCL5 (rs2280789) foram estatisticamente mais frequentes em indivíduos com fibrose cardíaca <15% quando comparados com o grupo com fibrose ≥15%. Diante do exposto concluimos que os polimorfismos analisados podem ser úteis como futuros biomarcadores para estadiamento e conduta terapêutica em pacientes com doença de Chagas. / Trypanosoma cruzi is an intracellular parasite and the agent that causes Chagas disease, which affects millions of people worldwide. Several factors and mediators are known to actively participate in the inflammation, fibrosis and tissue regeneration, which is triggered by T. cruzi within the host. The aim of this study was to evaluate the association of single nucleotide polymorphisms with clinical forms and rate of fibrosis in Chagas disease patients. The polymorphisms were analyzed by real-time PCR. The study consisted of 55 Chagas disease patients that were classified according to the clinical form of the disease, including 17 patients presenting the indeterminate form, 15 patients presenting the cardiac form without ventricular dysfunction and 23 patients presenting the cardiac form with ventricular dysfunction. The genotypes of CA of LGALS3 gene polymorphisms (rs4644 and rs4652); AG and GG of SOCS3 (rs4969170); CT and TT of IL-28B (rs12979860 and 8099917, respectively); AG, AG, CC, AG and AG of CLDN-1 (rs10212165, rs3909582, rs9865082, rs9880018 and rs9848283, respectively); and CC of CCL5 (rs2280789) were significantly more frequent in patients presenting the cardiac form compared to patients presenting the indeterminate form. Regarding the degree of fibrosis, the CC genotype of polymorphisms of the genes LGALS3 (rs4644 and rs4652); AA of SOCS3 (rs4969170); CC of rs12979860 and TT of rs8099917 of the IL-28B; AA of rs10212165 and AA, AG and GG of rs3909582, CC and CT of rs9865082, AG and GG of rs9880018 and AA of rs9848283 of the gene CLDN1; and CC of CCL5 (rs2280789) were statistically more frequent in patients presenting <15% cardiac fibrosis when compared to patients presenting fibrosis ≥15%. Taken together, our results suggest that the polymorphisms analyzed may be useful biomarkers for therapeutic management of patients with Chagas disease.

Pulmonary arterial hypertension (PAH) is a rare, devastating disease with no cure. Current treatment consists of a cocktail of vasodilators which relieve symptoms of PAH but do not treat the cause. Thus, there is a need for novel drugs that target the underlying pathological causes of PAH. PAH is a multi-factorial, but one key contributor is the pro-inflammatory cytokine IL-6 which stimulates pro-inflammatory and pro-angiogenic signalling mediated by the JAK/STAT pathway. One way in which IL-6 signalling via JAK/STAT is inhibited is via SOCS3 in a type of negative feedback loop whereby IL-6 induces transcription of SOCS3, which then attenuates further JAK/STAT signalling. SOCS3 can also be induced by cAMP. This is interesting as prostanoids, a type of drug used in the treatment of PAH due to its vasodilator effects and the only type to show any efficacy improving the life expectancy of PAH patients, acts by mobilising cAMP. Thus, prostanoid stimulation of cAMP could potentially limit IL-6 signalling via the induction of SOCS3. This is a novel mechanism of prostanoids which has not previously been considered. This study investigated the capability of prostanoids to limit the pro-inflammatory/pro-angiogenic effects of IL-6 that enable PAH to develop. Initial experiments confirmed that vascular endothelial cells responded to prostanoids which increased SOCS3 and limited IL-6 signalling activity. Further experiments utilising SOCS3 KO endothelial cell models demonstrated prostanoid inhibition of IL-6 signalling was due in part to SOCS3. In conclusion, this project has confirmed that prostanoids do limit the pro-inflammatory effects induced by IL-6 and that this is in part due to SOCS3. Although the exact mechanism is yet to be discovered, it will be beneficial in the treatment of PAH as it provides currently unexploited drug targets which can be considered for future PAH therapies. / British Heart Foundation

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A periodontite está relacionada à genética do hospedeiro, constituição do biofilme dental e fatores ambientais como o hábito de fumar. A metilação do DNA é um mecanismo de expressão genética que pode inibir ou silenciar a expressão do gene. Desta forma, vários pesquisadores têm se dedicado a estudar a influência genética sobre a suscetibilidade e/ou risco aumentado à doença periodontal. Estudos têm relatado associação entre vários biomarcadores epigenéticos com a inflamação periodontal. Considerando a hipótese de que existe associação do tabagismo com a metilação em genes relacionados à doença periodontal, o objetivo deste estudo foi verificar o padrão de metilação do DNA em células do epitélio oral de pacientes com periodontite crônica (CP) no promotor de um gene específico envolvido no controle da inflamação, como supressor da sinalização de citocinas (SOCS-1) em pacientes fumantes e não fumantes. O gene SOCS-1 é localizado no cromossomo 16p13.3, compostos por uma região amino-terminal, um domínio SH2 central e uma caixa SOCS. É um regulador negativo da via JAK / STAT. Inibe os efeitos biológicos de várias citocinas, incluindo IL-2, IL-3, IL-4, IL-6, interferão (INF) - &gamma; e INF- &alpha; / &beta;. Este foi um estudo caso-controle, comparando dois grupos, um grupo (teste) com consumo de 10 cigarros mínimos por dia, com diagnóstico de periodontite crônica e outro grupo controle que foram pacientes não fumantes com periodontite crônica. Para tal, DNA genômico foi purificado de células epiteliais bucais obtidas por meio de enxágue com sacarose 3%, por tempo único de coleta. O DNA foi modificado pelo bissulfito de Sódio e os padrões de metilação do DNA foram analisados com a técnica MS-PCR (Polymerase chain reaction). A análise estatística foi realizada pela plataforma estatística R version 3.3.2 Core Team (2016). Foi realizado Teste t de Student para amostras independentes e teste não paramétrico de Wilcoxon & Mann-Whitney para variáveis qualitativas; teste qui-quadrado e para a variável metilação, foi feito um teste exato de Fisher para testar a associação entre os grupos e a metilação. Os resultados indicaram que, para células epiteliais da mucosa bucal, a frequência de desmetilação no gene SOCS-1 é maior no grupo sem o hábito do fumo, em comparação ao grupo fumante. Foram detectadas diferenças no padrão de metilação entre os dois grupos. Ao estabelecer uma estimativa de risco relativo entre os grupos e a variável metilação, foi observado que pacientes fumantes têm 7,08 vezes (risco relativo) com um intervalo (1,95-51.46) de apresentar doença periodontal crônica, com um padrão de metilação no gene SOCS-1 / Periodontitis is related to host genetics, constitution of the dental biofilm and environmental factors such as smoking. DNA methylation is a mechanism of genetic expression that can inhibit or silence gene expression. In this way several researchers have been dedicated to study the genetic influence on the susceptibility and / or increased risk to periodontal disease. Studies have reported association between several epigenetic biomarkers with periodontal inflammation. Considering the hypothesis that there is an association between smoking and methylation in genes related to periodontal disease, the objective of this study was to verify the DNA methylation pattern in oral epithelial cells of patients with chronic periodontitis (ChP) in the promoter of a specific gene involved in the control of inflammation, as suppressor of cytokine signaling (SOCS-1) in smokers and nonsmokers patients. The SOCS-1 gene is located on chromosome 16p13.3 composed of an amino-terminal region, a central SH2 domain and a SOCS box. It is a negative regulator of the JAK / STAT path. It inhibits the biological effects of various cytokines, including IL-2, IL-3, IL-4, IL-6, interferon (INF) -&gamma; and INF-&alpha; / &beta; . This was an case-control type study, comparing two groups, a group with consumption of 10 minimum cigarettes per day, with a diagnosis of chronic periodontitis and another control group were non-smokers with chronic periodontitis. For this, genomic DNA was purified from oral epithelial cells obtained by rinsing with 3% sucrose, for a single time of collection. The DNA was modified by Sodium bisulfite and the methylation patterns of the DNA were analyzed with the MS-PCR technique (Polymerase chain reaction). Statistical analysis was performed by the statistical platform R version 3.3.2 Core Team (2016), Student\'s t-test was performed for independent samples and Wilcoxon\'s & Mann-Whitney non-parametric test for qualitative variables; chi-square test. For the methylation variable, an exact Fisher\'s test was performed to test the association between the groups and the methylation. The results indicated that, for oral mucosal epithelial cells, the frequency of demethylation in the SOCS-1 gene is higher in the non-smoking group as compared to the smoker group. statistically significant differences were detected in the methylation pattern between the two groups. When establishing an relative risk between the groups and the methylation variable, it was observed that smokers are 7.08 times (relative risk) of having chronic periodontal disease with a methylation pattern in the SOCS-1 gene

Chronic Myelogenous Leukemia (CML) is a blood cancer that arises when hematopoietic cells acquire an abnormal protein known as BCR-ABL. Current therapies for CML include drugs that inhibit BCR-ABL. However, these drugs only suppress the disease and do not cure it. One reason is that BCR-ABL drugs fail to kill the primitive population of CML cells, referred to as leukemia stem cells (LSCs), which are responsible for initiating and propagating CML. Since LSCs are not killed, the cancer is not cured and many affected patients eventually relapse. Recent studies suggest that LSCs are protected from current therapies by the bone marrow micro-environment where they reside. There, cytokine signaling molecules are present, which mediate processes that protect LSCs from BCR-ABL drugs. The stem cell factor (SCF) is one of these signaling molecules. It activates the receptor c-Kit located on the surface of LSCs, and this activation in turn allows proliferating LSCs to resist BCR-ABL drugs, even without prior exposure to these drugs, i.e., innate resistance is observed. In this thesis, the mechanism of this innate resistance is investigated, so that a suitable treatment strategy can be developed. To this end, a co-agent approach based on synthetic antibodies (sABs) is proposed to inhibit the receptor c-Kit, with the goal of disrupting its activation by the ligand SCF. This disruption should in turn block the SCF-mediated innate resistance, thus potentially restoring BCR-ABL drug apoptotic activity. The method for this disruption involves targeting the c-Kit structural susceptibility. Specifically, the sABs are designed via antibody phage display technology to target the D1–D2–D3 domains representing the SCF binding sites, hence preventing downstream pathway activation. The hypothesis is that, by blocking the SCF-mediated innate resistance, a suitable combination of such an sAB co-agent and a BCR-ABL drug should be conducive to suppressing LSCs, thereby providing a potential means to improve CML treatment. In addition, to assess the performance of the proposed treatment strategy, a set of in vitro tests is conducted, focusing on performance behaviors such as cell binding, cell death, and the progenitor inhibition. The experimental results support the hypothesis that the proposed combinatorial strategy is indeed a promising approach to mitigate the innate resistance, thus restoring BCR-ABL drug apoptotic activity.

Resumo: A expressão de citocinas inflamatórias é um processo estritamente regulado por mecanismos variados, incluindo o controle da sinalização intracelular e da atividade transcricional por inibidores endógenos, os quais são pouco estudados e compreendidos. Três grupos de proteínas: SHP, PIAS e SOCS inibem de maneira distinta e específica a transdução de sinais pela via JAK/STAT, bem como a atividade dos fatores de transcrição, eventos que modulam a expressão de diversas citocinas. As doenças periodontais estão associadas à inflamação persistente, com elevados níveis de citocinas proinflamatórias, no entanto praticamente não existem informações sobre a participação destes mecanismos de regulação nas diferentes condições clínicas periodontais. Os objetivos deste projeto incluíram avaliar a cinética de expressão das proteínas SOCS1 e SOCS3 e suas proteínas-alvo, STAT1 e STAT3, respectivamente, durante a evolução da doença periodontal. Foram utilizados 36 ratos Wistar divididos em 2 grupos: DP - doença periodontal induzida por 2 métodos: ligaduras ao redor dos 1os molares inferiores e injeções de 60 μg de LPS de E. coli no tecido gengival palatino dos molares superiores, 3x/semana; Grupo controle negativo - recebeu apenas injeções de PBS (veículo). Os ratos foram sacrificados 7, 15 e 30 dias após a indução da doença periodontal para avaliação histológica e análise macroscópica da perda óssea alveolar. A expressão de SOCS1 e SOCS3 e a ativação de STAT1 e STAT3 foram avaliadas nas biópsias gengivais por PCR em tempo real e Western blot. Ambos os modelos apresentaram significante e progressiva perda óssea dos 7 aos 30 dias. A inflamação foi evidente já no período de 7 dias em ambos os modelos, porém enquanto manteve-se similar nos demais períodos no modelo de indução por LPS, apresentou uma diminuição na severidade da inflamação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Inflammatory cytokine gene expression is a process strictly regulated by various mechanisms, including the negative regulation of signaling of cytokine receptors and of the activity of transcription factors such as STATs. These mechanisms involve endogenous proteins and are largely unknown, especially in periodontal diseases. Three groups of proteins, SHP, PIAS and SOCS modulate in a fairly specific manner JAK/STAT signaling and/or STAT activity. Periodontal diseases are infectious-inflammatory conditions of the supporting tissues of the teeth associated with increased levels of proinflammatory cytokines, but there are no information regarding the role of these endogenous mediators of JAK/STAT during its course. The aims of this study included the evaluation of the expression kinetics of inducible negative regulators and their target proteins during the course of experimentally induced periodontal disease. 36 Wistar rats were divided into two groups: PD - experimental periodontal disease induced by two methods: ligature placement around the first mandibular molars and E. coli lipopolysaccharide (LPS) injections into the palatal gingival tissues of the maxillary molars, 3x/week, and Negative Control group. Rats were sacrificed 07, 15 and 30 days after disease induction for histological evaluation of periodontal inflammation and macroscopic analysis of alveolar bone loss. SOCS expression and the activation status of STAT1 and STAT3 were evaluated in gingival biopsies by real time PCR and Western Blot. Both disease models presented significant progressive bone loss from 7 to 30 days. Inflammation was evident and similar for all the periods in LPS injected sites; however, a decrease on severity at the end of the experimental period was observed in the ligature model. There was a significant (p<0.05) increase on SOCS1 and SOCS3 gene expression in PD compared to control... (Complete abstract click electronic access below) / Orientador: Joni Augusto Cirelli / Coorientador: Carlos Rossa Junior / Banca: Carlos Ferreira dos Santos / Banca: Paulo Sergio Cerri / Mestre