Schistosomiasis caused by the parasitic helminth Schistosoma mansoni is a major health problem in tropical and subtropical regions. Its detection is crucial for patient management, evaluation of treatment, and monitoring of disease transmission, thus the development of novel diagnostic assays is of immense importance. The pathology of S.mansoni infection is characterised by formation of granulomatous lesions, hepatic fibrosis and portal hypertension. Liver fibrosis itself, regardless of the causative agent is an important health problem. The fact that there is no treatment for hepatic fibrosis other than transplantation in end stage liver failure emphasizes the importance of investigating the molecular basis of fibrogenesis and development of better therapeutic tools. MicroRNAs (miRNAs) are a class of short non-coding RNA that play important roles in disease processes in animals. Several miRNAs have been implicated in hepatic fibrogenesis; however, the expression profile and the role of miRNAs in S. mansoni infection are yet to be determined. This thesis focuses on the characterization of miRNAs in the liver and serum of mice during S.mansoni infection in order to determine their therapeutic and diagnostic potential in this disease. Profiling of miRNA expression in the liver of mice infected with S.mansoni revealed a set of mouse miRNAs that were differentially expressed in infected compared to naïve mice including miR-199a-3p, miR-199a-5p, miR-214 and miR-21, which have previously been associated with liver fibrosis in other settings. Further, inhibition of one of the up-regulated miRNAs, miR-199a-3p, in the liver upon S.mansoni infection resulted in reduced levels of collagen and other fibrosis related genes. Our results are consistent with a model where miRNA inhibition influences the clearance or reversion of hepatic stellate cells (HSCs) from a “myofibroblast-like” to an inactivated state. Thus, these results suggest miR-199a-3p inhibitor as potential therapeutic in treatment of liver fibrosis. miRNAs have been shown to be altered in disease process and are present in body fluids in a stable form, indicating that they can be used as novel diagnostic biomarkers. Studies of miRNAs in the circulation revealed that 5 of the mouse miRNAs altered in the liver were also significantly elevated in serum by 12 weeks post-infection. Sequencing of small RNAs from serum confirmed the presence of these miRNAs and further revealed 11 parasite-derived miRNAs that were detectable by 8 weeks post infection. Analysis of host and parasite miRNA abundance by qRTPCR was extended to the serum of patients from low- and high-infection sites in Zimbabwe and Uganda. The host-derived miRNAs failed to distinguish uninfected from infected individuals. However, analysis of three of the parasite-derived miRNAs (miR-277, miR-3479-3p and bantam) could detect infected individuals from low- and high-infection intensity sites with specificity/sensitivity values of 89%/80% and 80%/90%, respectively. Moreover, sequencing of small RNAs from serum revealed that specific tRNA fragments of 29-33 nt exist in mouse serum and occur at a >10 fold higher copy number than known microRNAs. The tRNA fragments appear to be the product of a specific cleavage event near the anti-codon loop, which has previously been associated with oxidative stress inside cells. We detected a clear bias in the abundance of 5’ versus 3’ products of tRNAGly(GCC), indicating specificity in the mechanism of stabilization of these products following cleavage. Our findings suggest that these specific tRNA cleavage products are either generated in, or exported to, serum and are protected against serum RNase activity by protein rather than encapsulation within vesicles. Further work is necessary to investigate the role and potential involvement of tRNA halves in cell-to-cell communication in oxidative stress induced by S.mansoni infection. In summary, this thesis reveals three major findings, which provide an important base for further studies of the role of miRNAs in S.mansoni infection and liver fibrosis. Firstly, it characterises host miRNAs dysregulated during S.mansoni infection in the liver and identifies a miR-199a-3p inhibitor as a potential anti-fibrotic agent. Secondly, it identifies parasite-derived miRNAs as novel markers of S. mansoni infection in the serum of both mice and humans, with the potential to be used with existing techniques to improve S.mansoni diagnosis. Lastly, it identifies and characterises novel tRNA-derived fragments in the serum, whose properties as biomarkers awaits further characterisation.

La galectine-3 (Gal-3) est biologiquement liée au processus d'inflammation et de fibrose dans l’insuffisance cardiaque (IC). Le système rénine-angiotensine-aldostérone (SRAA) a été largement démontré comme exerçant des effets profibrotiques, prohypertrophiques et pro-inflammatoires dans la pathophysiologie de l’IC. En outre, il existe des preuves expérimentales que Gal-3 et SRAA peuvent interagir dans le développement des dommages cardiovasculaires dans des modèles d’hypertension. Cependant, une telle interaction n'a pas été étudiée dans un contexte de dysfonction ventriculair gauche (VG). Nous avons donc cherché à tester les hypothèses suivantes: 1. La Gal-3 participe aux mécanismes de la fibrose cardiaque et du remodelage tissulaire médiés par l'aldostérone dans un modèle murin de dysfonction du VG; 2. Le niveau de Gal-3 est associé au développement du remodelage du VG et de la fibrose et peut prédire les effets des l'antagonistes neurohormonaux chez les patients atteints d'IC chronique due à une cardiomyopathie dilatée non ischémique (CMDNI). Méthode 1. Des souris mâles adultes atteintes d'hyperaldostéronisme cardiaque spécifique ont été soumises à des injections sous-cutanées d'isoprotérénol (200 mg/kg x 2/jour pendant deux jours), puis randomisées pour recevoir un placebo, un inhibiteur de Gal-3 (modified citrus pectin, MCP), un antagoniste de récepteurs aux minéralocortïcoides (canrenoate de potassium) ou l’association de deux (MCP+canrenoate) pendant 14 jours. 2. Nous avons recruté 150 patients avec un diagnostic de CMDNI. Tous les patients ont bénéficié d'une évaluation clinique et biohumorale, incluant le dosage de Gal-3, l'échocardiographie, et une imagerie par résonance magnétique cardiaque (IRM) contrastée pour l'évaluation des volumes et de la fibrose par le late gadolinium enhancement (LGE). Une plus petite cohorte de 70 patients a également reçu un dosage de soluble suppression of tumorigenicity protein 2 (sST2) à l’inclusion, et une IRM de suivi à 24 mois pour l'évaluation du remodelage inverse du VG, définie comme une augmentation de >10% de la fraction d’ejection du VG ou une diminution de 10% du volume diastolique du VG indexé. Résultats L’isoprotérénol a induit une diminution rapide et persistante de la fonction systolique du VG chez la souris, qui a été nettement améliorée par le traitement au MCP ou canrenoate. Le MCP et le canrenoate ont également prévenue l'hypertrophie et la fibrose cardiaques ainsi que l’augmentation de l'expression de gènes impliqués dans la fibrogénèse (Coll-1 et Coll-3) et l'infiltration de macrophages (CD-68 et MCP-1). L'utilisation combinée du MCP et canrenoate a entraîné des effets additifs sur l'hypertrophie cardiaque, l'inflammation et la fibrose, comparativement au MCP ou au canrenoate seul. Dans notre cohorte de patients avec CMDNI, la valeur médiane de Gal-3 était de 14,4 ng/mL; le LGE a été observée chez 106 patients. Les patients ayant un LGE positif avaient un taux de Gal-3 plus élevée que ceux qui n'en avaient pas (p=0,006). Après analyse multivariée suel la Gal-3, le sexe, l’ancienneté del la cardiopathie et la fraction d’éjection du ventricule droit sont des facteurs prédicteurs indépendants de la présence de LGE. L’IRM de suivi a montrée que 35 patients présentaient un remodelage inverse. L’analyse multivariée met en evidence Gal-3 comme un prédicteur indépendant du remodelage inverse contrairement au sST2. Conclusion La Gal-3 participe à des mécanismes de lésions myocardiques médiées par l'aldostérone dans un modèle murin de IC. De même, dans un cadre clinique de CMDNI, Gal-3 semble être associée à la fibrose du VG et à l'évolution du remodelage cardiaque, en identifiant éventuellement le sous-ensemble de patients avec une réponse plus prononcée à l'antagonisme neuro-hormonal pharmacologique. / BackgroundGalectin-3 (Gal-3) is biologically linked to the process of inflammation and fibrosis in heart failure (HF). The renin-angiotensin-aldosterone system (RAAS) has been largely demonstrated to exert profibrotic, prohypertrophic and proinflammatory effects in the pathophysiology of HF. Further, there is initial experimental evidence that Gal-3 and RAAS may interplay in the development of cardiovascular damage in hypertensive models. However, such interaction has not been investigated in the setting of left ventricular (LV) dysfunction and HF. We aimed therefore to test the following hypotheses:1. Gal-3 participates in the mechanisms of aldosterone mediated cardiac fibrosis and tissue remodeling in a murine model of LV dysfunction;2. Gal-3 level is associated with the development of LV remodeling and fibrosis and can predict the effects of neurohormonal antagonism in patients with chronic HF due to non-ischemic dilated cardiomyopathy (NIDCM).Methods1. Adult male mice with cardiac-specific hyperaldosteronism (AS) underwent isoproterenol subcutaneous injections (200 mg/kg x 2/day over two days), to be then randomized to receive placebo, a Gal-3 inhibitor (modified citrus pectin, MCP), an aldosterone antagonist (potassium canrenoate), or MCP+canrenoate for 14 days.2. We enrolled 150 patients with a diagnosis of NIDCM. All patients underwent a comprehensive clinical and biohumoral evaluation, including Gal-3 assay, 2-D echocardiography, and a contrast-enhanced cardiac magnetic resonance (CMR) for the assessment of LV volumes and fibrosis by the late gadolinium enhancement (LGE) technique. A smaller cohort of 70 patients also received soluble suppression of tumorigenicity protein 2 (sST2) assay at baseline as well as a follow-up CMR after 24 months for the evaluation of LV reverse remodeling, defined as a >10 percentage units increase in LV ejection fraction or a >10% decrease in LV end-diastolic volume indexed.ResultsIsoproterenol induced a rapid and persistent decrease in LV systolic function which was markedly improved by treatment with either MCP or canrenoate. MCP and canrenoate also reduced cardiac hypertrophy and fibrosis and the expression of genes involved in fibrogenesis (Coll-1 and Coll-3) and macrophage infiltration (CD-68 and MCP-1). The combined use of antagonists of Gal-3 and aldosterone resulted in enhanced effects on cardiac hypertrophy, inflammation, and fibrosis, when compared to MCP or canrenoate alone. In our cohort of NIDCM patients, median Gal-3 value was 14.4 ng/mL; LGE was detected in 106. Patients with LGE had higher Gal-3 than those without (p=0.006). Among univariate predictors of LGE, Gal-3 maintained its predictive value at multivariate analysis, together with sex, hypertension, disease duration and right ventricular ejection fraction. At follow-up CMR, 35 patients showed reverse remodeling. Gal-3, but not sST2 resulted as an independent predictor of LV reverse remodeling at multivariate analysis.ConclusionGal-3 participates in mechanisms of aldosterone-mediated myocardial damage in murine model of HF. Likewise, in the clinical setting of NIDCM, Gal-3 seems to be associated with LV fibrosis and to the evolution of cardiac remodeling, possibly identifying the subset of patients with a more pronounced response to pharmacological neurohormonal antagonism.

Accumulation of intracellular damage by reactive oxygen species accelerates biological aging, leading to the development of age-related lung diseases such as idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction and mitochondria-related oxidative stress has been implicated in the pathogenesis of many age-related diseases. Selective autophagic degradation of mitochondria (mitophagy) is critical to maintain a proper pool of the organelle and preserve cellular energy homeostasis. Oxidative stress resulting from age-dependent defects in the quality of proteins and degradation of mitochondria promotes alveolar epithelial cell damage potentiating lung injury. Our research found diminished autophagy corresponding with elevated levels of oxidized proteins and lipofuscin in response to lung injury in old and middle-aged mice compared to younger animals. More importantly, older mice exposed to lung injury are characterized by deficient mitophagic responses. The pro-fibrotic cytokine transforming growth factor beta 1 (TGFβ1) plays a pivotal role in driving fibroblast-to-myofibroblast differentiation (FMD), an important feature of pulmonary fibrosis. TGFβ1-mediated FMD is characterized by reduced autophagy flux, altered mitophagy and defects in mitochondrial function. In accordance, PINK1 expression is reduced in the aging murine lung and biopsies from IPF patients compared to controls. "nOur research also revealed a decline in mitochondrial protein deacetylase sirtuin 3 (SIRT3) expression in the lungs of aging mice. Low levels of SIRT3 transcripts were observed in two different animal models of pulmonary fibrosis. SIRT3 expression was reduced in fibrotic regions of lung tissues from patients with fibrotic diseases. We demonstrated that down-regulation of SIRT3 by TGFβ1 promotes acetylation of major oxidative stress response regulators, such as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), and that resveratrol induced SIRT3 expression and ameliorated acetylation changes induced by TGFβ1. Knockdown of SIRT3 expression by siRNA exacerbated TGFβ1-induced FMD. By contrast, promotion of SIRT3 expression attenuated the effect of TGFβ1 on myofibroblast differentiation. Finally, SIRT3-deficient mice were more susceptible to pulmonary fibrosis in response to bleomycin and had increased collagen deposition compared to control mice. Collectively, our research indicates that an age-related decline in autophagy, SIRT3 expression, and mitochondrial homeostasis may contribute to the promotion and/or perpetuation of pulmonary fibrosis. / Meredith L Sosulski

Meredith L Sosulski

Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

Thesis (Ph. D.)--Ohio State University, 20043. / Title from first page of PDF file. Document formatted into pages; contains xiv, 115 p.; also includes graphics. Includes abstract and vita. Advisor: Clay B. Marsh, Dept.of Pathology. Includes bibliographical references (p. 102-115).

Fibronectin (Fn) is an adhesive extracellular matrix protein assembled by fibroblasts into fibrils within ECMs of developing and remodeling tissues. Fn is sensitive to mechanical forces exerted by contractile cells, and can alter its structural conformations in response to mechanical strain within Fn fibrils. We developed probes (both peptide and antibody) to Fn to detect mechano-sensitive perturbations of Fn conformation. Probes were characterized for their binding characteristics (affinity, epitope, mechano-sensitivity) and validated on multiple in vitro and in vivo ECM models. Furthermore, we showed that the mechano-sensitive H5 antibody that we have developed have utility in detection of early molecular signatures of fibrosis in vivo in a mouse model of pulmonary fibrosis. Using the H5 antibody, we also report detection of a conformational switch within the integrin binding FnIII9-10 region. Modulation of Fn’s integrin switching behavior may help in the development of controllable “smart” biomaterials, as well as to the development of conformation-specific imaging probes to detect early molecular signatures of tumor and fibrotic ECMs.

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