Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive and severe interstitial lung diseases (ILDs) for which there is no cure. IPF is characterized by an excessive accumulation of fibroblasts which secrete an abundance of extracellular proteins such as collagen. These processes lead to repetitive tissue scarring and fibrosis in the lung parenchyma. As a result, lungs become rigid limiting oxygen intake and gas exchange. Once diagnosed, IPF is fatal within 2-3 years. There is no known cause or proven treatment that significantly improves outcomes. Although the cause is unknown, the current model of IPF suggests that an overactive epithelial repair mechanism caused by genetic and epigenetic factors as well as environmental exposures is responsible for the chronic fibrosis and scarring characteristic of IPF. The transforming growth factor beta (TGF-B) signaling pathway has been implicated as a major contributor in activating this chronic fibrosis. An upstream activator of the TGF-B pathway, avB6, has been identified as a potential therapeutic target. My collaborators in Dr. David Baker's lab at the University of Washington have created a novel avB6 integrin inhibitor (BP2_disulf) whose efficacy in improving IPF outcomes has yet to be tested. In my study, I test the ability of BP2_disulf to combat IPF through the use of the standard IPF murine model and translatable end points like non-invasive uCT scans, pulmonary function tests, bronchoalveolar lavage fluid (BALF) profiles, and histology. With these methods, I demonstrate that intraperitoneal injection of BP2_disulf in bleomycin-injured mice has the ability to decrease rate of fibrotic progression and pulmonary function decline compared to mice treated with bleomycin alone. These results prove that BP2_disulf is a promising therapeutic not only for IPF but other ILDs as well. Further efficacy validation and investigation into an aerosolized delivery method will advance this drug to clinical trials and make it accessible to those in need.

Les niveaux de chlorure sont rigoureusement régulés par des canaux de chlorure tels que le régulateur transmembranaire de mucoviscidose (CFTR), la famille de canaux CLC ou les canaux de chlorure activés par le calcium. Un dérèglement des concentrations de chlorure ou du transport de ce dernier a été rapportée pour être lié à plusieurs maladies telles que la mucoviscidose, la myotonie, l'épilepsie, l'hyperekplexie ou la surdité. Toutes ces maladies appartiennent à la famille des pathologies qui présentent un déficit de la fonction lysosomale et qui sont caractérisées par un défaut du processus autophagique.Les cellules épithéliales pulmonaires des malades atteints de mucoviscidose montrent également un défaut d’autophagie. L’association de deux produits : la cysteamine et l’epigallocathecin gallate (EGCG) permet de restaurer la fonction autophagique de ces cellules et améliore les symptômes de la maladie. Nous avons tenté d'améliorer cette combinaison en criblant des inducteurs de l'autophagie pour leur capacité d’interaction avec la cystéamine et pour une meilleure efficacité de traitement. Nous avons ainsi trouvé que l'amiodarone, de manière similaire à l'EGCG, était capable d'engager une interaction coopérative avec la cystéamine pour stimuler l'autophagie dans les lignées cellulaires. De plus, l'amiodarone s’est révélé relativement efficace pour restaurer l'expression d’une protéine Del 508 CFTR mature et fonctionnelle dans les cellules épithéliales.Dans la deuxième partie de cette thèse, nous avons exploré les relations entre la modulation des concentrations intracellulaires des ions chlorure et l'autophagie. Trois approches complémentaires ont été utilisées (i) l’appauvrissement / réduction du chlorure dans le milieu de culture des cellulaires (ii) la réduction de l'expression du gène CLCN7 codant pour le seul transporteur de chlorure connu pour être présent dans les lysosomes et (iii) l’utilisation de transporteurs synthétique des ions chlorure. Nous avons montré que la modulation de la concentration des ions Cl- régulait l'autophagie. Cela a été observé lorsque nous avons déplété le chlorure du milieu de culture cellulaire ou lorsque nous avons aboli le gradient de chlorure en utilisant des transporteurs de chlorure synthétique (SCTs). Nous avons également montré que les STCs modifiaient la structure et la fonction des mitochondries. De plus, nous avons montré que les antioxydants restauraient la fonction mitochondriale et inhibaient la stimulation de l'autophagie, ce qui permet de voir sous un jour nouveau le rôle que le chlorure pourrait jouer dans la signalisation de différentes voies cellulaire de réponse au stress. / Chloride levels are stringently regulated by chloride channels such as cystic fibrosis transmembrane regulator (CFTR), the CLC family of channels or calcium activated chloride channels. A dysregulation of chloride concentrations or transport has been reported to be linked to several diseases including cystic fibrosis (CF), myotonia, epilepsy, hyperekplexia or deafness. All these diseases belong to the family of lysosomal storage pathologies which are characterized by a defect in the autophagic process.The pulmonary epithelial cells of CF patients show a defect of autophagy. The combination of two products: cysteamine and epigallocathecin gallate (EGCG) has been shown to restore the autophagic function of these cells and to improve the symptoms of the disease. We have tried to enhance this combination by screening inducers of autophagy for their ability to interact with cysteamine and for better treatment efficacy. We found that amiodarone, similarly to EGCG, was able to engage in a cooperative interaction with cysteamine to stimulate autophagy in cell lines. In addition, amiodarone has been found to be relatively effective in restoring expression of a mature and functional Del F508 CFTR protein in epithelial cells.In the second part of this thesis, we explored the relationships between the modulation of intracellular chloride concentration and autophagy. Three complementary approaches were used (i) the depletion/reduction of chloride in the cell culture medium (ii) the reduction of the expression of the CLCN7 gene encoding the only chloride transporter known to be present in lysosomes and (iii) the use of synthetic carriers of chloride ions. We have shown that the modulation of the concentration of Cl- modulates autophagy. This was observed when we depleted chloride from the cell culture medium or when we abolished the chloride gradient using Synthetic Chloride Transporters (SCTs). We have also shown that SCTs modify the structure and function of mitochondria. In addition, we have shown that anti-oxidants restore mitochondrial function and inhibit the stimulation of autophagy, allowing us to see in a new light the role that chloride could play in different stress response pathways.

La mucoviscidose (ou cystic fibrosis, CF) est une maladie caractérisée par une inflammation pulmonaire chronique qui contribue à la dégradation progressive de l’épithélium des voies aériennes des patients. Les glucocorticoïdes (GC) représentent un outil essentiel pour le traitement du patient mais leur efficacité et leur rapport bénéfice/risque restent cependant controversés. Les effets secondaires provoqués par l’administration de ces molécules pourraient être diminués par l’utilisation de leur protéine d’adressage, la corticosteroid-binding globulin (CBG). L’objectif de ce travail était d’étudier l’expression de la CBG chez les patients CF, afin d’optimiser leur traitement anti-inflammatoire par GC. Nous avons montré, dans un premier temps, que la synthèse hépatique de CBG était augmentée, tandis que son taux plasmatique était conservé chez les patients CF, comparé aux patients non-CF. Dans un second temps, nous nous sommes intéressés à l’expression de la CBG au niveau pulmonaire. L’inflammation chez les patients CF étant principalement pulmonaire, l’expression locale de CBG à ce niveau pourrait moduler l’efficacité du GC, en le recaptant. Nos données montrent que l’expression de cette CBG pulmonaire est diminuée chez les patients CF. Les études sur des modèles in vitro hépatiques et pulmonaires n’ont pas permis d’expliquer les résultats obtenus et ont souligné la limite des modèles et des outils à disposition. Le maintien de la concentration plasmatique de CBG et la diminution de la CBG pulmonaire chez les patients CF suggèrent ainsi que la CBG pourrait être utilisée comme outil thérapeutique dans le contexte de la mucoviscidose, afin d’optimiser le traitement par GC. / Cystic fibrosis (CF) is characterized by a chronic pulmonary inflammation, responsible of the progressive degradation of the airways epithelium. In CF, glucocorticoids (GC) are widely used but their efficiency and benefit/risk ratio are still discussed. The side effects, caused by the administration of these molecules, might be decreased by the use of their delivery protein, corticosteroid-binding globulin (CBG). The aim of the work was to study the expression of CBG in CF patients, in order to optimise their anti-inflammatory treatment by GC. First, we showed that the hepatic synthesis of CBG was increased while its plasmatic level was preserved in CF patients, compared to non-CF. Second, we were interested by the expression of CBG at the pulmonary level. The inflammation in CF patients being primarely pulmonary, the local expression of CBG in the lung could modulate the efficiency of GC through recapture. Our data showed that this expression of this pulmonary CBG was decreased in CF patients. The studies conducted in vitro on hepatic and pulmonary models did not explained our results and highlighted the limit of the models and tools at our disposal. The maintained plasmatic concentration of CBG and the decrease of pulmonary CBG in CF patients suggest that CBG might be useful as a therapeutic tool in the CF context, in order to optimise the GC treatment.

The purpose is trying to classify the Liver Fibrosis stage using Gadoxetic Acid-EnhancedMR Images.  In the very beginning, a method proposed by one Korean group is being examined and trying to reproduce their result. However, the performance is not as impressive as theirs. Then, some gray-scale image feature extraction methods are used. Last but not least, the hottest method in recent years - ConvolutionNeural Network(CNN) was utilized. Finally, the performance has been evaluated in both methods. The result shows that with manual feature extraction, the Adaboost model works pretty well that AUC achieves 0.9. Besides, the AUC of ResNet-18 network - a deep learning architecture, can reach 0.93. Also, all the hyperparameters and training settings used on ResNet-18 can be transferred to ResNet-50/ResNet-101/InceptionV3 very well. The best model that can be obtained is ResNet-101which has an AUC of 0.96 - higher than all current publications for machine learning methods for staging liver fibrosis.

Les maladies fibroprolifératives se caractérisent par l’accumulation de constituants de la matrice extracellulaire en réponse à une agression chronique et répétée conduisant à la destruction de l’architecture tissulaire. Les myofibroblaste, sous l’influence du TGFβ, représentent les cellules effectrices majoritaires dans ce processus. Les miARN, régulateurs négatifs de l’expression génique, interviennent dans de nombreux mécanismes physio-pathologiques dont la fibrose tissulaire mais leur mécanismes d’action et la synergie potentielle entre miARN co-régulés au sein d’un même cluster restent mal compris. Nos travaux ont consisté à caractériser l’implication du cluster miR-199/214 généré à partir du LncARN DNM3os, dans la fibrose pulmonaire. Nous avons montré que les miARN de ce cluster participent à l’activation et à la différenciation des fibroblastes en myofibroblastes via la régulation des voies canoniques et non canoniques du TGF-β. Ce cluster agit également en tant qu’inhibiteur de la réparation épithéliale. In vivo, l’inhibition de l’un de ces « fibromiRs », a permis, dans un modèle murin, de réduire significativement les lésions de fibrose. Par ailleurs, de par leur présence et leur stabilité dans les fluides biologiques, les miARN représentent également une nouvelle classe de biomarqueurs diagnostiques ou pronostiques non invasifs. Nous avons ainsi montré que les niveaux sériques de miR-21-5p étaient augmentés chez les patients présentant une fibrose rénale sévère. Ces travaux soulignent l’importance des miARN dans la pathogénèse des maladies fibroprolifératives et montrent qu’ils représentent de nouvelles cibles thérapeutiques ou des biomarqueurs non invasifs. / Fibrotic diseases are characterized by the accumulation of extracellular matrix components in response to chronic aggression leading to the destruction of tissue architecture. Myofibroblasts, controlled by TGFβ, are central effectors in this process. MiRNAs, negative regulator of gene expression, are involved in many patho-physiological mechanisms, including tissue fibrosis but their mechanisms of action and the potential synergistic activity between co-regulated miRNAs within the same cluster remain poorly understood. Our work consisted in characterizing the involvement of the miR-199/214 cluster, generated from LncRNA DNM3os, in pulmonary fibrosis. We have shown that these miRNAs are involved in the activation and the differentiation of fibroblasts to myofibroblasts through the regulation of canonical and non-canonical TGF-β pathways. This cluster also acts as an inhibitor of epithelial repair. In vivo, the inhibition of one of this “FibromiR”, significantly decrease fibrosis, in a murine lung fibrosis model. In addition, because of their presence and their stability in biological fluids, miRNAs also represent a new class of non-invasive diagnostic or prognostic biomarker. We showed that serum levels of miR-21-5p were increased in patients with severe kidney fibrosis. These studies highlight the importance of miRNAs in pathogenesis of fibrotic disorders and show that they represent new therapeutic targets or non-invasive biomarkers.

Background: Liver fibrosis induced by non-alcoholic fatty liver disease causes peri-interventional complications in morbidly obese patients. We determined the performance of transient elastography (TE), acoustic radiation force impulse (ARFI) imaging, and enhanced liver fibrosis (ELF) score for fibrosis detection in bariatric patients. Patients and Methods: 41 patients (median BMI 47 kg/m2) underwent 14-day low-energy diets to improve conditions prior to bariatric surgery (day 0). TE (M and XL probe), ARFI, and ELF score were performed on days -15 and -1 and compared with intraoperative liver biopsies (NAS staging). Results: Valid TE and ARFI results at day -15 and -1 were obtained in 49%/88%and 51%/90%of cases, respectively. High skin-to-liver-capsule distances correlated with invalid TE measurements. Fibrosis of liver biopsies was staged as F1 and F3 in n = 40 and n = 1 individuals. However, variations (median/range at d-15/-1) of TE (4.6/2.6–75 and 6.7/2.9–21.3 kPa) and ARFI (2.1/0.7–3.7 and 2.0/0.7–3.8 m/s) were high and associated with overestimation of fibrosis. The ELF score correctly classified 87.5%of patients. Conclusion: In bariatric patients, performance of TE and ARFI was poor and did not improve after weight loss. The ELF score correctly classified the majority of cases and should be further evaluated.

Background Epidural fibrosis (EF) is defined as nonphysiological scar formation, usually at the site of neurosurgical access into the spinal canal, in intimate vicinity to and around the origin of the radicular sheath. From the very onset, EF behaves as a reparative inflammation causing, as a rule, symptoms of characteristic nature and clinical course (pain). Treatment of epidural fibrosis causing failed back surgery syndrome (FBSS) by neuromadulation technique is very expensive. Finding of suitable parameters for the indication of treatment is therefore very important. Aims The study is based on evidence of the importance of epidural fibrosis for the development of chronic pain. Research is also focused on the comparison of the range fibrosis and the effect of stimulation (spinal cord stimulation - SCS). The goal is to find a suitable selection factor for the indication of neuromodulation. Methods I. A double-blind prospective study was conducted to investigate a cohort of 200 patients requiring surgical treatment for intervertebral disc hernia (hernia disci intervertebralis). The patients were randomly and blindly divided into 2 groups, one on peroperatively applied local doses of a mixture containing corticosteroids, the other without such medication. All the requirements of a double-blind...

Vitamin K belongs to the family of fat-soluble vitamins, which is not determinated in clinical laboratories. It is a cofactor necessary for posttranslational γ-carboxylation of glutamyl residues in selected proteins such as the osteocalcin, and procoagulation factors II, VII, IX, X. Vitamin K deficient individuals appear to have more undercarboxylated proteins, which are functionally defective. Lack of this vitamin has been associated with risk of developing osteoporosis and cardiovascular diseases. The aim of this work was to develop and validate the HPLC method and the LC-MS/MS method for determination of three vitamin K's forms - vitamin K1, MK-4 and MK-7 in serum. After successful validation of both methods, patient samples and healthy population samples were measured. There were measured 350 patient samples by HPLC method. These samples were divided into two groups - patients with diagnostic of osteoporosis and patients without osteoporosis. We measured 946 samples by LC-MS/MS method. Samples were divided into groups: patients with osteoporosis, patients without osteoporosis, healthy population, patients with osteopenia and patients with cystic fibrosis. The reference range of vitamin K in healthy population was obtained by LC-MS/MS method. The next aim was to compare the effectiveness of...

Cardiovascular disease is the leading cause of death with over 600,000 deaths per year. A key feature of heart failure is over stimulation of the beta-adrenergic receptors. Over stimulation of these receptors leads to changes in contractility of the heart, which can eventually lead to myocardial remodeling such as cardiomyocyte cell death (apoptosis). Prolonged activation and injury of the heart stimulates fibroblasts to repair the injured site and can lead to stiffening and scar formation. These changes in the heart are heightened in post-menopausal women as estrogen is depleted. Clinical trials have shown that estrogen has cardioprotective effects through its receptors: estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Though estrogen has been shown to be cardioprotective, studies have shown that hormone replacement therapy had little to no benefit to postmenopausal women with heart disease. These discrepancies lead to the need for more studies on how estrogen can protect the heart against failure. We therefore, examined the effects of estrogen deficiency and its role on cardiac structure and function following chronic β-adrenergic stimulation. Female mice (4 months of age) were treated with isoproterenol (ISO) (400μg/kg/h, subcutaneously) for 7 days one month after bilateral ovariectomy (OVX) at 3 months of age. Echocardiography and pulse wave doppler analysis was performed to examine cardiac function. Animals were then euthanized and hearts were isolated and paraffin embedded for histology analysis. Fibrosis was analyzed using Masson’s trichrome staining and apoptosis was analyzed with TUNEL assay. Ovariectomy did result in significant compromised cardiac function and ISO treatment exacerbated these results. Results from the echocardiography exam showed that ISO increased left ventricular diameter and that ovariectomy increased the diameter in a similar manner. Ovariectomized mice treated with ISO showed even greater increases in left ventricular diameter. The Doppler pattern results also showed a significant decrease in contraction time in the ISO and OVX+ISO groups compared to the OVX group alone. Surprisingly, Masson’s trichrome staining showed no significant change between the SHAM and ISO treated groups. The low amount of fibrosis could be attributed to the length of the ISO treatment. One-month post-ovariectomy may not be long enough to cause significant estrogen loss for the heart with effects on fibrosis. Extending the length of time post-ovariectomy before beginning isoproterenol treatment will be important to study the effects of estrogen loss on heart failure over time. We will analyze cardiac apoptosis and hypertrophy as further indicators of cardiac remodeling following ovariectomy and chronic beta-adrenergic stimulation. The results of this work can provide new information on heart disease in post menopausal women and alternative drug targets.

Cardiovascular disease (CVD) is the leading cause of death in the United States. A common feature in most cardiac pathologies is the dysregulation of beta-adrenergic receptors (β-AR) and changes in the extracellular matrix (ECM). The ECM maintains strength and normal organization of cardiac tissue, while fibrosis (connective tissue scarring) is necessary for repair of damaged cardiac tissue. However, the dysregulation of the ECM leads to a number of cardiac disease pathologies. Osteopontin (OPN) is a protein with diverse biological functions in regulating the ECM such as bone resorption and calcification, wound healing, cell adhesion, cell survival, and apoptosis. OPN is expressed at low levels in the heart but increases with injury by promoting collagen synthesis, cardiac fibroblast growth, and adhesions to ECM proteins. Furthermore, as the heart ages, increases in ECM reorganization leads to cardiac damage and failure. Several studies have examined the role of OPN in the heart, but to date, no studies exist on the role of OPN in response to β-AR signaling and cardiac remodeling or the role that aging plays in this response. The goal of this study was to examine the effects of OPN on cardiac ECM remodeling following chronic beta-adrenergic stimulation. We proposed that OPN expression increases cardiac remodeling and dysfunction following ISO treatment in the aging heart evidenced by increased fibrosis. For this study, young (4 months) and middle age (14 months) mice with (WT) and without (KO) the OPN gene were treated with isoproterenol (ISO) for 28 days. Echocardiography was used to assess cardiac function. Mice were euthanized, and the hearts were analyzed for fibrosis using Masson’s Trichrome Staining. Results showed ISO increased fibrosis in the WT-ISO, but not KO-ISO compared to the respective controls (SHAM, no ISO treatment) for the middle age mice (p≤0.05). Furthermore, the aged WT-ISO group exhibited a 3-fold increase in fibrosis compared to the young WT-ISO group. Results from echocardiography will be analyzed and we expect to see compromised cardiac function in the WT-ISO groups compared to KO-ISO groups. OPN is currently being examined as a potential biomarker in heart failure. The results from this study will provide new insight on changes in the cardiac vasculature in the aging heart following injury and the role OPN plays in this process.