L'alcool est une des principales causes de maladie du foie en Europe avec peu d'options thérapeutiques. Parmi les consommateurs d'alcool, seul certains patients vont évoluer vers des formes sévères d'atteinte hépatique et le microbiote intestinal a été identifié comme cofacteur de cette susceptibilité individuelle. Il interagit avec le foie notamment via la production de métabolites bactériens tels que les acides biliaires secondaires.L'objectif de ce travail a été dans un premier temps d'analyser le microbiote intestinal, au sein d’une cohorte de patients alcooliques à différents stades de la maladie alcoolique du foie, et d’étudier sa relation avec les acides biliaires. Dans un deuxième temps, ce projet avait pour but d’étudier s’il était possible d'améliorer l'atteinte hépatique liée à l'alcool, en modulant le microbiote intestinal, dans un modèle animal de maladie alcoolique du foie.Les patients avec une atteinte hépatique sévère liée à l'alcool ont un profil d’acides biliaires plus hydrophobe, et donc plus toxique, associé à une dysbiose et à des modifications des fonctions bactériennes. Ces modifications des fonctions bactériennes participent à la gravité de la maladie. La modulation du microbiote par la pectine, un prébiotique, permet de prévenir mais également de faire régresser les lésions hépatiques chez la souris. Les effets protecteurs de la pectine sont corrélés avec le métabolisme du tryptophane.Ce travail de thèse montre que le microbiote intestinal et ses métabolites sont une cible thérapeutique potentielle dans la maladie alcoolique du foie. Il ouvre la porte à la réalisation d'essais cliniques chez l'homme dans lesquels nous pourrions limiter la progression des lésions hépatiques liées à l'alcool en contrôlant le microbiote intestinal grâce à l’utilisation de la pectine ou du métabolisme du tryptophane. / Alcohol is one of the main causes of alcoholic liver disease in Europe with few therapeutic options. Among alcohol consumers, only a part of these patients will develop severe liver lesions. This individual susceptibility is driven by intestinal microbiota. Intestinal microbiota interacts with the liver through the production of bacterial metabolites including the secondary bile acids.The aim of my project, was firstly to study the relationship between the intestinal microbiota and the bile acids composition depending on the severity of alcoholic liver disease in a cohort of alcoholic patients. Secondly, I assessed the improvement of alcohol induced liver lesions by changing the intestinal microbiota in a mouse model of alcoholic liver disease.Patients with a severe form of alcoholic liver disease display a higher hydrophobic bile acid pool, more toxic, associated with a specific dysbiosis and changes in the bacterial functions. Changing the intestinal microbiota by using pectin, a prebiotic, prevents and reverts alcohol induced liver injury in mice. These protective effects of pectin involve changes in the tryptophan metabolism.In conclusion, these studies highlight that the intestinal microbiota and its metabolites are potential therapeutic targets for alcoholic liver disease. Moreover, pectin as an alimentary product could be proposed in the management of alcoholic liver disease in humans.

The development of checkpoint immunotherapy has been a paradigm shift in the treatment of cancer, leading to dramatic improvement in treatment outcomes across a broad range of tumor types. Nevertheless, our current understanding of the tumor immune microenvironment and mediators of resistance to therapy are limited. The recent development of high-throughput single-cell RNA-Sequencing (scRNA-Seq) technology has opened up an unprecedented window into the transcriptional states of distinct tumor-infiltrating immune and stromal cells. However, even this technology has its biological limitations, with very high levels of data dropout induced by low total mRNA molecules and capture efficiency. This thesis explores the application of a transcriptional regulatory protein activity inference approach to single-cell data in order to resolve gene dropout and more deeply characterize upstream drivers of cell state within the micro-environment of several distinct tumor types. To this end, algorithms for inference of protein activity, drug sensitivity, and cell-cell interaction have been adapted to scRNA-Seq data, along with an approach for querying enrichment of single-cell-derived population marker gene sets patient-by-patient in larger bulk-RNA-Seq cohorts. By applying these tools systematically, we have identified distinct cellular sub-populations associated with clinical outcome in different tumor types, including a novel population of C1Q+/TREM2+/APOE+ macrophages associated with post-surgical tumor recurrence in clear cell renal carcinoma, a sub-population of fibroblasts associated with improved response to immunotherapy in head and neck squamous cell carcinoma, tumor cell subpopulations with distinct inferred drug sensitivities in cholangiocarcinoma and prostate cancer, as well as tumor-specific regulatory T-cells (Tregs), active as a mechanism of immunotherapy resistance across a range of tumor types. In ongoing clinical trials from both primary and metastatic prostate cancer as well as clear cell renal carcinoma, we are able to assess which of these populations are enriched in non-responders to checkpoint immunotherapy. The proteomic master regulators of each of these single-cell types have direct utility as potential biomarkers for treatment response, but they may also be therapeutically modulated as novel targets for combination immunotherapy, potentially improving treatment response rates and treatment outcomes in future clinical trials. Finally, this thesis also presents a discovery-to-validation platform to accelerate micro-environment-directed drug repurposing in the context of immunotherapy resistance and rapid CRISPRko validation of novel therapeutic targets. This platform has been developed specifically to validate newly identified master regulators of tumor-specific immunosuppressive regulatory T-cells (Tregs), resulting in discovery of low-dose gemcitabine as a tumor-specific Treg-modulating drug synergistic with anti-PD1 checkpoint immunotherapy and TRPS1 as a proteomic master regulator with clinically significant effect on tumor Treg-infiltrating and tumor growth rate. However, the platform itself may be readily extended in future work to prioritize agents against immunosuppressive macrophage and fibroblast populations for clinical development and trials. As we have discovered, different cancers have different populations of cells driving therapy response and resistance. Taken together, the analytical and validation tools presented in this thesis represent an opportunity to tailor future immuno-therapies at the single-cell level to particular tumor types and to individual patients.

<p>Doktorska disertacija razmatra solubilizaciju holesterola pomoću okso derivata žučnih kiselina, upoređuje okso derivate žučnih&nbsp; kiselina&nbsp; sa hidroksi-derivatima istih u solubilizaciji holesterola i ispituje uticaj okso derivata na vijabilnost ćelijske membrane.&nbsp; Takođe, ispituje pKa vrednost različitih okso-derivata žučnih kiselina sa ciljem da se odredi kiselinska konstanta ovih slabih organskih kiselina. Cilj određivanja pKa vrednosti jeste determinacija rastvorljivosti žučnih kiselina. Kada se primenjuju oralno u raznim farmaceutsko-tehnolo&scaron;kim formulacijama, one se primenjuju u obliku soli, koje su rastvorne u vodi. Međutim, u kiseloj sredini želuca može doći do taloženja žučne kiseline i do daljeg sprečavanja delovanja soli žučne kiseline kao solubilizatora određenih farmacutski aktivnih supstanci. Doktorska disertacija ispituje i kritičnu micelarnu koncentraciju me&scaron;ovitih micela natrijumovih soli 3 žučne kiseline (holne, deoksiholne i 7-oksodeoksiholne kiseline) i natrijum-dodecil-sulfata u različitim molskim udelima na temperaturama od 0 do 50&deg;C pomoću spektrofluorifotometra pirenskom metodom.</p> / <p>The PhD thesis discusses solubilization of cholesterol by using oxo derivatives of bile acids and compares it&nbsp; with the hydroxy-derivatives of the same in the solubilization of cholesterol, and examines the impact of the oxo derivatives to the viability of the cell membrane. Also, the pKa value of different&nbsp; tested oxo-derivatives of&nbsp; bile acids is determined. The aim of determining the pKa values is to determine the solubility of bile acids. When administered orally in various pharmaceutical-technological formulations, they are applied in the form of salts, that are soluble in water. However, the acidic medium of the stomach may cause precipitation of a bile salt and further prevent the action of bile acid salts as a solubilizers of specific pharmaceutically active substances. The PhD&nbsp;&nbsp; thesis examined the the critical micelle concentration of the mixed micelles of sodium salt of 3 bile acid (cholic, deoxycholic, and 7-oksodeoksiholne acid) and sodium dodecyl sulphate at various temperatures, the mole fractions ranging from 0 to 50 &deg;C using the method of spectrofluoriphotometry by pirene as a probe molecule.</p>

<p>Surfaktanti imaju značajnu primenu u farmaciji i medicini. Ove supstance se primenjuju u farmakoterapiji, koriste se za solubilizaciju hidrofobnih lekova, a pojedina ispitivanja pokazuju da mogu unaprediti bioraspoloživost određenih aktivnih supstanci. U poslednje vreme se sve vi&scaron;e pažnje posvećuje ispitivanju sme&scaron;a surfaktanata, po&scaron;to je utvrđeno da sistemi dva ili vi&scaron;e surfaktanta često pokazuju poželjnija svojstva od pojedinačnih surfaktanata za aplikaciju u farmaciji i medicini. U ovoj disertaciji su ispitivani binarni sistemi osam anjona žučnih kiselina i dva nejonska surfaktanta (polisorbat 40 i polisorbat 80). Ciljevi su određivanje vrednosti kritičnih micelarnih koncentracija ispitivanih sme&scaron;a surfaktanta, utvrđivanje međudejstva između različitih surfaktanta u njihovim me&scaron;ovitim micelama, kao i ispitivanje uticaja stukture ispitivanih surfaktanata na fizičko-hemijske karakteristike me&scaron;ovitih micela. Rezultati pokazuju da ispitivane sme&scaron;e imaju znatno niže vrednosti kritičnih micelarnih koncentracija od anjona žučnih kiselina. Sme&scaron;e anjona žučnih kiselina i polisorbata 40 imaju manje vrednosti eksperimentalnih kritičnih micelarnih koncentracija, od izračunatih, idealnih, vrednosti, &scaron;to ukazuje na postojanje sinergističkih interakcija u me&scaron;ovitim micelama. Sme&scaron;e anjona žučnih kiselina i polisorbata 80 imaju uglavnom veće vrednosti kritičnih micelarnih koncentracija od idealnih vrednosti, &scaron;to može biti posledica postojanja antagonističkih interakcija između gradivnih jedinica me&scaron;ovitih micela. Vrednosti interakcija, koje dovode do neidealnog pona&scaron;anja sistema surfaktanata, su određene računanjem vrednosti interakcionog parametra, &beta;_1,2, prema regular solution theory. Sistemi anjona žučnih kiselina i polisorbata 40 imaju negativne vrednosti interakcionog parametra, dok sistemi anjona žučnih kiselina i polisorbata 80 imaju uglavnom pozitivne vrednosti interakcionog parametra. Poređenjem fizičko-hemijskih parametara me&scaron;ovitih micela je utvrđeno da postojanje privlačnih međudejstava između hidrofilnih delova različitih surfaktanata najverovatnije potiče od vodoničnih veza koje se formiraju između hidrofilnih grupa anjona žučnih kiselina i polioksietilenskih delova. Pozitivne vrednosti &beta;_1,2 parametra su najverovatnije posledica sterno krute cis dvostruke veze oleinske kiseline u molekulu polisorbata 80, usled čega se lipofilni deo ovog nejonskog surfaktanta teže pakuje u jezgru me&scaron;ovitih micela. Pretpostavlja se da zbog toga dolazi do formiranja dimera anjona žučnih kiselina u me&scaron;ovitim micelama u kojima se javljaju odbojne interakcije između negativno naelektrisanih karboksilatnih grupa.</p> / <p>Surfactants have important application in pharmacy and medicine. These substances are applied in pharmacotherapy, they are used for hydrophobic drug solubilisation, and certain investigations indicate they can improve bioavailability of certain active substances. Lately, investigations of surfactant mixtures have gained a lot of attention, since it was found that systems of two or more surfactants often show more desirable properties than the individual surfactants, for application in pharmacy and medicine. In this dissertation, binary systems of eight bile acid anions and two nonionic surfactants (polysorbate 40 and polysorbate 80) were investigated. The aims were to determine values of critical micelle concentrations of investigated surfactant mixtures, interactions between different surfactants in their mixed micelles, and to investigate the influence of the structure of investigated surfactants on physico-chemical characteristics of mixed micelles. The results indicate that investigated mixtures have significantly lower values of critical micelle concentrations than bile acid anions. Mixtures of bile acid anions and polysorbate 40 have&nbsp; lower values of experimentally obtained critical micelle concentrations than the calculated, ideal, values, indicating the existence of synergistic interactions in mixed micelles. Mixtures of bile acid anions and polysorbate 80 have mainly higher values of critical micelle concentrations than the ideal values, what could be due to the existence of antagonistic interactions between building units of mixed micelles. The values of the interactions, attributing to the nonideal behaviour of the surfactant systems were obtained by calculating the values of the interaction parameter, &beta;1,2 , according to the regular solution theory. Systems of bile acid anions and polysorbate 40 have negative values of the interaction parameter, while systems of bile acid anions and polysorbate 80 have mainly positive values of interaction parameter. By comparing the physico-chemical parameters of mixed micelles, it was determined that existence of attractive interactions between hydrophilic parts of different surfactants most probably originates from the hydrogen bonds, which are formed between hydrophilic groups of bile acid anions and polyoxyethylene parts. Positive values of &beta;1,2 parameter are most probably due to sterically rigid cis double bond of oleic acid in polysorbate 80 molecule, causing the lipophilic tail of this nonionic surfactant to pack less easily in the core of mixed micelles. It is hypothesised that this influences formation of dimers of bile acid anions in mixed micelles, where repulsive interactions emerge between negatively charged carboxylate groups.</p>

Bile acids are the end products of cholesterol metabolism and are important biological surfactants. The curved shape of their chains allows the cyclization of molecules, and the formation of a supramolecular structure. The goal of this thesis was to study the electrochemical and adsorption behavior of selected bile acids: lithocholic, deoxycholic and cholic acids. The measurements were carried out in the medium Brittonův-Robinson buffer:methanol in the ratio 9:1 using cyclic voltammetry and AC voltammetry methods and measuring the dependence of the differential capacitance Cd on the applied potential E. A hanging mercury drop electrode was used as a working electrode. The measurements showed that bile acids are adsorbed on the surface of the electrode and organizing themselves in self assembled monolayers (SAM). In our case we have observed formation of 2D condensed layers as specific form of SAM. Transfer techniques were used to demonstrate bile acid adsorption. A study of the behavior of lithocholic acid as a function of different pH values showed that only at pH 10.0 to 12.0 2D 2D condensation occurs, i. e. that at pH values in the range of 2.0 to 9.0 it is another type of adsorption. On AC voltammograms, there are a maximum of two areas in which peaks occur: the first is around -0.2 V and the...

This master's thesis contains a study of electrochemical processes of selected bile acids possessing 7 hydroxyl group (cholic, chenodeoxycholic and −muricholic). The measurements were performed on boron−doped diamond electrode in the non-aqueous medium of acetonitrile and perchloric acid (water content 0.55 %) by cyclic voltammetry. It is known that the electrochemical activity of 7 bile acids is increased by a dehydration reaction between perchloric acid and the 7 bile acid. The subject of the study was the stability of the voltammetric response of chemically activated bile acids in the region of negative potentials. It was found that the presence of oxygen in the measured solution is an important factor for obtaining the cathodic signal of 7 bile acids. It probably performs a regenerative function; the product of the electrochemical reduction is re-oxidized in its presence, which leads to an increase in the voltammetric response. At the same time, it is important that the direction of the scan in cyclic voltammetry first proceeds to positive values. A potential of +2.0 V (vs. Ag/AgNO3 in acetonitrile) must be reached for the HO● radicals to be formed. It is these radicals that presumably lead to the formation of the product(s) of bile acids electrochemical oxidation that can be subsequently...

Mobile applications are software that extend the functionality of our smartphones by connecting us with friends and a wide range of other services. Android, which is an operating system based on the Linux kernel, leads the market with over 2.6 million applications recorded on their official store. Application developers, due to the ever-growing innovation in smartphones, are compelled to release new ideas on limited budget and time, resulting in the deployment of malicious applications. Although there exists a security mechanism on the Google Play Store to remove these applications, studies have shown that most of the applications on the app store compromise privacy or pose security-related risks. It is therefore essential to investigate the security risk of installing any of these applications on a device. The objectives are to identify methods and techniques for assessing mobile application security, investigate how attributes indicate the harmfulness of applications, and evaluate the performance of K Nearest Neighbors(K-NN) and Random forest machine learning models in assessing the security risk of installing mobile applications based on information available on the application distribution platform. A literature analysis was done to gather information on the different methods and techniques for assessing security in mobile applications and investigations on how different attributes on the application distribution platform indicate the harmfulness of an application. An experiment was also conducted to examine how various machine learning models perform in evaluating the security risk associated with installing applications, based on information on the application distribution platform. Literature analysis presents the various methods and techniques for mobile application security assessment and identifies how mobile application attributes indicate the harmfulness of mobile applications. The experimental results demonstrate the performance of the aforementioned machine learning models in evaluating the security risk of installing mobile applications. In conclusion, Static, dynamic, and grey-box analysis are the methods used to evaluate mobile application security, and machine learning models including K-NN and Random forest are suitable techniques for evaluating mobile application security risk. Attributes such as the permissions, number of installations, and ratings reveal the likelihood and impact of an underlying security threat. The K-NN and Random forest models when compared to evaluate the security risk of installing mobile applications based on information on the application distribution platform showed high performance with little differences.

This thesis focuses on the importance of intermediate products of biosynthetic and catabolic pathway of cholesterol. The aim of the first part of the thesis is mainly to investigate, whether statins (HMG- CoA reductase inhibitors) possess antitumor properties and to compare the differences in antitumor potential of individual statins. The other part of the thesis aims at the utilization of 7α-hydroxycholest-4-en-3-one (C4), a promising marker of cholesterol 7α-monooxygenase (CYP7A1) activity and bile acid malabsorption. We demonstrated antitumor effect of statins on an experimental model of pancreatic cancer. Individual statins, however, differed significantly in their efficacy, depending on their physico-chemical properties. Our data suggests, that the most likely (but not the only) mechanism of antitumor effect of statins is decreased prenylation of signaling proteins, especially Ras protooncogene. We set up a reliable method for measurement of C4, which facilitated our research in CYP7A1 regulation. We demonstrated, that promoter polymorphism -203A>C might affect CYP7A1 activity, that diurnal variability of CYP7A1 activity might be triggered by insulin, and that insulin resistance in patients with non-alcoholic fatty liver disease impedes the feedback regulation of CYP7A1, which may lead to disease...

Presented work is focused on heme metabolism with the main interest in bile pigments. Recent data indicate that bilirubin is not only a waste product of the heme catabolic pathway, but also emphasize its important biological impacts, including possible antiproliferative effects. Until today metabolism of bilirubin has not been completely elucidated, which has prevented detailed evaluation of its potential anticancer action. The aim of this study was to clarify some aspects of heme catabolism with respect for antiproliferative properties of its products. Based on the fact that bilirubin potently affects carcinogenesis of the intestine, we initially investigated not properly known bilirubin metabolism by intestinal bacteria. We studied bilirubin neurotoxic effects in hyperbilirubinemic Gunn rats - its distribution in the brain tissue and its degradation during pathological conditions, such as severe newborn jaundice or Crigler-Najjar syndrome. Possible approaches to improve the treatment of severe unconjugated hyperbilirubinemias, combination of the phototherapy and human albumin administration were also investigated. The main reason of these studies was the fact that mechanisms of neurotoxic effects of bilirubin are predominantly identical with those, by which bilirubin inhibits cancer cells growth....

The gut microbiota plays a major part in maintaining the health of a human host. Countless of crucial functions in the body, including immune responses, cell signaling and energy metabolism to name a few, are conducted by the gut microbiota and its metabolites. Accordingly, it is of interest to gain knowledge on what can alter the gut microbiota, as these alterations by extension can give rise to adverse health effects. In this study, the impact of polychlorinated biphenyl (PCB)- and organochlorine pesticide (OCP) exposure on tricarboxylic acid (TCA) cycle metabolites, short-chain fatty acids (SCFAs) and bile acids, as well as other polar and semi-polar metabolites, which are all related to the gut microbiota, were investigated. An in vitro fermentation of faecal samples exposed to a PCB/OCP mixture was performed, and liquid chromatography-time of flight mass spectrometry (LC-qToF-MS) targeted and non-targeted approaches were applied to the extracts. The results obtained suggested that PCBs and OCPs most likely have an effect on the levels of several features of the gut metabolome with either increased or decreased levels upon exposure. Bile acids and TCA metabolites appear to follow a trend of decreasing levels, while no apparent effects could be seen for the SCFAs. Furthermore, distinct concentrations of the PCB/OCP mixture appear to induce different changes in gut microbiota functioning, which highlights the importance of performing dose-response studies when exploring biological effects of these compounds. The identification of different metabolite profiles during fermentation also allows for the possibility of further investigation of potential biomarkers to assess PCB/OCP exposure.