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...

Chapter 1. General Introduction This chapter gives an introduction to supramolecular organo/hydrogels and the related bile acid chemistry touching upon the gelation properties of the bile acid derivatives. Diverse applications of the supramolecular gels are illustrated with several examples. In the concluding section of this chapter, a brief introduction on the semiconductor nanocrystals is provided. Finally, the content of the thesis is outlined. Chapter 2. Bile Acid Derived Novel Organo/hydrogelators Part 1. Bile Acid Derived Organo/hydrogelators With a Basic Side Chain Cationic analogues of bile acids which showed remarkable gelation properties in water were reported from our laboratory. This led us to investigate the aggregation behaviour of some of the lithocholic and deoxycholic acid derivatives having a basic side-chain. Figure 1. Bile acid based organo/hydrogelators containing a basic side-chain. In this part, an organogelator 1 and a hydrogelator 2 derived from parent bile acids have been described with respect to their gelation properties, morphology, thermal and mechanical stability of the gels. The organo/hydrogels were shown to be responsive to acid-base stimuli as the organogel formed only in the protonated state and the hydrogel formed in the neutral form of the tertiary amines. The xerogel fibres obtained from the organogel were found to be solid-like and stable up to 200 oC as confirmed by variable temperature polarizing optical microscopy. The non-fluorescent organogel was doped with a fluorescent dye (coumarin 153) to design a novel dye-organogel composite material which was investigated with laser scanning confocal fluorescence microscopy showing the dye molecules were uniformly deposited on the organogel fibres. Part 2. Serendipitous Organogelation by Dimeric Bile Acid Esters This section highlights our work on the organogelators based on a number of dimeric esters consisting of different bile acid units. Figure 2. The three different dimeric bile acid esters as organogelators. In this part, three bile acid derived dimeric esters (1, 2 and 3) were shown to possess organogelation properties in aromatic and halogenated aromatic solvents. We studied the morphological features and rheological properties of these organogels. Next, the organogel matrix was exploited to generate and stabilize gold nanoparticles and prepare AuNP/gel hybrid material. Chapter 3. Cholate Hydrogels and Soft Gel-nanoparticle Hybrid Materials Sodium cholate does not form gel in water under any condition as compared to other sodium salts of other bile acids such as sodium deoxycholate and lithocholate which show pH-dependent gelation behaviour. Figure 3. Metal cholate hydrogels derived from sodium cholate and a variety of metal ions. In this chapter, super hydrogelation of sodium cholate induced by a variety of metal ions (Ca2+, Cu2+, Co2+, Zn2+, Cd2+, Hg2+ and Ag+) is highlighted with respect to their morphology and mechanical strength/stability. The calcium cholate supramolecular system showed the presence of helically twisted nanofibres which were utilised in the synthesis of soft hybrid materials containing metal (Au and Ag) and metal sulphide (CdS, ZnS, HgS, etc.) nanoparticles. Chapter 4. Cadmium Deoxycholate and Highly Luminescent CdSe Nanocrystals Bile acid derivatives have very high chemical and thermal stability owing to the presence of a rigid steroidal nucleus. We explored the possibility of utilizing the bile salt derived from Cd as a metal complexes as precursor to high quality nanocrystals (NCs) which can only be accessed at high temperatures (>200 oC). Figure 4. Synthesis of high quality CdSe NCs from cadmium deoxycholate. In this chapter, the synthesis of high quality CdSe nanocrystals is discussed using a novel bile acid based precursor: cadmium salt of 7-deoxycholic acid, which has high thermal stability and can be conveniently used at very high temperatures (>300 oC) required for the synthesis of high quality nanocrystals. Syntheses were done both by ‘injection’ and ‘non-injection’ modes. The as-prepared nanocrystals have high photoluminescence quantum yield, multiple excitons, narrow size-distributions and zinc blende/wurtzite crystalline cores. Appendix. Steroidal Thiols in Design of Novel Quantum dot (QD)/Gel Hybrid Materials Bile acid derived steroidal thiols were reported to be efficient capping agents for silver and gold nanoparticles from our laboratory. So, we wanted to check whether they could stabilize the semiconductor nanocrystals as well. Figure 5. Steroidal thiols as stabilizers of semiconductor quantum dots. In this short report, we describe the efficient capping by bile acid derived thiols of group II-VI semiconductor nanocrystals/quantum dots (QDs) (CdS, CdSe). After synthesizing the thiol capped QDs, we tried to disperse the capped nanoparticles into the gel fibres. The hybrid gels showed the presence of nanoparticles inside the fibres as observed by transmission electron microscopy, although the photoluminescence of the QDs was very low in the gel matrix, which might be due to the inefficient surface passivation of the nanoparticles in the gel.

Diabetes Mellitus has continued to drastically affect the health of the world and many complications can prove fatal. As long as this metabolic disease persist, research discoveries will need to continue to be made so that patient outcomes and healthcare are dramatically enhanced. In recent years, GLP-1 has been the topic of conversation for diabetes research, due to its promising effects in promoting insulin sensitivity. Furthermore, bile acids and their receptors (TGR5 & FXR) have shown promise in their actions in the regulation of GLP-1, and thus glucose homeostasis. Here we have shown the detection and increased expression of TGR5 and GLP-1, and decreased expression of FXR in diabetic mouse intestinal mucosa tissues. We have also shown the detection and increased expression of these receptors in STC-1 cells. More importantly we have linked the connection of increased glucose concentration (hyperglycemia) to increased TGR5 activation to increased GLP-1 release, thus leading to increased insulin sensitivity and altered diabetic outcomes.

Research activities towards the self-assembly of small organic molecules building blocks which lead to form supramolecular gel has increased extensively during the past two decades. The fundamental investigations of the morphological properties and the mechanical properties of these supramolecular gels are crucial for understanding gelation processes. Most supramolecular gelators were discovered by serendipity, but nowadays ratiional design of new gelators has become somewh at feasible. As a consequence, an increasing number of multi stimuli-responsive and functional molecular gels are reported, offering great prospects with myriads of applications includ ing drug delivery and smart materials as shown in scheme 1. Scheme 1 Part 2: Synthesis of semiconductor nanocrystals In the last two decades, the synthetic development of semiconductor col loidal nanocrystals has been extended from the adjustment of their size, shape, and composition of the particles at the molecular level. Such adjustments of nanocrystals at the molecula r level might open different fields of applications in materials and biological sciences. I n this chapter, the concept of the shape contr ol synthesis of colloidal nanocrystals with a narrow size distribution, and the synthesis of composition dependent alloy type mat erials are described (Scheme 2). Scheme 2 Chapter 2: Synthesis of luminescent semiconductor nanocrystals Part 1: Cadmium deoxycholate: a new and efficient precursor for high ly luminescent CdSe nanocrystals This part demonstrates the sy nthesis of Cadmium deoxycholate (CdDCh2), an efficient Cd-precursor for the synthesis of high quality, monodisperse, multi color emittting CdSe Scheme 3 nanocrystals, while maintaining their high photoluminescent quantum efficiency (Scheme 3). The high thermal stability of CdDCh2 (decomposition temperature: 332 °C) was utilized to achieve high injection and growth temperatures (∼300 °C) for the syntheesis of red emitting nanocrystals with a sharp f ull width at half maximum (FWHM) and multiple excitonic absorption features. We believe that CdDCh2 can be useful for the prreparation of other nanomaterials such as CdS, CdTe and CdSe@CdS core-shell QDs. Part 2: Ligand mediated exccited state carrier relaxation dynamics of Cd1-xZnxSe1-ySy NCs derived from bile salts Bile salts of Cadmium and Zinc provide a convenient and inexpensive single step synthetic route for highly photoluminescent and stable semiconductor nanocrystals (NCs). The high thermal stabilities of Cadmium and Zinc deoxycholates (CdDCh2 and ZnDCh2) allowed us to fine-tune the synthesis of the NCs at high temperatures while maintaining the monodispersity, crystallinity and reproducibility (Scheme 4). Organic capping agent induced lattice strain affects the excited Scheme 4 state relaxation processes of the NCs. The analysis of photoluminescence decay profiles revealed that the average lifettime decreased with the increasing lattice strain of the NCs. A kinetic stochastic model of photoexcited carrier relaxation dynamics of NCs was employed to estimate the values of the radiative recombination rates, the photoluminescence quenching rates and the non-radiative recombination rates of the NCs. These data showed that the non-radiative relaxation rates and the numbeer of surface trap states increased with the incrreasing lattice strain of the NCs. Such types of NCs can have great potential in nonlinear optics, photocatalysis and solar cells. Chapter 3: Synthesis of organic-inorganic hybrid materials Part 1: Hierarchical self-assembly of photoluminescent CdS nanoparticles into bile acid derived organogel: morphological and photophysical properties In this part a strategy towards integrating photoluminescent semiconductor nanoparticles into a bio-surfactant derived organoggel has been reported. A facially amphiphilic bile thiol was used for capping CdS nanoparticless (NPs) which were embedded in a gel derived from a new bile acid organogelator in order to furnish a soft hybrid material (Scheme 5). The presence of CdS NPs in a well-ordered 1D array on the organogel network was confirmed using microscopic Scheme 5 techniques. Photophysical stuudies of the gel–NP hybrid revealed resolved excitation and emission characteristics. Time resolved spectroscopic studies showed that the average lifetime value of the CdS NPs increased in the gel state compared to the sol phase. A kinetic model was utilized to obtain quantitative information about the different decay pathways of the photoexcited NPs in the sol and gel states. Part 2: A novel strategy towards designing a CdSe quantum dot–metallohydrogel composite material This section describes an efficiient method to disperse hydrophobic CdSe quaantum dots (QDs) in an aqueous phase using cetyltriimethylammonium bromide (CTAB) micelles without any surface ligand exchange. The water soluble QDs were then embedded in the 3D self-assembled fibrillar networks (SAFINs) of a hydrogel showing homogeneous dispersibility as eviidenced by Scheme 6 optical and electron microscopico techniques (Scheme 6). The photophyssical studies of the hydrogel–QD from composite are reported for the first time. These composite materials may have potential applications in biology, optoelectronics, sensors, non-linear optics and materials science. Part 3: Photophysical aspectts of self-assembled CdSe QD-organogel hyybrid and its thermoresponsive properties A luminescent hybrid gel was constructed by incorporating CdSe quantuum dots (QDs) in a facially amphiphilic bile acid derived dimeric urea organogel throough non-covalent interaction between ligands capped on QDs surface and hydrophobic pockets of the gel (Scheme 7). The optical transparency of the hybrid materials and the dirrectionalities of the QDs in the gel medium were confirmed by photophysical and microscopic studies. The detailed excited state dynamics of the QD–organogel hybrid has been reported for the first time with the help of lifetime analysis and a kinetic decay model, and thee data revealed that the average lifetime of the QDs decreased in the gel medium. The reversible thermoresponsive behavior of the QD doped organogel was investigated by steady-state fluorescence spectroscopy. W e believe that the results obtained herein provides a route to develop a thermoresponsive system for practical application, especially because of the spatial assembly between soft organic scaffolds and colloidal QDs. Scheme 7 Part 4: In-situ formation of luminescent CdSe QDs in a metallohydrogel: a strategy towards synthesis, isolation, storage and re-dispersion of the QDs A one step, in-situ, room temperature synthesis of yellow luminesce nt CdSe QD was achieved in a metallohydrog el derived from a facially amphiphilic bile salt, resulting in a QD-gel hybrid (Scheme 8). T he ordered self-assembly and homogeneous distribution of the CdSe QDs in the hydrogel network was observed from optical and electro n micrographs. The different excited state behav iors of the hybrid were revealed for the fir st time using time resolved spectroscopy. Ad ditionally, we described the successful isolation of the photoluminescent CdSe QDs from the gel followed by their re-dispersion in an organic solvent using suitable capping ligands. Scheme 8 Chapter 4: Facially a mphiphilic bile acid derived meta llohydrogel: an efficient template for th e enantioselective Diels-Alder reactio n An enantioselective Diels-Ald er reaction mediated by a facially amphiphilic bile acid derived metallogel scaffold has been a chieved (Scheme 9). Different hydrophobic domains present in Scheme 9 the gel appear to facilitate the enantioselective reaction. Various spectro scopic and electron microscopic techniques were employed to understand the possible reasons for the stereoselectivity in the gel. Subsequently, different counter anion s dependent rate accelerations and induced enantioselectivity in the ZnCh2 gel were studied in detail. These preliminary results of the non-covalent based supramolecular heterogeneous catalysis offer new possibilities for using metallogels as nanoreactors for different stereoselective reactions.

Bile acid-based cation sensors involving through space photo-induced electron transfer (PET) processes have been synthesized. In this approach, appropriate known fluorophores and aza crown ether receptor units were attached on a suitable bile acid scaffold. A through space photo induced electron transfer from N-atom of the aza 18-crown-6 to the excited pyrene was responsible for quenching of the pyrene fluorescence. A fluorescence enhancement was observed with the addition of K+ due to the inhibition of fluorescence quenching by PET mechanism. In order check the relationship between the sensitivity and the molecular structure of the sensors, four different molecules with different geometries were synthesized. The changes in the fluorescence spectra for different sensors were recorded in MeOH. The binding constants calculated by curve fitting showed that while the binding constants did not significantly vary, the sensitivities were different depending on the structure of the sensors. The modular nature of the sensor design was verified by changing the receptor module from aza-18-crown-6 to aza-15-crown-5, keeping other parts of the sensor same, to prepare a sodium selective sensor using the same principle. Fluorescence titration in MeOH confirmed the Na+ selective sensing in the presence of K+. The modular design concept was further extended by replacing the fluorophore pyrene to a coumarin derivative. Coumarin sensors showed a behavior similar to that of the pyrene sensors. In order to check the possibility of sensing metal ions in water, non ionic surfactant, Triton X-100 was chosen to dissolve the sensor in water. Fluorescence titration of the sensors showed a desired selective fluorescence enhancement with the particular metal ions. Merrifield resin and water swellable Tentagel® was used to immobilize the sensor to fabricate reusable sensor beads for detecting the metal ions in non polar solvent and water respectively. Fluorescence enhancements of the sensor beads with the metal ions confirmed the process in the immobilized solid state. K+ and Na+ selective sensor beads successfully demonstrated the fluorescence enhancement with the respective cations. This general strategy can be extended to fabricate other sensors for practical uses.

Bile acids in the small intestines of animals serve to breakdown fats and fatsoluble vitamins. Most of the bile acids are reabsorbed into the enterohepatic circulation, but approximately five percent of these bile acids pass into the large intestine. These bile acids are swiftly deconjugated by the bacterial population, and then subjected to further intestinal bacterial chemical modifications. The most significant of these modifications are 7α-dehydroxylations which form secondary bile acids (deoxycholate and lithocholate). Much research has illuminated the 7α-dehydroxylation pathway: of particular interest is the bile acid inducible operon, for which Clostridium scindens VPI 12708 serves as the model organism. There is a lack of knowledge on how this operon is regulated, so the goal of this project was to create a genetic construct consisting of upstream regulatory elements, a bile acid inducible promoter, and a ϐ- glucuronidase reporter. Cloning strategies utilized PCR to amplify desired DNA fragments and sewing methodology to combine DNA fragments. DNA fragments were ligated into plasmids and transformed into competent E. coli. Transformants were evaluated for the desired reporter gene fusion by blue/white screening, additional PCR, and/or restriction digestion. The bile acid inducible promoter was successfully amplified, and the upstream sequence and uidA (ϐ- glucuronidase) reporter was demonstrated. However, no E. colitransformants were demonstrated to possess the baiP-uidA gene fusion. The project strategy is plausible and data regarding the bile acid inducible promoter are greatly needed.

Chapter 1. An Overview of Bile Acid Science This chapter deals with an overview of bile acid science (cholanology) compiling elevant literature review, covering bile acid chemistry, biosynthesis, bile salt evolution, physiology and medicinal values. Figure 1. (a) Digestive system; (b) enterohepatic circulation and (c) cholic acid Bile acids are the end products of cholesterol metabolism, secreted in the liver and stored in the gall bladder (Figure 1). They are normally conjugated with glycine (75%) or taurine (25%). Because of their facially amphiphilic nature, bile salts tend to form micellar aggregates in aqueous solution. They have remarkable ability to transform lamellar array of lipids into mixed micelles. All primary bile acids seem to have three features in common: (1) They are major products of cholesterol metabolism; (ii) they are secreted into the bile largely in a conjugated form and (iii) the conjugates are membrane impermeable, water soluble, amphiphilic molecules. Recent advances in molecular biology have greatly accelerated the knowledge relating to the significance of bile salts in a number of physiological functions. The new role of bile salts as pheromones and ligands for nuclear hormone receptors has been discussed. Chapter 2. Pythocholic Acid: A Major Constituent of Python’s Bile and 16α-Hydroxycholic Acid: A Minor Constituent of Avian’s Bile The first chemical synthesis of pythocholic acid (major constituent of python’s bile) and 16α-Hydroxycholic acid (a minor constituent of avian’s bile) were accomplished starting from cholic acid with overall yields of 5.0% and 5.5%, respectively. A biomimetic remote functionalization strategy was utilized as a key step to achieve the selective chlorination at C-17. Dehydrochlorination of 17-chlorosteroid resulted in the Δ16 olefin. Hydroboration-oxidation of the Δ16 olefin followed by the selective oxidation of the pentol under TEMPO mediated oxidation resulted in an ε-lactone. Hydrolysis of the lactone using 5% KOH in MeOH furnished the 16α-Hydroxycholic acid. On the other hand, selective oxidation of 7-OH of the lactone was achieved using N-bromosuccinimide in acetone/H2O to yield the 7-keto lactone. The ketolactone when subjected to the Huang-Minlon modification of the Wolf-Kishner reduction furnished pythocholic acid. Pythocholic acid showed unusual aggregation behavior and high cholesterol solubilization ability, compared to other trihydroxy bile acids. Chapter 3. 16-Epi-pythocholic acid: An Unnatural Analogue of Pythocholic Acid The synthesis of 16-epi-pythocholic acid, an unnatural analogue of pythocholic acid, was accomplished starting from cholic acid. Cholic acid was converted to Δ8-14) olefin using ZnCl2 in refluxing acetone. Methylation followed by isomerization in CHCl3 by passing dry. HCl at -78 oC resulted in the Δ14 olefin. Allylic oxidation using Na2Cr2O7.2H2O in the presence of N-hydroxysuccinimide in acetone furnished the enone. Selective reduction of the olefin using Pd/C-H2 resulted in 16-Epi-pythocholic acid the 16-keto steroid. NaBH4 reduction of this ketone in MeOH/THF (2:1 v/v) followed by hydrolysis produced the 16-OH bile acid. Analysis of spectral data confirmed that it is a 16β-epimer of pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid). Critical micellar concentration and cholesterol solubilization properties were studied. Chapter 4. Low Molecular Mass Organogelators Derived from Simple Esters of Cholic Acid This chapter begins with an introduction to low molecular mass organogelators and highlights their applications. Serendipitous gelation of a number of organic solvents by allyl cholate and the design of related simple esters of cholic acid are discussed. A series of simple and easily accessible esters of bile acids were prepared. Ethyl cholate and propyl cholate were found to immobilize a variety of organic solvents like benzene, toluene, xylene, mesitylene, 1,2-dichlorbenzene (DCB) and chlorobenzene (Figure 2). The morphology of the xerogels was well characterized using SEM, AFM and polarizing optical microscopy (POM) techniques, Which revealed the presence of highly entangled self-assembled 3D-fibrillar network (SAFINs). The fiber diameter was found to vary between 300-500 nm. Direct imaging of the collapse of this fibrillar network and direct observation of the evolution of nanofibers was achieved for the first time using variable temperature POM techniques. FT-IR studies, X-ray powder diffraction and variable temperature POM studies revealed the resemblance of SAFINs to the bulk solid. Formation of helical fibrillar network was observed in SEM images and the existence of chiral aggregates was confirmed by induced circular dichroism experiment using achiral Reichardt’s dye as the chromophore. Chapter 5. Ambidextrous Gelators Derived from Spacer Linked Bile Acid Derivatives Based on our observation of simple esters of cholic acid as organogelators a rational design of a series of spacer linked dimers and tripodal derivatives were carried out. Some of these molecules formed highly transparent gels in solvents like haloarenes, anisole, xylene and dibromoalkanes. These molecules also showed rapid gelation in DMF/H2O and DMSO/H2O mixtures in varying proportions of water and the co-solvent. These types of gelators are known as ambidextrous gelators. The xerogels were characterized using SEM, TEM and POM techniques and the presence of highly entangled 3D-fibrillar network (Figure 3) was observed. XRPD showed crystalline nature of bulk solid, whereas the xerogels were shown to lose their crystalline nature. (For figures and structural formula pl see the pdf file.)

<p>U disertaciji je ostvarena sineza amida i oksazolina žučnih kiselina, kao i njihovih alkil i alkilidenskih derivata polazeći od holne kiseline. Ipitano je pona&scaron;anje različitih okso derivata žučnih kiselina u uslovima Grignard-ove i Wittig-ove reakcije. Ispitana je biolo&scaron;ka aktivnost odabranih sintetizovanih jedinjenja</p> / <p>Synhesis of bile acid amide and oxazoline derivatives, and their alkyl and alkylidene derivatives was accomplished starting from cholic acid. Also, chemical behavior of different bile acid oxo derivatives in Grignard and Wittig reaction was investigated. Biological activity&nbsp; of selected synthesized compounds was evaluated.</p>

Les acides biliaires sont des composants naturels du tractus gastro-intestinal. La hausse du taux d’acides biliaires dans l’intestin est associée à une carcinogenèse de l’appareil digestif. L’homéostasie des acides biliaires est maintenue par le contrôle de l’expression des gènes impliqués dans le métabolisme tels que SHP, FGF19 et CYP-7A1 par le récepteur nucléaire FXRα. FXRα agit comme facteur de transcription en réponse à l’interaction directe des acides biliaires. Par contre, plusieurs évidences tendent à démontrer le rôle central de FXRα dans la carcinogenèse hépatique. Les récepteurs de la famille EGFR/ErbB sont des récepteurs tyrosine kinase également activés par les acides biliaires, dont la surexpression est associée au développement de plusieurs cancers. Ainsi, le projet vise à déterminer l’impact de la signalisation des récepteurs EGFR/ErbB sur la réponse transcriptionnelle de FXRα. Nous avons identifié un mécanisme d’inhibition de l’activité transcriptionnelle de FXRα et de l’expression de ses gènes cibles par l’activation des récepteurs EGFR/ErbB par leur ligands HRG et EGF et par l’expression du mutant constitutivement actif ErbB2-V659E dans les cellules hépatiques. Nous avons montré que ce processus dépend de la signalisation par la voie MAPK. On observe également que l’activation de FXRα diminue la prolifération des cellules cancéreuses du foie alors que celle du récepteur ErbB2 augmente cette prolifération. Ainsi, cette étude nous a permis d’établir un nouveau mécanisme de l’impact délétère de la suractivation des récepteurs EGFR/ErbB sur la prolifération des cellules cancéreuses du foie qui implique une inhibition du potentiel transcriptionnel du récepteur aux acides biliaires FXRα. / Bile acids are natural components of the gastrointestinal tract. An increase of bile acid levels in the intestine is associated with the carcinogenesis of the digestive system. Bile acid homeostasis is maintained by the nuclear receptor FXRα which regulates the expression of specific genes involved in metabolism such as SHP, FGF19 and CYP-7A1. In this way, FXRα acts as a transcription factor following bile acids binding, allowing its transcriptional activation. On the other hand, several studies established the central role of FXRα activation in liver carcinogenesis. EGFR/ErbB receptors are a family of tyrosine kinase receptors that can be regulated by bile acids. Overexpression of EGFR/ErbB receptors is associated with several cancers. Thus, the project aims to examine the impact of EGFR/ErbB receptors signaling on FXR transcriptional potential. We identify that EGFR/ErbB activation by their ligands HRG and EGF and by the expression of the constitutively active mutant ErbB2-V659E inhibits FXR transcriptional activity and expression of its target genes in liver cells. We demonstrate that this process is dependent on the MAPK signaling pathway. We also show that FXR activation decreases proliferation of liver cancer cells while activation of ErbB2 increases this cellular response. Thus, this study identifies a new mechanism of the deleterious impact of EGFR/ErbB receptors overactivation on liver cancer cells proliferation, involving the inhibition of the transcriptional potential of the bile acid receptor FXRα.

Grâce à de récentes percées scientifiques, certains médiateurs clés dans divers états pathologiques ont été identifiés et de nouveaux composés thérapeutiques ont été développés pour les inhiber. Bien que très efficaces, ces composés possèdent souvent des propriétés physico-chimiques incompatibles avec celles du corps humain et deviennent, donc, difficiles à formuler. Au cours des dernières décennies, les systèmes de vectorisation de médicaments ont été étudiés comme une solution potentielle promettant une augmentation de la concentration du médicament au site d'action tout en atténuant les problèmes de stabilité et de solubilité. Plus particulièrement, les polymères ont démontré un succès en tant que matière première dans la conception de ces formulations. Cependant, un obstacle majeur à leur développement clinique est le faible niveau d’encapsulation du principe actif. Afin de remédier à cette limitation, les travaux présentés dans cette thèse se sont concentrés sur l'utilisation de copolymères blocs en forme d'étoile et à base d'acide cholique pour faciliter l'encapsulation. Divers principes actifs aux propriétés physico-chimiques variables ont été encapsulés dans nos systèmes polymères, témoignant ainsi de leur grande efficacité et ceci à travers une large gamme de médicaments. Dans un premier temps, les propriétés physico-chimiques de notre système ont été étudiées. Les copolymères bloc sont composés d'un noyau d'acide cholique (CA) sur lequel le poly (allyl glycidyl éther) (PAGE) et le poly (éthylène glycol) (PEG) sont polymérisées séquentiellement pour donner lieu au CA-(PAGE-b-PEG)4 amphiphiles à quatre branches. De plus, le bloc PAGE a été fonctionnalisé pour porter des groupements amines primaires. Les effets de la longueur du bloc PEG et des groupements amines sur le comportement thermosensible des polymères dans l'eau ont été examinés. Cette thermosensibilité a aussi été étudiée en présence de diverses concentrations de sels. Il a été découvert que l'augmentation de la longueur du PEG augmente la température du point de trouble. De même, la fonctionnalisation des blocs PAGE pour porter des groupements amines a augmenté le point de trouble en l'absence de sel, mais a significativement diminué en présence de sel. Cette observation a été attribuée au « salting-out » des polymères. Dans un second temps, employée comme un médicament hydrophobe modèle, la doxorubicine (Dox) a été encapsulée à l’aide de nos copolymères blocs CA-(PAGE-b-PEG)4. Dans ce cas, les interactions polymère-médicament régissant l’encapsulation de la Dox ont été étudiées. Plus précisément, les interactions hydrophobes et électrostatiques ont été comparées pour leur influence sur la charge de médicament à l'intérieur des copolymères blocs. Une charge élevée de Dox a été obtenue à l’aide des interactions électrostatiques par rapport aux interactions hydrophobes avec ou sans la présence d'acide oléique comme co-tensioactif. De plus, les interactions électrostatiques conféraient au système de relargage une réactivité au pH permettant ainsi un relargage de la Dox en présence d’un pH acide. Les copolymères blocs ont présenté une bonne biocompatibilité lors d’essai in vitro. Les nouveaux copolymères blocs en étoile et à base d'acide cholique ont montré un grand potentiel en tant que vecteurs de relargage de médicaments pour l’encapsulation de la Dox. Pour démontrer l’étendue de l’application de notre système, des petits acides ribonucléiques interférant (pARNi) ont été encapsulés à l’aide des copolymères blocs CA-(PAGE-b-PEG)4 où le PAGE a été fonctionnalisé pour porter des groupements amines. Les pARNi sont des composés thérapeutiques hydrophiles chargés négativement et nécessitant une méthodologie d’encapsulation différente de celle utilisée pour la Dox. Les groupements allyles du bloc PAGE ont été fonctionnalisés pour porter des amines primaires ou tertiaires. Également, l'acide folique a été greffé sur l'extrémité de la chaîne PEG pour augmenter l'absorption cellulaire. Les (CA-PAGE-b-PEG)4 fonctionnalisés avec des amines primaires ou tertiaires ont présenté une forte complexation des pARNi. Des agrégats micellaires uniformes ont ainsi été obtenus. De plus, des lipides ont été ajoutés comme co-tensioactifs pour aider à stabiliser les nanoparticules dans les milieux de culture cellulaire. Ces systèmes micellaires mixtes avaient une charge élevée de pARNi et une absorption cellulaire améliorée avec une augmentation concomitante de la transfection des pARNi dans des cellules modèles de HeLa et HeLa-GFP, respectivement. Les résultats présentés dans cette thèse témoignent du grand potentiel de l'utilisation de copolymères blocs en forme d'étoile et à base d'acide cholique dans la conception de systèmes de vectorisation de médicaments. Ces résultats offrent des conclusions pertinentes sur les différents paramètres clés contrôlant l’efficacité des systèmes de vectorisation des médicaments à base de polymères pouvant être traduits dans d'autres systèmes. Les stratégies développées ici aideront grandement au développement des systèmes de vectorisation de médicaments et accéléreront potentiellement leur évolution vers la clinique. / Recent scientific breakthroughs have fostered the identification of key mediators of various diseased states while permitting the development of novel therapeutic compounds to address them. Although very potent, these compounds often possess physico-chemical properties that are incompatible with those of the human body and are becoming increasingly difficult to formulate. In the recent decades, drug delivery systems have been studied as a potential solution in the formulation of these therapeutic compounds promising improved accumulation at the site of action while mitigating issues of stability and solubility. Most notably, polymers have shown tremendous success as starting material in the design of these drug formulations. However, one major hurdle curtailing their clinical translatability is their low drug loading levels. In an effort to address this limitation, the work presented in this thesis focused on the use of cholic acid-based star-shaped block copolymers for the encapsulation of active pharmaceutical ingredients with varying physico-chemical properties thereby demonstrating their successful application to a broad range of compounds. First, the physico-chemical properties of our proposed system were studied. The block copolymers are composed of a cholic acid (CA) core onto which poly(allyl glycidyl ether) (PAGE) and poly(ethylene glycol) (PEG) are polymerized sequentially to afford an amphiphilic CA-(PAGE-b-PEG)4 with four branches. The PAGE block was further functionalized to bear pendant amine groups. The effects of PEG length and of the amine groups on the thermoresponsive behavior of the polymers in water at various salt concentrations were examined. It was discovered that increasing the length of PEG increases the cloud point temperature. Similarly, functionalizing the PAGE blocks to bear pendant amine groups increased the cloud point in the absence of salt, but significantly decreased the cloud point in the presence of salt. This observation was attributed to the salting-out of the polymers. Acting as a model hydrophobic drug, doxorubicin (Dox) was first encapsulated using our proposed CA-(PAGE-b-PEG)4 block copolymers. In this case, the polymer-drug interactions driving the loading of Dox was studied. Specifically, hydrophobic and electrostatic interactions were compared for their influence on the drug loading inside the block copolymers. A high loading of Dox was achieved vis electrostatic interactions compared to hydrophobic interactions with or without the presence of oleic acid as a cosurfactant. Also, the electrostatic interactions conferred a pH responsiveness to the system where the Dox remained encapsulated at physiological pH but was released in acidic pH. The block copolymers displayed good biocompatibility in vitro. The new functionalized star block copolymers based on cholic acid showed great potential as drug delivery carriers for the loading of Dox. To demonstrate the widespread application of our proposed system, small interfering RNA (siRNA) was loading using the CA-(PAGE-b-PEG)4 block copolymers where PAGE was functionalized with amine. siRNA is a hydrophilic, negatively charged therapeutic compound necessitating a different loading methodology than that used for Dox. The allyl groups of PAGE were functionalized to bear primary or tertiary amines and folic acid was grafted onto the PEG chain end to increase cell uptake. (CA-PAGE-b-PEG)4 functionalized with either primary or tertiary amines show high siRNA complexation. Uniform micellar aggregates were obtained. Lipids were added as co-surfactants to help stabilize the nanoparticles in the cell culture media. The mixed micelles had high siRNA loading and improved cell uptake with a concomitant increase in siRNA transfection in HeLa and HeLa-GFP model cells, respectively. The results presented in this thesis, demonstrate the feasibility of using cholic acid-based star-shaped block copolymers in the design of drug delivery systems and offers insights into key parameters controlling their efficacy which can be translated to other polymer-based systems. The strategies developed herein will greatly aid in the development of drug delivery systems and potentially accelerate their progress into the clinic.