Brucellosis is a chronic, recurring disease caused by the bacterium Brucella abortus, along with other species of the genus Brucella, and is one of the most common bacterial zoonosis worldwide. The bacteria preferentially infect and reside within host macrophages, causing an undulant fever, joint pain, and other flu-like symptoms, in addition to more severe problems like hepatosplenomegaly and endocarditis. Brucella infection is most often acquired via inhalation through the respiratory route, or via consumption of unpasteurized dairy products. Although ingestion is a major route of infection, the transcriptional response of B. abortus during oral infection remains poorly characterized. In this project, RNA sequencing was used to discover genes with the greatest transcriptional changes in B. abortus subjected to deoxycholate, a host bile acid encountered by bacteria during oral infection. Gene deletion strains of B. abortus were then created and tested for susceptibility to pH and bile acid stress, along with their ability to invade and replicate within macrophages. If the genes of interest are important for the oral infection process, B. abortus strains lacking these genes will likely be more susceptible to pH and deoxycholate stress and may exhibit attenuation in the macrophage infection model. Determination of genes important for the oral infection process would further elucidate the molecular mechanisms by which B. abortus invades the host, and could help lead to future treatments and novel therapeutics. / Master of Science / Brucellosis, caused by the bacterium Brucella abortus, is a zoonotic disease, meaning that humans can acquire the illness from animals. Once infected, sufferers of brucellosis experience a chronic, recurring fever that repeatedly rises and falls. Additionally, the disease can cause enlargement of the spleen and liver, and can sometimes cause inflammation of the valves within the heart. Although B. abortus can infect a host through many routes of entry (inhalation, accidental injection, etc), patients are often infected through the consumption of contaminated, unpasteurized dairy products. The genes utilized by B. abortus during oral infection have not been well characterized, so it is not well known what mechanisms B. abortus uses to survive the pH and bile acid stresses it faces in the host stomach and intestines. This research examines which genes are increasingly or decreasingly utilized by B. abortus when it is subjected to deoxycholate—a bile acid stress used to simulate the host small intestine. Genes that exhibited the largest change in expression upon deoxycholate exposure were then chosen for further study: new strains of B. abortus lacking these genes of interest were created to determine if the gene deletion decreased the bacteria's ability to survive acid and deoxycholate stress, along with its ability to infect host macrophages, a type of white blood cell. If deletion of these genes weakens the ability of B. abortus to survive and infect, then these genes likely have a role during the oral infection process. By further elucidating which genes are used by B. abortus to survive host defenses and infect via the oral route, one could then create new medicines that are more effective at inhibiting the mechanisms needed by B. abortus for successful infection and persistence within the host.

Shigella are bacteria that are responsible for millions of infections and hundreds of thousands of deaths every year. The emergence of antibiotic resistant Shigella adds to the potentially devastating effect that these bacteria can have on human health. Shigella flexneri utilize specialized molecular machinery called the Type III secretion system to infect humans and cause disease. Research of this machinery promises to provide the knowledge, tools, and direction for the development of new avenues to combat shigellosis. This dissertation presents studies of two Shigella proteins, invasion plasmid antigens C and D (IpaC and IpaD). These proteins are part of a syringe and needle like protein structure that allows Shigella to secrete proteins directly into the host that hijack host cells to benefit support Shigella infections. IpaC and IpaD are part of a protein tip complex that is directly involved in these Shigella-host (e.g. human) interactions. We have advanced the biochemical tools for the in vitro study of IpaC by utilizing a new way to isolate it. This purification methodology allowed us to look at one of IpaC’s main roles, to interact with the host cell membranes. We examined IpaC’s role and tried to identify the parts of IpaC responsible for some specific interactions. We found that the parts of IpaC we believed were responsible were not but that the composition of the membrane IpaC is interacting with is more important than we previously believed. Finally, we examined a rare part of IpaD structure to determine its role. We determined that this rare feature is required for IpaD to sense Shigella’s host environment and prepare the bacteria to infect, making a promising target for anti-infective treatments against Shigella infections. Our findings advance the understanding of key molecular mechanisms that are required for Shigella virulence. We expect that our findings will aid future researchers as the pursuit for new treatments for shigellosis continues.

Transparent electrodes made from metal oxides suffer from poor flexibility and durability. Highly transparent and electrically conductive thin films based on carbon nanotubes (CNTs) were assembled as a potential indium tin oxide (ITO) replacement using layer-by-layer (LbL) assembly. The ultimate objective of this dissertation work is to produce CNT-based assemblies with sheet resistance below 100 Omega/sq and visible light transmission greater than 85 percent. The alternate deposition of positively charged poly(diallyldimethylammonium chloride) [PDDA] and CNTs stabilized with negatively charged deoxycholate (DOC) exhibit linear film growth and thin film properties can be precisely tuned. Ellipsometry, quartz crystal microbalance, and UV-vis were used to measure the growth of these films as a function of PDDA-CNT bilayers deposited, while TEM, SEM, and AFM were used to visualize the nanostructure of these films. Following a literature review describing potential ITO substitutes and LbL technology, the influence of CNT type on optoelectronic performance of LbL assemblies is described. Three different types of nanotubes were investigated: (1) multiwalled carbon nanotubes (MWNTs), (2) few-walled carbon nanotubes (FWNT), and (3) purified single-walled carbon nanotubes (SWNTs). SWNTs produced the most transparent (>85 percent visible light transmittance) and electrically conductive (148 S/cm, 1.62 kOmega/sq) 20-bilayer films with a 41.6 nm thickness, while MWNT-based films are much thicker and more opaque. A 20-bilayer PDDA/(MWNT DOC) film is approximately 103 nm thick, with a conductivity of 36 S/cm and a transmittance of 30 percent. In an effort to improve both transparency and electrical conductivity, heat and acid treatments were studied. Heating films to 300 degree C reduced sheet resistance to 701 Omega/sq (618 S/cm conductivity, 38.4 nm thickness), with no change in transparency, owing to the removal of insulating component in the film. Despite improving conductivity, heating is not compatible with most plastic substrates, so acid doping was investigated as an alternate means to enhance properties. Exposing SWNT-based assemblies to HNO3 vapor reduced sheet resistance of a 10 BL film to 227 Omega/sq. Replacing SWNTs with double walled carbon nanotubes (DWNTs) provided further reduction in sheet resistance due to the greater metallic of DWNT. A 5 BL DWNT film exhibited the lowest 104 Omega/sq sheet resistance (4200 S/cm conductivity, 22.9 nm thickness) with 84 percent transmittance after nitric acid treatment. DWNT-based assemblies maintained their low sheet resistance after repeated bending and also showed electrochemical stability relative to ITO. This work demonstrates the excellent optoelectronic performance, mechanical flexibility, and electrochemical stability of CNT-based assemblies, which are potentially useful as flexible transparent electrodes for a variety of flexible electronics.

The inner surface of the stomach is lined by a mucous membrane known as the gastric mucosa. The integrity of the gastric mucosa is critical for protecting the stomach from the low pH and proteolytic environment within the lumen. Both clinically and experimentally, exposure of gastric mucosal cells to bile salts is known to cause injury. Bile salts present in duodenogastric reflux are thought to play a significant role in gastric ulcer formation and alkaline gastritis. In vitro, studies using physiologic concentrations of the secondary bile salt, deoxycholic acid, indicate that bile salts can induce apoptosis in cultured human gastric epithelial cells in a caspase-dependent manner. Therefore, there is interest in developing approaches that can protect gastric cells from bile salt-induced damage. It has been shown that induction of the stress protein, heme oxygenase-1, can provide protection against apoptosis. Therefore, the objective of this study was to test the hypotheses that heme oxygenase-1 expression could be induced in human gastric epithelial cells and that furthermore; this would provide protection from deoxycholic acid-induced apoptosis. Heme oxygenase-1 expression was induced pharmacologically or by introduction of a plasmid expressing heme oxygenase-1 into the gastric epithelial cell line, AGS. Induction of heme oxygenase-1 prior to challenge with deoxycholate reduced apoptotic-associated morphological changes, DNA fragmentation, the appearance of oligonucleosomes in the cytoplasm, and activation of caspase-3 and caspase-9. Based on these results, it was concluded that expression of heme oxygenase-1, or the introduction of its products, can provide protection to human gastric epithelial cells against sodium deoxycholic acid induced-apoptosis.

La leishmaniose viscérale est une maladie tropicale négligée et létale en l’absence de traitement. L’Amphotéricine B (AmB) est une molécule efficace mais sa forme conventionnelle, Fungizone®, conduit à une toxicité limitant les doses tandis que les formulations lipidiques moins toxiques telles que l’Ambisome® sont très coûteuses. Ainsi, le besoin de nouvelles formes thérapeutiques à la fois non toxiques et peu coûteuses subsiste actuellement. Dans ce travail, nous avons étudié deux solutions possibles : la Fungizone® chauffée (H-AmB) et une microémulsion chargée en AmB (MEAmB). En ce qui concerne la microémulsion, une forme lyophilisée est souhaitable afin de s’affranchir des risques d’hydrolyse et de contamination microbienne. Les objectifs de la thèse étaient de développer les deux nouvelles formes, d’évaluer la toxicité et l’efficacité de H-AmB et MEAmB contre Leishmania donovani (souche LV9) in-vitro et in-vivo et également d’optimiser la lyophilisation de la microémulsion.La microémulsion MEAmB est composée de gouttelettes sphériques dont le diamètre moyen est proche de 35 nm et a montré un comportement rhéologique de type newtonien. L’analyse spectroscopique de H-AmB a révélé la formation de super-agrégats qui sont moins toxiques que d’autres états d’agrégation. La MeAmB ainsi que l’H-AmB ont montré une activité antiparasitaire équivalente à celle d’AmBisome® sur les formes axénique et intramacrophagique de L. donovani. L’indice de sélectivité pour ces deux formulations est élevé, en contraste avec celui de la Fungizone® native. De plus, à la différence d’AmBisome®, elles ont montré une activité importante sur des souches résistantes à l’AmB. L’activité anti-leishmanienne in-vivo des nouvelles formulations est comparable aux formulations de référence. De même, aucune différence significative des marqueurs de toxicité rénale et hépatique n’a pu être observée. Ainsi, l’H-AmB et la MEAmB pourraient être considérées comme des traitements alternatifs de la leishmaniose viscérale, avec l’avantage d’être moins onéreuses à produire que l’Ambisome®.Afin d’optimiser le procédé de lyophilisation de la MEAmB, un plan d’expérience a été mis en œuvre. Ainsi, la taille des gouttelettes est minimisée par l’utilisation de 5% de maltose comme cryoprotectant, avec une température de congélation de -80°C et un temps total de lyophilisation de 24h. Par ailleurs, aucune modification significative de la teneur en AmB n’a été observée après la lyophilisation. Ainsi, la MEAmB lyophilisée est stable et pourrait éviter la dégradation due à la présence d’eau. / Visceral leishmaniasis is a neglected tropical disease that can be fatal if left untreated. Amphotericin B (AmB) is effective in the treatment of this disease, but the conventional formulation, Fungizone® has dose-limiting toxicity while the less toxic lipid-based forms such as Ambisome® are expensive. Therefore, the need for new therapeutic systems remains. In this respect, the heating of the Fungizone® formulation (H-AmB), and the development of a microemulsion (ME) containing AmB (MEAmB) are possible solutions. In addition, it is desirable to remove water from microemulsion systems in order to reduce instability due to microbiological contamination and hydrolysis. Therefore, the objective of this work was to develop and to evaluate the activity and toxicity in vitro and in vivo of H-AmB and MEAmB against Leishmania donovani (strain LV9) and, furthermore, to optimize a lyophilized microemulsion system containing AmB. Rheological, size and morphology studies showed that MEAmB presented average droplet sizes of 35 nm, a Newtonian behavior and spherical morphology. Spectroscopic characterization of H-AmB showed the formation of superaggregates, which are less toxic than the other states of aggregation. In-vitro evaluation on both the axenic and intramacrophagic amastigote forms showed that H-AmB and MEAmB showed similar activity to Ambisome®. A high selectivity index of H-AmB and MEAmB was observed compared with unheated Fungizone®. Furthermore, both new formulations showed high activity against AmB-resistant strains compared with Ambisome®. In-vivo experiments designed to evaluate their activity and toxicity did not reveal significant differences in activity between the new and reference formulations. There were no significant differences either in indicators of renal and hepatic toxicity. Therefore, both H-AmB and MEAmB can be used as an alternative for the treatment of LV9, presenting an advantage over Ambisome® in their lower costs of production. Therefore, a complete experimental design was performed in order to optimize the lyophilisation of the microemulsion system. It was observed that microemulsions with smaller droplet sizes were obtained using maltose as a cryoprotectant at a concentration of 5%, with freezing at -80 ° C, and a lyophilization process period of 24 h. Furthermore, it was observed that ME containing AmB showed no significant changes in drug content before and after the lyophilization process. Therefore, in its lyophilized form, the ME can remain stable and avoid degradation due to the presence of water.

Chapter 1: Functional and Responsive Supramolecular Gels In this chapter ‘supramolecular gels’ derived from small organic molecules with molecular mass of typically less than 2000 daltons are discussed. Representative examples of various low molecular weight gelators based on their natural availability and also divergent functionalities are mentioned (Scheme 1). Scheme 1 Advances in the recent years have been very rapid in the field of supramolecular chemistry of gels giving rise to ‘Tunable responsive gels’. Control of the gel property in a reversible fashion has been the highlight of responsive gels. A few of the gels which are responsive towards various stimuli such as pH, photoirradiation, cations, anions, neutral species have been discussed. Advances and scope of supramolecular gels in various applications have also been mentioned in detail with respective examples. Utilities of supramolecular gels in synthesis of nanostructures, in biology and medicine, enzyme recognition, catalysis etc are discussed. (Scheme 2). Chapter 2: Charge transfer triggered organogels of bis(bile acid)anthracene conjugates and 2,4.7-trinitrofluorenone. In this chapter the study involves the synthesis of a special class of anthracene based steroidal derivatives. The appending of two amphiphilic bile acid units imparts a unique hydrophobic/ hydrophilic balance on the chromophore. The 2,3-didecyloxyanthracene (DDOA) was reported to be a gelator of various organic solvents but none of the three bile acid derivatives of anthracene synthesized was a gelator on its own. It was also observed that dialkoxy (propyl, heptyl, decyl) derivatives of anthracene formed strong charge-transfer gels in the presence of 2,4,7-trinitrofluorenone (TNF). The addition of electron deficient TNF to the steroidal derivatives of anthracene resulted in the gelation of some specific organic solvents. The driving force behind the gel formation resulted from the charge-transfer (CT) interaction between the electron rich anthracene and electron deficient fluorenone. Figure 1. Chemical structures of 2,3-bis(bile acid)anthracenes and TNF (centre), a scanning electronic microscopy image of xerogels prepared from bis(deoxycholyl)anthracene and TNF (left) and a photograph of the gel of bis(deoxycholyl)anthracene and TNF in n-octanol. Thermochromic property (during sol to gel phase transition), absorption and variable temperature fluorescence measurements supported CT interaction. Thermal stability studies and dynamic rheology experiments confirmed that CT gels were thermally most stable and mechanically stronger with equi-molar amounts of the two components. Stiffness values obtained from rheological experiments also suggested that the gels were viscoelastic solids. Chapter 3(A): Tb(III) sensitization in an organogel matrix: Selective luminescence quenching by an aromatic nitro derivative In this chapter the discovery of metallo organogel formation by mixing methanolic solutions of Tb(OAc)3 and sodium deoxycholate (NaDCh) has been explored. Sensitization of Tb3+ was observed by doping micromolar quantities of 2,3-dihydroxynaphthalene (DHN). Mechanical properties of Tb3+-DCh gels were investigated by rheology at three different ratios of Tb3+ and DCh. It was observed that increasing in the Tb3+ to DCh ratio increased the mechanical property of the gels. Time delayed emission spectra were recorded with increasing concentration of DHN and luminescence increase was noticed in a linear fashion. Importance of gel matrix was demonstrated by measuring the Tb3+ luminescence at fixed concentration (5 mM) with/without DHN in the solution and gel media. Figure 2:: Schematic representation of Tb3+ sensitization by DHN. Photograph (right)) of the Tb3++-DCh (5/15 mM) gels with (a) 50 µM DHHN (b) No DHHN under UVV (365 nm). Sensitization by an electron rich chromophore created interest in us to dope relatively electron deficient compounds into the gel matrix for possible quenching off Tb3+-luminescence. Among the electron deficient analytes screened included 1,5-difluro-2,4-diinitrobenzenne (DFDNB)), 2,4 dinitrophenol (DNPPh), p-nitrobenzaldehydde (p-NB), 2,4,6-trinitrootoluene (TTNT) and 22,4,7¬trinitrofluuorenone (TTNF). Microscopy studies such as AFM, TEMM and SEMM revealed highly entangled fibrous network in the morphology of Tb3+--DCh xerogel. Solid state luminescence experiments suggested that sensiitization was observed in the xerogels and extent of sensitization was comparable to that of the gel state. Xerogel soaking studies inferred the strong adherence of the DHNN to the gel fibres. Chapter 3(B): Anion dependent structural, morphological and mechanical features of Ln(III)-Cholate gels In this chapter the counter anion influence on various aspects of hydrogels has been discussed. It has been reported from our laboratory that mixing of aqueous solutions of sodium cholate (15 mM) and various lanthanide acetates (5 mM) followed by sonication resulted in either transparent or transluscent gels. Unsurprisingly we found that aqueous solutions of lanthanide nitrates and lanthanide chlorides also formed hydrogels upon mixing with sodium cholate (Figure 33). Dried films of Tb3++-cholate and Eu3+-cholate gels prepared from their respective nitrate salts displayed birefringent structures under polarizing optical microscopy (POM). But no significant textures of any type were observed in the case of gels prepared from either chloride or acetate salts. Figure 3:: Photographs of the hydrogels prepared by mixing of aqueous solutions of various salts Tb33+ and Eu3+ with sodium cholate solutions. Scanning electron microscopic images exhibited fibrous structures for all the xerogels in the morphology. Atomic force microscopy and transmission electron microscopy measurements revealed helical morphology for xerogels prepared from nitrate salts where as flat tape-like cross linkage was observed for chloride or acetate based xerogels. Anion effect on mechanical properties was significant in the sense that gels prepared from acetate salts displayed highest mechanical strength followed by nitrate based gels which were stronger than that of chloride based gels. Titration of sodium cholate solution with various lanthanide salt solutions gave the direct evidence of thee pH variation as a function of the anions. Figure 4: TEM images of xerogels prepared from gels of nitrate salts of Tb3+ (left) and Eu3+ (right) Chapter 4: Design, synthesis a nd study of bile acid ‘click’ conjugates of perylene bisimides (PBIs) and naphthalene bisimides (NBIs) In this chapter the synthesis of novel bile acid derivatives of perylene and naphthalene bisimides is discussed. The ‘click’ chemistry procedure was used to link bile acid groups on to the chromophores. Azide derivatives of PBIs and NBIs were prepared inn 3 step methods which were coupled to propargyl esters of bile acids by following standard ‘click chemistry’ protocols to achieve the target molecules (Scheme 3). Scheme 3 The studies conducted mainly focused on Cholic acid (CA) conjugates of PBIs and NBIs. Steady state absorption and emission studies of CA conjugates were performed in 10% MeOH/DCM system. POM and fluorescence images showed red emissive aggregates in case of PBI films. TEM measurements revealed uniform aggregate sizes for both the films of PBI(CA)2 and NBI(CA)2. SEM and AFM (Fig 5) studies exhibited spherical aggregates of diameter around 100-200 nm for PBI(CA)2 films where as aggregates of diameter around 500-700 nm were observed for NBI(CA)2 films. Figure 5: AFM images and their corresponding height profiles of PBI films (left) and NBI films (right)

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.