This thesis describes a study of the aggregational behaviour of egg yolk lecithin (EYL), a natural lecithin, and bile salt mixtures especially with respect to an increase of the ionic strength of the solvent. Mixtures of two amphiphiles with very different spontaneous curvature as EYL lecithin and bile salt form mixed micelles and vesicles in aqueous solution. Their properties have been well-studied under physiological conditions, i.e. 150 mM electrolyte concentration and pH 7- 8, while other conditions are still hardly explored. Upon increasing ionic strength the formed structures and the transitional pathways (micelles, coexistence of micelles and vesicles, and vesicles) change the generated structures completely from those observed under physiological conditions. We quantitatively determined these structures formed in a broad range of electrolyte concentrations with various scattering techniques, x-ray, light and neutron scattering and calorimetry. With calorimetry, phase diagrams in the EYL and bile salt concentration phase plane were determined at various ionic strength ranging from physiological salt concentration to up to 1000 mM. Additionally a new electrochemical approach using functionalised electrodes, i.e. sensitive and selective to bile salt, and thus to control the bile salt concentration in solution (concentrations below the critical micellar concentration (cmc)) was attempted, since bile salt removal or injection drives the micelle-to-vesicle or the vesicle-to-micelle transition, respectively, of the mixed aggregational system of EYL/bile salt. Although this control was not achieved within the framework of this thesis, promising results show directions for future experiments.

Bile salts are supramolecules with amphiphilic properties. Bile salts form aggregates in aqueous solutions. The primary aggregates of bile salts are hydrophobic and the secondary aggregates which form at higher concentrations are relatively hydrophilic. Among bile salts, sodium deoxycholate (NaDC) has been known to form hydrogels at pHs close to the neutral pH and within a certain concentration range. The aim of this work was to provide insight into the properties of a NaDC hydrogel as a supramolecular gel system through three different projects. Pyrene is a hydrophobic polycyclic aromatic compound which was used as a fluorescent probe and the guest for these projects. 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate (DiD) is another fluorescent compound which was used as another guest. The objective of the first project was to understand the mobility of a small guest molecule in NaDC gel in the presence of cucurbit[6]uril (CB[6]) compound as an additive for the gel. Cucurbit[n]urils are macrocyclic compounds with a hydrophobic cavity and two hydrophilic portals. The presence of CB[6] provides another binding site for pyrene in addition to the primary aggregates of the bile salts. The results showed that the mobility of the guest from water and CB[6] to the bile salts network happens when the temperature is raised. The release of the guest back into the water happens when the gel is cooled. The objective of the second project was to investigate the effect of surfaces with different hydrophilicity on the NaDC gel properties. The results of fluorescence correlation spectroscopy (FCS) experiments revealed that either the hydrophilicity of the surface does not affect the NaDC gel network or the FCS is insensitive to the structural changes induced by the hydrophilicity of the surface. These experiments depicted that the aggregates involved in the gel’s network are the same as those formed in the aqueous solutions. Moreover, results of the steady-state and time-resolved fluorescence experiments showed that the bulk gel properties are not affected by the hydrophilicity of the surface. The objective of the last project was to determine the effect of ions on NaDC gel properties. The results showed that cations with different charge density have different effects on the gel formation and properties. The presence of inorganic salts with a monovalent cation leads to the formation of a kinetically favored gel sample within a few hours after sample preparation. The extension of the network occurs overtime and a thermodynamically stable gel forms a couple of days after sample preparation. / Graduate / 2020-10-20

Phospholipid molecules self-assemble to form bilayers that are poorly soluble in an aqueous solvent. Phospholipids may, however, be readily dissolved by mixing with a bile salt or amphiphilic drug surfactant that forms mixed surfactant/phospholipid micelles. Mixed bile salt/phospholipid micelles play an important role in the digestion of fats in the gastrointestinal tract as well as solubilizers of water-insoluble drugs and other drug delivery applications. The ability of surfactants to dissolve phospholipids largely depends on the chemical structure of both surfactant and phospholipid. While bile salt and amphiphilic drug surfactants, with a rigid chemical structure, are good solubilizers of phospholipids, conventional surfactants, with a flexible aliphatic hydrocarbon tail, are poor solubilizers. In addition, the chemical structure of phospholipids, such as tail lengths and charge number, or the fraction of a cosurfactant, for instance cholesterol, is expected to influence the ability to form mixed micelles. In this paper, the aggregation behaviour and mixed micelle formation of the phospholipid dimyristoyl phosphatidylglycerol (DMPC) and two different surfactants: the anionic surfactant sodium dodecyl sulfate (SDS) and the amphiphilic drug surfactant Sodium fusidate (SF, similar structure to that of bile salt), have been studied, and the transition from micelles to bilayers has  been determined for the different surfactants, as well as the size and structure of micelles and bilayers close to the points of transition. The self-assembly of the mixed micelles of surfactants/phospholipids have been investigated using surface tension measurements, refractive index increment and static and dynamic light scattering (SLS and DLS). The results suggest that the transition from micelles to bilayers are found to exist in the following range of bile salt/phospholipid compositions: For SF, 70-75 mol % phospholipid in the micelle was determined to be the point of transition, whilst 20-30 mol % for SDS. As the mole fractions of DMPC increased for both mixtures, the samples became turbid, which indicates the transition of micelles to bilayers. An exact value for molar ratio of transition might not be possible to determine from this study, but instead a, somewhat wider, range of values. In spite of this, a clear trend and difference between the two surfactants was observed.

Giardia duodenalis est le protozoaire responsable de la giardiose, la parasitose intestinale la plus répandue dans le monde. Cette infection se caractérise par une malabsorption intestinale, des diarrhées, une perte de poids et des douleurs abdominales intenses chez l’Homme et de nombreux mammifères. Par ailleurs, cette maladie dont l’impact en santé publique et vétérinaire est reconnu, peut entraîner d’importantes déficiences nutritionnelles en particulier chez les sujets jeunes. L’infection est causée par l’ingestion de kystes de Giardia duodenalis (syn. G. lamblia, G. intestinalis) présents dans les aliments ou l’eau contaminée. Infectieux à très faibles doses, ces kystes survivent pendant plusieurs semaines dans l’environnement et sont résistants aux différents désinfectants. Suite au dékystement, la forme végétative de Giardia, le trophozoïte, adhère à l’épithélium intestinal au niveau des parties supérieures de l’intestin grêle et se multiplie, causant les symptômes. Cette phase se termine par un nouvel enkystement et l’excrétion de kystes par les fèces. Le nombre croissant d’infections liées à la contamination de l’eau potable, à l’émergence de souches résistantes aux médicaments disponibles, à la fréquence des échecs thérapeutiques et à l’importance des effets secondaires associés aux traitements font de cette maladie un sujet d’actualité de plus en plus préoccupant qui nécessite le développement de traitements alternatifs. Il est désormais bien établi que le microbiote et/ou certaines souches de bactéries probiotiques ont un impact bénéfique dans la giardiose. En particulier, la bactérie probiotique Lactobacillus johnsonii La1 (LjLa1) a un rôle protecteur contre la croissance de Giardia in vitro et in vivo. Nous avons cherché dans ce travail de Thèse à décrypter les mécanismes moléculaires associés à l’effet inhibiteur des facteurs sécrétés par LjLa1. Nous avons montré qu’in vitro, LjLa1 agissait en libérant des enzymes de type Bile Salt Hydrolases (BSH) qui modifient alors des composants de la bile non-toxiques pour le parasite (sels biliaires conjugués) en des composants toxiques (sels biliaires déconjugués). Les 3 gènes BSH codés dans le génome de LjLa1 ont été clonés chez Escherichia coli et les protéines taguées histidine purifiées pour étudier leurs propriétés biochimiques et biologiques. Obtenues sous forme active, nous avons pu en définir les spécificités de substrats et montrer qu’elles sont capables d’inhiber significativement la croissance de G. duodenalis in vitro et in vivo, dans un modèle murin de la giardiose (souriceaux OF1 non sevrés). En parallèle, nous avons identifié, à l’issue d’un large criblage de souches de lactobacilles selon leur activité anti-Giardia in vitro, une souche probiotique aux effets inhibiteurs comparables à ceux de LjLa1 : Lactobacillus gasseri CNCM-4884. Administrée in vivo dans le modèle murin de la giardiose, cette souche a réduit de 93% la charge parasitaire dans l'intestin grêle des nouveaux nés et a également réduit de façon significative le nombre de kystes libérés dans l’environnement, permettant ainsi de réduire la transmission de Giardia. Des travaux parallèles ont été réalisés au cours de ce projet de Thèse, notamment le développement d’outils de moléculaire pour l’expression hétérologue de molécules d’intérêt en santé animale chez divers lactobacilles. Le développement de ces « vecteurs mucosaux » permettra à terme de proposer une stratégie de surexpression de BSH par les lactobacilles afin d’accroitre l’activité BSH in vivo, et renforcer ainsi l’élimination du parasite. Ces résultats permettent de proposer de nouvelles pistes thérapeutiques originales contre les giardioses humaines et animales, basées sur l’utilisation de lactobacilles probiotiques ou sur les activités BSH qui en sont dérivées. Ces traitements offrent alors une alternative sérieuse aux antibiotiques et permettront de pallier aux actuels fréquents échecs thérapeutiques. / Giardia duodenalis is a protozoan parasite responsible for giardiasis, the most common intestinal parasitic disease worldwide. This infection is characterized by intestinal malabsorption, diarrhea, weight loss and abdominal pain in humans and various mammalian species. Besides, this disease has a high veterinary and public health impact, leading to important nutritional deficiencies in young subjects. The infection is caused by the ingestion of food or water contaminated with infectious cysts of the parasite. Giardia cysts can survive for several weeks in the environment and are highly resistant to disinfectants. Giardia excysts in the intestinal tracts of its host and replicates under the trophozoite stage causing the symptoms. Trophozoites adhere to the intestinal epithelium of the small intestine and multiply, causing the symptoms. The cycle ends by a new encystment and infectious cysts are released in environments with feces. The increasing number of giardiasis cases, mainly due to water contaminations, the emergence of parasite strains resistant to drugs and therapeutic failures, make research on alternative therapeutic strategies and treatments highly needed. Nowadays, it is well known that the microbiota and probiotics play an important role in protection against this parasite. For instance, the probiotic strain Lactobacillus johnsonii La1 (LjLa1) prevents the establishment of Giardia in vitro and in vivo. In this thesis, we have tried to point out the molecular mechanism(s) involved in this inhibitory effect(s). We showed in vitro that LjLa1 was releasing "Bile Salt Hydrolases" (BSH) – like activities that modify some components of bile (conjugated bile salts) into toxic compounds (deconjugated bile salts) for Giardia. We have cloned and expressed each of the three bsh genes present in the genome of LjLa1 in Escherichia coli in order to study their enzymatic and biological properties. Two BSH were obtained as recombinant active enzymes and biochemical tests showed that they have distinct substrate specificities despite similar predicted 3D structures. Moreover, these two BSHs of LjLa1 exhibited anti-giardial effects in vitro and in vivo in a murine model of the giardiasis (OF1suckling mice), comforting the hypothesis of the biological role of active BSH, derived from probiotics, against Giardia. A wide collection of diverse lactobacilli strains was screened to assess their effectiveness to also display both anti-giardial and BSH activities. This screening allowed the identification of several strains exhibiting strong anti-giardial effects such as Lactobacillus gasseri CNCM I-4884. In a murine model of giardiasis, this strain dramatically reduced the parasite burden in the small intestine of treated animals and significantly reduced the number of cysts in the colon, which could contribute to blockage of parasite transmission in environments. Additional studies were realized in parallel in order to explore the potency of lactobacilli to exert beneficial effects on health. For this, molecular tools were successfully developed in various lactobacilli strains to express and deliver therapeutic molecules at mucosal surfaces. The development of these tools will further allow the overexpression of BSH by lactobacilli to increase their in vivo BSH-activity and strengthen the elimination of the parasite. Altogether, this thesis work proposes new original therapeutic strategies against human and animal giardiasis, based on the use of BSH-positive lactobacilli strains or recombinant BSH- derived from probiotic strains, to counteract the frequent therapeutic failures, offering a serious alternative to antibiotics.

Diagnosis of acute pancreatitis (AP) is erroneous in up to one third of patients when based on clinical criteria and elevated serum amylase values. Furthermore, according to autopsy reports fatal pancreatitis remains clinically undiagnosed in 22 to 86 % of hospitalised patients. Consequently, search for better methods for the diagnosis of AP seems not only justified but urgent. The pancreas secretes an nonspecific lipase, the carboxylic ester hydrolase (CEH) with molecular properties different from other pancreatic secretory enzymes. These differences may imply that sites and rates of clearances from blood of pancreatic enzymes differ. Except for the pancreas this enzyme is secreted from the lactating mammary gland with milk. A sensitive and reproducible sandwich-ELISA for quantitative determination of CEH was developed. When establishing referent values it was noted that in individuals aged 20 to 65 years serum concentrations of CEH did not depend on age, gender, the time of the day or duration from food intake to blood sampling, or use of nicotine. The mammary gland did not contribute significantly to basal serum levels of CEH; enzyme levels in lactating women or women with mammary tumours were identical to those of the reference population. Seventy percent of patients with the diagnosis AP, based on elevated serum amylase levels and abdominal pain, had elevated CEH values. Among the patients with elevated amylase alone a probable cause of pancreatitis was lacking in the majority of patients. Contrastingly, a likely cause of AP could be identified in all patients presenting with abdominal pain and elevated CEH levels alone. These findings suggested that an elevated CEH level indicated AP more reliably than an elevated amylase level. In patients with AP diagnosed by contrast enhanced computed tomography (CECT) alone, or combined with histopathological diagnosis, serum CEH levels were elevated on admission in all but one patient, and in all within the next 24 h. Furthermore, in patients with severe pancreatitis CEH levels remained at a raised level from the second to at least the 10:th day following admission, whereas a significant decrease was noted in patients with mild pancreatitis. In contrast, serum amylase values were higher in patients with mild pancreatitis during the observation period than in those with severe pancreatitis. CEH levels were higher in patients with three or more Ranson signs than in those with less than three signs from the first day after admission. CEH levels were within referent range in 164 patients without known pancreatic disease admitted due to abdominal emergency conditions, or due to planned surgery for chronic extrapancreatic gastrointestinal diseases, and 16 patients having CECT without pathological findings in the pancreas. This suggests that AP can be excluded with very high degree of probability in presence of non-elevated CEH levels. A sandwich ELISA for determination of Guinea pig CEH and a model for graded pancreatitis in the same species were developed. CEH levels showed proportional to severity of inflammation, thus confirming previous clinical observations. CEH levels in bile were proportional to inflammation, while it was absent in urine. Amylase levels in urine were identical regardless of severity of inflammation, but low in bile. These results suggested differences in sites and rates of clearance between the two enzymes. Seemingly elevated CEH levels allowed identification of clinically significant pancreatitis following ERCP, which amylase levels did not. The presented studies have shown that quantitative determination in serum of CEH by the described method is a more reliable test for the diagnosis of AP than determination of amylase activity. The differences between CEH and amylase are, at least partly, due to differences in molecular properties determining rates and routes of clearances of the two enzymes from serum. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser.</p> / digitalisering@umu.se

No description available.

Chapter 1: Introduction to the luminescent properties of lanthanides Luminescence properties of trivalent lanthanides have been explored extensively over the past few decades owing to their unique properties. Lanthanides emission is known to be due to intra-configurational f-f transitions. Because the partially filled 4f shell is well shielded from its 26 environment by the closed 5sand 5pshells, the ligands in the first and second coordination sphere perturb the electronic configurations of the trivalent lanthanide ions only to a very limited extent. This leads to interesting properties such as long lifetimes, sharp line-like emissions etc. which in turn make lanthanides very attractive choice for commercial optical applications. Despite this, the scope of applications remained limited because of the low molar extinction coefficient values of the forbidden lanthanide f-f transitions. However, this problem has been successfully addressed by complexing the lanthanide ion with suitable ligands which can sensitize it resulting in a significant increase in the emission intensity (so called “antenna effect”). The strategy worked very well and resulted in widespread applications of lanthanides form biology to optoelectronics. This chapter discusses elementary ideas regarding the mechanism of sensitization and relevant examples that traces various applications of such lanthanide complexes from the current literature. Chapter 2: A self-assembled Europium Cholate hydrogel: a novel approach towards lanthanide sensitization Luminescent lanthanides can be of great value in a number of possible applications but their scope is limited by their intrinsic low molar absorptivities. Though this problem can be circumvented by complexing the lanthanide ion with suitable chelating ligands to improve the luminescence properties drastically, the design of such systems often involves meticulous planning and laborious synthetic steps to obtain a ligand suitable for the job. It is therefore desirable to have a simpler version of a sensitizing system that does not require the complexities of a chelating ligand but can sensitize trivalent lanthanides with comparable efficiency. It was observed in our group that divalent metal ions (Ni2+, Zn2+, Cu2+, Coetc.) form hydrogels on addition of sodium cholate. We extended to obtain hydrogels of trivalent lanthanides. Furthermore, when the gel was doped with pyrene, a ten-fold increase in the intensity of Eu(III) emission was observed (Fig 2). Thus we established a unique way to sensitize lanthanides in a hydrogel media by non-coordinating chromophores. The approach was completely modular in nature and avoids any laborious synthesis. We also tried other derivatives of pyrene as sensitizers and found that 1-pyreneboronic acid also caused similar sensitization of Eu(III). Fig 2. (a) Schematic representation of the sensitization process (the arrangement of molecules in the gel fiber is arbitrary). Eu-cholate (5 mM/15 mM) gel (a) normal light and (b) 354 nm UV excitation in the presence of 6 μM pyrene Further studies revealed, that 2,3-dihydroxynapthalene (DHN) can sensitize Tb(III) in a similar hydrogel. We also demonstrated Tb(III) to Eu(III) energy transfer process occurring in the gel when doped with DHN. This allowed us to achieve a hydrogel system with tunable luminescence properties (by varying relative ratios of Tb(III) and Eu(III) ). When the effect of divalent metal ions on such energy transfer processes were explored, it was observed that the luminescence from the composite gel of Tb(III)/ Eu(III) is tunable by Zn(II) and through proper manipulation of concentrations one can obtain white light emitting gel (Fig 3). Fig 3. Effect of Zn(II) (from left to right 0 mM, 2.8 mM, 11.3 mM) on Tb3+ (4.5 mM)/Eu3+ (0.11mM)/ sodium cholate (13.6 mM) gels. b) Tb/Eu/Zn-cholate gel (Tb3+ (4.4 mM), Eu3+ (0.11 mM), Zn2+ (7.4 mM), NaC (13.6 mM, DHN 0.2 mM) under 365 nm UV lamp (c) CIE 1931 diagram depicting the luminescence as white (black spot). Chapter 3. A “Pro-Sensitizer” based Sensing of Enzymes using Tb(III) Luminescence in a Hydrogel matrix This chapter descirbes design and realisation of a sensor system based on Tb(III) luminescnece for the detection of enzymes. The idea involved synthesizing a covalently modified DHN molecule by attaching appropriate enzyme cleavable units. We coined the term “pro-sensitizer”to describe the modified molecule which would not sensitize Tb(III) in the gel matrix but when proper enzymes are applied the free form of DHN would be released triggering a luminescence response from Tb(III). This would enable us to monitor the acitivities of the particular enzyme by examining the luminescence intensity enhancement with time (Fig 4) Fig 4. A “pro-sensitizer” based approach to detect different types of enzymes in a hydrogel matrix through Tb(III) luminescence. We applied the idea to develop a novel luminogenic gel probe for inexpensive and rapid detection of three different hydrolases, lipase, β–glucosidase and α-chymotrypsin. The corresponding “pro-sensitizer”for each enzyme were synthesized (Fig 5).The sensing technique depends on the gel matrix to provide the nessesary platform for lanthanide sensitization. Thereofore, it enjoys an edge over the contemporary techniques that typically involve specially designed and synthesized multidentate chelating ligands for this purpose. We also determined important kinetic parameters of all the enzymes, thus enabling us to have a better insight into the activity of the enzymes in the hydrogel matrix. Fig 4. Pro-sensitizers molecules for (1) lipase, (2) β-glucosidase and (3)α-chymotrypsin Chapter 4. A novel approach towards templated synthesis of lanthanide trifluoride nanoparticles Nanomaterials with excellent optical properties have been of special interest. Lanthanide derived nanoparticles, owing to their unique physical properties, provide an excellent choice for applications such as biolabels, lasers, optical amplifiers, and optical-display phosphors. Several types of lanthanide nanoparticles or nanocrystals are reported in the literature such as Nd2O3, Eu2O3, Gd2O3, Tb2O3, and Y2O3. Among them lanthanide fluoride nanoparticles have emerged as the best choice because of their low phonon energy, and thus minimum quenching of emissive Lnions thereby allowing maximum efficiency for several optical applications. In previous literature precedence, LnF3 nanoparticles were typically synthesized following conventional approaches which necessitate use of high temperatures, high pressures (hydrothermal techniques) and capping ligands. In this chapter, we demonstrated a simpler synthesis of LnF3 nanoparticles at ambient temperatures without the requirement of added capping agents. The room temperature synthesis of LnF3 was unprecedented and was achieved simply by diffusing NaF solution through the hydrogels of corresponding Ln-cholate gels. The nanoparticles were characterized by transmission electron microscopy (TEM) and by powder XRD analysis which established the presence of very small (3-4 nm) nanoparticles mono-dispersed uniformly over the the gel matrix (Fig 6). The LnF3 containing xerogels of Tb(III) and Eu(III) cholate gels were also shown to be highly emissive. Fig 6. HRTEM images of a) TbF3, b) GdF3, c) NdF3 and d) DyF3 in their corresponding gel media.

According to the American Heart Association, cardiovascular disease (CVD) is the leading cause of death in the U.S., representing about 20-30% of all deaths every year in the U.S. Major risk factors for developing CVD include high blood lipid and LDL-cholesterol levels. A large number of heart attacks and strokes could be prevented by controlling these factors through lifestyle modifications and diet interventions. Epidemiological evidence shows that consumption of dry or common beans (Phaseolus vulgaris L.) has positive effects on reducing blood LDL-cholesterol and lipid levels. These health benefits are mainly attributed to the high content of dietary fiber (DF) of beans, including soluble and insoluble DF (SDF and IDF). Some proposed mechanisms to explain the cholesterol and lipid-lowering effects of DF are related to the physico-chemical properties (e.g. viscosity) of DF, and involve binding to bile salts (BS) in the small intestinal to prevent BS re-absorption which further promote cholesterol catabolism and delay lipid digestion. Nevertheless, the precise mechanisms are not fully understood yet. In addition, cooking and processing operations, and in particular high-hydrostatic pressure (HHP) processing, can modify the composition, structure and functional properties of foods; however, whether HHP affects the ability of beans to interfere with different aspects of lipid digestion remains unknown. The overall goal of this research is to understand how common beans and HHP processing impact the ability of beans to bind BS and influence lipid digestion in vitro. The specific objectives are 1) to evaluate the effect of HHP treatments (and compared it with conventional cooking (HT)) on the thermo-rheological and functional properties of dry beans; 2) to identify the impact of major bean components on the in vitro BS-binding ability of beans, the role played by the bean matrix and how this is affected by HHP processing; 3) to investigate how bean (micro)structure and fiber fractions, as well as HHP processing of dry beans, influence lipid digestion in vitro. Results showed that HT caused complete starch gelatinization and protein denaturation of beans, while HHP treatments induced partial or no starch gelatinization and a lower degree of protein denaturation, which resulted in enhanced protein solubility and emulsifying activity/stability. It was observed that, while HT treatment reduced the capacity of bean flours to retain BS because of severe disruption of the bean cell wall integrity, protein matrices, and starch granules, HHP treatments maintained or enhanced BS retention, possibly by promoting the formation of starch/protein/fiber networks able to entrap BS. Furthermore, by using an in vitro dialysis-based digestion model combined with viscosity measurements and thermal analysis, it was shown that the interaction between bean tissue materials and primary BS was not only related to viscosity but also involved hydrophobic linkages. The contribution of IDF and proteins (other than SDF) to retain BS was also significant. There was a different binding preference of beans to four primary BS with sodium glycochenodeoxycholate, the more hydrophobic BS, showing the largest retention levels while sodium taurocholate being the least effectively retained BS by beans. Diverse sequences of the same processing operations showed distinct impacts on BS-retention by dry beans. By means of an in vitro digestion model simulating conditions in the upper gastrointestinal tract, bean flours delayed the digestion of extrinsic lipids to a higher extent, compared to isolated IDF and SDF. Furthermore, HHP treatment and less severe mechanical disintegration maintained the ability of beans to modulate lipid digestion, which suggests the importance of bean structural integrity in reducing the lipolysis rate and extent by beans. Overall, this research work shows that HHP processing is a promising minimal processing technology to produce bean flours with improved functionality. It also highlights the importance of considering the structure of foods, and not just their nutrient content, when evaluating potential health impacts. This knowledge could be applied to develop a range of bean-based ingredients and formulations with desirable health benefits. This work can be extended to research the influence of beans on the gut microbiota and profile of secondary BS and short-chain fatty acids, which are also closely related to cholesterol and lipid metabolism. / Doctor of Philosophy / According to the American Heart Association, cardiovascular disease (CVD) is the leading cause of death in the U.S., representing about 20-30% of all deaths every year in the U.S. Around the world, millions of people are struggling to control the risk of CVD. Major risk factors for developing CVD include high blood lipid and LDL-cholesterol levels. A large number of heart attacks and strokes can be prevented by controlling the major risk factors through lifestyle modifications and diet interventions. Epidemiological evidence shows that consumption of dry beans (Phaseolus vulgaris L.) has positive effects on reducing blood LDL-cholesterol and lipid levels. These health benefits are mainly attributed to the high content of dietary fiber (DF) in beans. DF is carbohydrate polymers that are not hydrolyzed by the endogenous enzymes in humans. However, some of them (water-soluble DF) could increase viscosity and retain the absorption of bile salts (BS) in the small intestinal. The BS retention or the binding of BS could promote more cholesterol convert to BS (thus reduce cholesterol levels) and decrease lipid digestion. Therefore, due to the increased viscosity and BS retention ability of DF, dry beans could help to reduce the blood cholesterol and lipid levels and further help to prevent CVD. Moreover, different cooking and processing method could also affect the composition, microstructure and functional properties of foods. The purpose of this research was to determine how common beans and high hydrostatic pressure (HHP) (compared with hydrothermal (HT)) processing, a non-thermal processing, influence the ability of dry beans to retain bile salts and modulate lipid digestion in vitro. This study showed that severe HT treatment disrupted the bean cell wall integrity severally and reduced the BS retaining the efficiency of dry beans, while HHP treatment, produced minimally processed beans, improved the application properties of dry beans and maintained/enhanced BS-retention by dry beans. It also showed that the whole bean matrix (other than soluble DF) also contributes to retain BS and modulate lipid digestion, indicating the importance of retaining intact food structures. The integrity of bean structures through HHP treatment and less severe mechanical treatment could help to retain the ability of dry beans to reduce lipid digestion. These findings suggest that dry beans, with a high content of dietary fiber and resistant starch, have significant health benefits related to lowering cholesterol and lipid levels. Increasing the consumption of dry beans would definitely help to improve overall health. HHP, as a non-thermal processing technology, showed the potential to produce minimally processed bean products with enhanced health benefits and diverse application properties. This study could be extended through continuing research into the influence of beans on the gut microbiota, which are also closely related to the cholesterol and lipid metabolism regulation.

Experiments with both quantitative and qualitative factors always complicate the selections of experimental settings and the statistical analysis for data. Response surface methodology (RSM) provides the systematic procedures such as the steepest ascent method to develop and improve the response models through the optimal settings of quantitative factors. However the sequential method lacks of exploring the direction of the maximum increase in the response among the qualitative levels. In this dissertation the optimal designs for experiments with both qualitative and quantitative factors are investigated. Focused on the second-order response surface model for quantitative factors, which is widely used in RSM as a good approximation for the true response surface, the approximate and exact D-optimal designs are proposed for the model containing the qualitative effects. On spherical design regions, the D-optimal designs have particular structures for considering the qualitative effects to be fixed or random. In this study, the exact D-optimal designs for a second-order response surface model on a circular design region with qualitative factors are proposed. For this model, the interactions between the quantitative and qualitative factors are assumed to be negligible. Based on this design region, an exact D-optimal design with regular polygon structure is made up according to the remainder terms of the numbers of experimental trials at each qualitative levels divided by 6. The complete proofs of exact D-optimality for models including two quantitative factors and one 2-level qualitative factor are presented as well as those for a model with only quantitative factors. When the qualitative factor has more than 2 levels, a method is proposed for constructing exact designs based on the polygonal structure with high efficiency. Furthermore, a procedure for minimizing the number of support points for the quantitative factors of exact D-optimal designs is also proposed for practical consideration. There are no more than 13 support points for the quantitative factors at an individual qualitative level. When the effects between the quantitative and qualitative factors are taken into consideration, approximate D-optimal designs are investigated for models in which the qualitative effects interact with, respectively, the linear quantitative effects, or the linear effects and 2-factor interactions of the quantitative factors or quadratic effects of the quantitative factors. It is shown that, at each qualitative level, the corresponding D-optimal design consists of three portions as a central composite design but with different weights on the cube portion, star portion and center points. Central composite design (CCD) is widely applied in many fields to construct a second-order response surface model with quantitative factors to help to increase the precision of the estimated model. A chemical study is illustrated to show that the effects of the qualitative factor interacts with 2-factor interactions of the quantitative factors are important but absent in a second-order model including a qualitative factor treated as a coded variable. The verification of the D-optimality for exact designs has become more and more intricate when the qualitative levels or the number of quantitative factors increase, even when the patterns of the exact optimal designs have been speculated. The efficient rounding method proposed by Pukelsheim and Rieder (1992) is a model-free approach and it generates an exact design by apportioning the number of trials on the same support points of a given design. For constructing the exact designs with high efficiencies, a modified efficient rounding method is proposed and is based on the polygonal structure of the approximate D-optimal design on a circular design region. This modification is still based on the same rounding approach by apportioning the number of trials to the concentric circles where the support points of the given design are standing on. Then a regular polygon design will be assigned on the circles by the apportionments. For illustration, the exact designs for a third-order response surface model with qualitative factors are presented as well as those for the second-order model. The results show that nearly D-optimal designs are obtained by the modified procedure and the improvement in D-efficiency is very significant. When the factors with the levels selected randomly from a population, they are treated as with random effects. Especially for the qualitative effects caused by the experimental units that the experimenter is not interested in, one should consider the model with random block effects. In this model, the observations on the same unit are assumed to be correlated and they are uncorrelated between different units. Then the mean response surface is still considered as second-order for quantitative factors but the covariance matrix of the observations is different from the identity matrix. In the fourth part of this dissertation, the locally D-optimal designs on a circular design region are proposed for given the value of the correlations. These optimal designs with the structures based on the regular polygons are similar to the D-optimal designs for the uncorrelated model.

Chapter 1. Introduction This chapter is an overview on the literature of self-association of small organic molecules. The chapter is presented in four parts. First, an introduction to aggregation of small molecules is given with the emphasis on micelles and gels(Parts 1 and 2) In part 3, a short overview is given on bile acid based aggregates and their applications. Lastly, the content of the thesis is outlined. Chapter 2. Solution properties of novel cationic bil salts: A structure-aggregation property study Scheme 1: Structures of Cationic bile salts(Refer PDF File) Bile Salts are biosurfactants and they are known to form micelles in aqueous medium. We studied the micellar properties of cationic bile salts(Scheme 1) and compared with their natural (anionic) counterparts. A serendipitous discovery of the gelation of a cationic bile salt(4) led us to investigate the aggregation properties of this new class of cationic hydrogelators. This chapter highlights the recent efforts on the study of side chain structure-aggregation property relationship of cationic bile salts. Bile acid analogues with a quaternary ammonium group(Scheme 1, compounds 2, 3, 4, 6, 8 and 12) on the side chain were found to efficiently gel aqueous salt solutions. Some of the cationic bile salts gelled water alone and many of them gelled aqueous salt solutions even in the presence of organic co-solvents(≤ 20%) such as ethanol, methanol, DMSO and DMF. A strong counter ion dependent gelation was observed. These gels showed interconnected fibrous networks. Unlike natural anionic bile salt gels(reported for NaDC, NaLC), the cationicgels reported here are pH independent. Cationic gels derived from DCA showed more solid-like rheological response compared to natural NaDC gels studied earlier by Tato et al. A strong structure(side-chain) andcounter-ion dependent flow of the cationic bile salt gels was seen. Chapter 3. Applications of cationic bile salts and their aggregates Cationic bile salts are useful in many ways. We have studied some of the applications of cationic bile salts(discussed in chapter 2) and their aggregates in this chapter. The chapter is presented in three parts. Part 1. Interaction of Cationic bile salts and DNA The bile acid amphiphilicity is believed to help the DNA binding process of polyamines. This has prompted us to study the DNA-bile salt binding interaction of bile salts. The binding of cationic bile salts has been expressed in terms of C50 values, which were determined from the plot of fluorescence of ethidium bromide bound DNA vs. bile salt concentration(Fig 1) The C50 values for cationic bile salts were estimated to be about 1.2 mM. Fig1: A plot of fluorescene of ethidium bromide bound DNA against bile salt concentration (Refer PDF File) Part 2. Cholesterol solubilization and crystallization studies in aqueous bile salt solutions. Dihydroxy bile salt micelles are well known for cholesterol dissolution(e.g. UDCA and CDCA). We studied the dissolution of cholesterol in the cationic bile salt micelles(of 21-25) and the results are discussed in this part. Scheme 2: Cationic bile salt chlorides studied for cholesterol dissolution (Refer PDF File) A powder dissolution method was used to study the solubility of anhydrous cholesterol in cationic bile salt solution. These cationic bile salt micelles can dissolve cholesterol to the same extent as the taurine conjugates of bile acids, but lesser than the natural anionic bile salts(Fig.2) Addition of PC(Phosphatidylcholine) to cationic bile salt micelles enhanced the micellar cholesterol solubilization. Fig 2:Cholesterol dissolution in cationic bile salt solutions(Refer PDF File) The crystal nucleation time of cholesterol did not change significantly by adding 5-30 mM of the cationic bile salts. The bile salt analogues did, however, attenuate cholesterol crystallization to a similar extent at all concentrations studied. All these effects wer comparable to those fo cholic acid. Part 3. Hydrogels as a reaction vessel for photodimerization Bile salt micelles have been shown to control the product selectivity in photochemical reactions. The dynamic nature of the bile salt micelles results in differential effects on reaction selectivity. The photodimerization of acenaphthylene(sheme 3) was studied in micellar and hydrogel medium(e.g. NaDC, 22, 28, etc.) The ratio of anti- to synphotodimer was found to be greater in gel bound state than in solution. Substitution on the CAN ring did not show larger variation on the product distribution from solution gel. Scheme 3: Photodimerization of acenaphthylene(Refer PDF File) Chapter 4. Bile acid derived sulfur analogues in designing novel materials. Part 1. A simple approach towards nanoparticle-gel hybrid material. Scheme 4: Scheme for the synthesis of thiols derived from bile acids (Refer PDF File) Our interest in bile acid based gelator molecules led us to explore the synthesis and properties of bile analogues with the side chain carboxylic acid replaced by a thiol(Scheme 4) to stabilize metal NPs. We reasoned that the specific self-aggregation modes of facially amphiphilic bile units would enable a metal NP capped by such a thiol to “lock” onto a gel fiber derived from a structurally related gelator molecule. AuNPs stabilized by 38-40 were obtained by the NaBH4 reduction of homogeneous methanolic solutions of the thiol and gold salt. These steroid capped nanoparticles were found to stay dispersed in a gel of 28, thus providing a simple approach to obtain gel-nanoparticle hybrid. A photograph of the hybrid material and their morphology are shown in Fig 3.(Refer PDF File) Chart 1: Structure of the gelator used for designing a hybrid material(Refer PDF File) Part 2. Gelation of aromatic solvents using sulfur analogues of bile acid A few of the sulfur derivatives were serendipitously fouond to gel organic solvents (Fig 4). Thiol 38 formed stable gels at room temperatures while the disulphide 36 formed stable gels below 5º C. The aggregation properties, morphology, and the melting profiles of gels of disulfides and thiols derived from bile acids have been highlighted in this part. Fig 4: A photograph of the gels derived from 38(Refer PDF File) (For Figures and Molecular Formula Pl refer the Original Thesis)