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Metabolic coupling between glial cells and neurons is essential for neuronal function. It is a well-conserved and vital feature of the bilaterian nervous system as well. Under normal and adverse conditions, glial cells act as a major metabolic hub fueling neuronal oxidative metabolism by producing lactate or ketone bodies. I now ask the question of whether such metabolic coupling is only necessary for preserving brain homeostasis or if it could also have implications in decision making such as food choice behavior. Choosing an appropriate food source is key for the survival of an organism. Carbohydrates are the preferred source of energy and thus evaluation of their nutritive content is essential. Several studies have demonstrated that Drosophila melanogaster larvae and adults, as mammals, can distinguish between nutritious and non-nutritious carbohydrates independent of their taste. Two groups of neurons, Diuretic Hormone 44 (Dh44)-expressing neurons and gustatory receptor 43a (Gr43a)-expressing neurons, have been implicated in postprandial sugar sensing in adult flies. Gr43a- expressing neurons are narrowly fine-tuned for sensing fructose in both adults and larvae. Nonetheless, in the larva, central nervous system (CNS) Gr43a neurons have been shown to act as the main sugar sensor. This raises the question of how CNS fructose-sensing neurons are involved in sensing non-fructose dietary sugars. To decipher this post-ingestive mechanism, I used frustrated total internal reflection (FTIR) - based larval tracking to investigate larval food choice behavior. The results present in this thesis suggest that besides Gr43a-expressing neurons, glial cells are indispensable for sensing nutritive non-fructose dietary sugars such as glucose and sorbitol. I show that post-ingestive carbohydrate sensing involves carbohydrate conversion into fructose locally in the glial cells. Glia-derived fructose then enables Gr43a-dependent postprandial carbohydrate sensing in the CNS and drives carbohydrate preferences with the help of the downstream signalling peptide corazonin. Thus, in post-ingestive carbohydrate sensing, the glial cells act as a master metabolite sensor and provide a fructose stimulus to neurons to regulate behavior.:Table of Contents Abstract 1 Zusammenfassung 2 1. Introduction 3 1.1. External Nutrient Sensing in mammals and Drosophila melanogaster 4 1.1.1. Taste detection in mammals 4 1.1.2. Taste detection in Drosophila melanogaster 6 1.1.3 Sweet and Umami Taste in mammals 8 1.1.4. Sweet Sensing in Drosophila melanogaster 8 1.1.5. Bitter Taste in mammals 9 1.1.6. Bitter Sensing in Drosophila melanogaster 9 1.1.7. Sour, Carbonation, Fatty acid and Salty taste in mammals 9 1.1.8. Amino acid, Fatty acid and Salt Sensing in Drosophila melanogaster 10 1.2 Post-ingestive nutrient sensing in mammals and Drosophila melanogaster 11 1.2.1. Post-ingestive amino acid sensing in mammals 11 1.2.2. Post-ingestive amino acid sensing in Drosophila melanogaster 12 1.2.3. Post-ingestive lipid sensing in mammals 13 1.2.4. Post-ingestive carbohydrate sensing in mammals 13 1.2.4.1. The role of the nervous system in post-ingestive carbohydrate sensing mammals 16 1.2.5. Post-ingestive carbohydrate sensing in Drosophila melanogaster 16 1.2.5.1. The role of the nervous system in post-ingestive carbohydrate sensing in flies 18 1.2.5.2. The role of the nervous system in post-ingestive carbohydrate sensing in larvae 19 1.3 The cellular architecture of the larval nervous system 20 1.3.1 The Blood-brain barrier (BBB) and carbohydrate transport 23 1.4 Aim of study 25 2. Experimental Procedures 26 2.1 Materials 26 2.1.1 Chemicals 26 2.1.2 Media 27 2.1.3 Buffer and Solution 27 2.1.4 Antibodies 28 2.1.5 Flystocks 29 2.2 Methods 33 2.2.1 Fly genetics 33 2.2.1.1 Maintenance and crosses 33 2.2.2 Immunohistochemistry & Microscopy 33 2.2.2.1 Immunohistochemistry of larval filets 33 2.2.2.2 Confocal Microscopy 33 2.3 Experimental Design 34 2.3.1 Studying food choice in Drosophila larvae 34 2.3.1.1 Concept 34 2.3.1.2 Experimental setup 36 2.3.1.3 Analysis 36 2.3.1.4 MATLAB Script 38 3. Results 40 3.1. Frustrated total internal reflection based two-choice assay (TCA) 40 3.1.1 Investigating larval sugar preference 40 3.2 The role of diuretic hormone 44 (Dh44) neurons in post-ingestive glucose sensing in third instar larvae 47 3.2.1 Immunohistochemical analysis of Dh44 in third instar larval brains 47 3.2.2 Role of Dh44 in glucose sensing 49 3.3 The role of gustatory receptor 43a (Gr43a) neurons in post-ingestive nutritive carbohydrate sensing in third instar larvae 51 3.3.1 Immunohistochemical analysis of Gr43a in third instar larval brains 51 3.4. Investigating the role of polyol pathway in post-ingestive carbohydrate sensing 56 3.4.1 Identifying the key polyol pathway enzyme in Drosophila melanogaster 56 3.4.2 Examining RNAi mediated neuronal and glial knockdown of polyol pathway enzymes in postprandial sugar sensing 57 3.4.2.1 CG9436 57 3.4.2.2 CG6084 and CG10863 60 3.4.2.3 Sodh-2 66 3.5 Determining the glia subtypes vital for conducting polyol pathway 69 3.6 The role of the blood brain barrier (BBB) in post-ingestive carbohydrate sensing 74 4. Discussion 80 4.1. Gr43a is the only sugar sensor in Drosophila larvae 80 4.2. Polyol Pathway is crucial for glucose and sorbitol sensing 82 4.3. Glia, the master metabolic sensor 84 4 4. Transport over BBB: a prerequisite for postprandial carbohydrate sensing85 4.5. Proposed model for larval post-ingestive nutritive carbohydrate sensing... 86 5. References 89 6. Abbreviation List 100 7. Appendix 103

Low-carbohydrate, high-protein (LCHP) diets have been shown to induce weight loss and beneficial changes in blood lipids that suggest cardiovascular disease risk reduction; however, LCHP diets have not been adequately investigated for health effects on the skeleton. A randomized trial to determine the effects of a LCHP diet on bone mineral status, biomarkers of bone turnover, indicators of acid-base balance, calcium homeostasis and fasting lipids in healthy pre-menopausal women was conducted. Women, aged 32 - 45 y, with a body mass index between 25-41 kg/m2 were randomized into one of two diet groups: LCHP (n = 13) or high-carbohydrate, low-fat (HCLF) (n = 12). Anthropometric (body weight, lean mass, fat mass) and bone mineral density (BMD) and content (BMC) measures and markers of lipid metabolism were taken at weeks 0, 6, and 12. Measures of acid-base balance, protein metabolism, and calcium homeostasis were conducted at weeks 0, 1-4, 6, and 12. Serum osteocalcin was analyzed at weeks 0, 1, 2, 6, and 12, while urinary NTx was analyzed at weeks 0, 1 and 2. Weight loss was significant at the end of 12 weeks in both diet groups (P < 0.05) but there was no Diet x Time interaction. Total proximal femur BMD was lower in the LCHP group (P < 0.05) compared to the HCLF group by week 12. Femoral neck BMC decreased in the LCHP diet group (P < 0.05), whereas total forearm BMC increased (P < 0.05) in the HCLF diet group by week 12 of the study. Serum osteocalcin showed significant main effects of diet (P < 0.05) and time (P < 0.0001), but a Diet x Time interaction was not observed. Urinary NTx exhibited no main diet effect, time effect or Diet x Time interaction at weeks 1 or 2. Urinary pH was lower in the LCHP group compared to the HCLF group throughout the study (P < 0.0001). Urinary calcium excretion was higher in the LCHP group and lower in the HCLF group (P < 0.0001) compared to baseline values at all intervals of the study. Urinary phosphorus excretion exhibited a significant diet effect (P < 0.001) and time effect (P < 0.002), while no Diet x Time interaction was observed. Total cholesterol, high-density and low-density lipoprotein cholesterol, and triacylglycerol concentrations did not differ between diets during the study. In conclusion, a LCHP diet appears to stimulate bone loss, while a HCLF diet appears to attenuate bone loss in healthy pre-menopausal women undergoing 12 weeks of weight loss. / Ph. D.

Four multiparous Holstein cows, averaging 44 days in milk and previously fitted with rumina1 and duodenal cannulae, were utilized in a 4 x 4 Latin square design to evaluate effects of dietary nonfibrous carbohydrate (NFC) and rumen undegradable protein (RUP) on metabolic and lactational parameters. Diets were isonitrogenous (17.4% CP) and structured in a 2 x 2 factorial arrangement with levels of NFC at 41 or 47% of dietary OM and RUP at 27 or 41% of dietary CP with inclusion of corn gluten meal, fish meal, and feather meal to increase dietary RUP. When cows were fed diets containing 47% NFC, intake of DM, OM, NFC, and N increased (P ± .05) while those containing 41 % NFC increased (P±.01) intake of NDF and ADF. Ruminal digestibility coefficients were not affected leading to greater quantities of NFC digested resulting in suppressed pH (P ±.01) and acetate (P ±.05) and increased propionate (P ± .05), Diets containing 27% RUP increased (P ±.01) ruminal ammonia and plasma urea nitrogen (N) with 41 % NFC eliciting higher ammonia N concentrations at each RUP level. Bacterial yields (averaging 220 g/d) were slightly higher when 41% NFC diets were consumed with no dietary effects on efficiency. Flow of ammonia N to the small intestine (SI) increased (P ± .01) with 27% RUP. When compared to 41% NFC, diets containing 47% NFC increased (P ± .05) flow of methionine (38.6 vs. 32.5 g/d). Fifty percent of the essential amino acids entering the SI were of bacterial origin. Diets containing 41% RUP elicited lower total tract digestion of N while enhancing that of hemicellulose. Total tract digestion of NFC and ADF was greater when 41% NFC diets were consumed. Yields of milk (38.7 vs. 37.7 kg/d) and lactose (1.94 vs 1.88 kg/d) increased with 27% RUP. Efficiency of production was increased by both 41% NFC and 27% RUP. Total and whey N in milk increased (P 5 ±.05) while milk urea N decreased (P ± .01) with higher (41%) dietary RUP. Increased (P ± .05) absorption of N (490.4 vs. 461.6 g/d) at the SI occurred with the 47 vs. 41 % NFC diets, respectively, with no effects observed on that retained or excreted in the urine or milk. Although supply of methionine to and the absorption of N from the S1 was greater with the 47% NFC diets, no positive effects were observed on nitrogen balance or milk production. Rather, dietary RUP provided greater promise in that lower dietary RUP increased milk production and efficiency while increased RUP enhanced milk N content. Therefore, economics regarding feed costs as well as price for milk and milk components may largely dictate preferential dietary RUP content. Abbreviation key: ADC = apparent digestion coefficient, CGM corn gluten meal, CRN = carbon hydrogen nitrogen, CHO = carbohydrate, CNCPS = Cornell Net Carbohydrate and Protein System, MUN = milk urea nitrogen, N = nitrogen, NAN = nonammonia N, NANMN = nonammonia nonmicrobial N, NFC = nonfibrous carbohydrate, NPN = nonprotein N, PUN = plasma urea N, RDP = rumen degradable protein, RUP = rumen undegradable protein, SI = small intestine, TDC = true digestion coefficient, 41:27 = 41% NFC and 27% RlUP, 47:27 = 47% NFC and 27% RUP, 41:41 = 41% NFC and 31% RUP, 47:41 = 47% NFC and 41% RUP. / Ph. D.

Woody plants are the most abundant renewable resources on the earth. From the paper we consume to the house we live in, our daily lives rely heavily on woody plants. Over the past decades, enormous efforts have been expended to improve the utilization of fiber and wood. For example, much research has been conducted to develop environmentally benign, and economically feasible techniques for pulp and papermaking. The economical conversion of wood to useful sugars and alcohol has also been the subject of intensive research. Investigations aimed at the genetic manipulation of wood growth to better meet our needs are also underway. Nonetheless, harsh pulping and bleaching conditions are still required in the pulp and paper industry, and the bioconversion of polysaccharides in biomass to alcohol is still too expensive. An argument could be put forth that a major reason for this is the lack of basic knowledge concerning the structural and biochemical characteristics of the plant cell wall. The three major polymeric components of plant cell walls, cellulose, hemicellulose and lignin, are intimately associated with one another. Cellulose is associated with hemicellulose via non-covalent linkages, whereas lignin is theorized to be associated with cellulose and hemicellulose via both covalent and non-covalent linkages. The nature of associations between wood polymers is still poorly understood. However, it is these intimate associations that make delignification difficult, and make the bioconversion of polysaccharides to alcohol inefficient. It is also believed that the linkages between lignin and polysaccharides are responsible for the reduced digestibility of grasses by ruminants. Besides cellulose, hemicellulose and lignin, there are many secondary metabolites such as lignans, neolignans, tannins and terpenoids. The structures of lignans and neolignans are analogous to the interunits of lignin. Lignin is considered an optically inactive polymer, whereas lignans and neolignans are optically active small molecules. Although it has been proposed that the biosynthesis of lignin, lignans and neolignans are via the same oxidative coupling mechanism, it is still unclear that how the plant cell wall differentiates the formation of lignans, neolignans and lignin. How and why plant cell wall generates so many lignans and neolignans having broad structural variation is also unknown. As a matter of fact, it is still uncertain which enzymes are actually involved in the biosynthesis of lignin. A better understanding of biosynthetic pathways of lignin, lignans and neolignans is a prerequisite for the genetic manipulation of plant growth. Investigations described in this dissertation were an effort to better understand the fundamental aspects of covalent linkages between lignin and hemicellulose in wood. Enantiomeric synthesis of neolignans provides a tool for investigating the optically active nature of neolignans, and may be helpful to study the biosynthetic pathways of neolignans. Chapter | describes chemical structures of wood components and the biosynthesis of lignin, lignans and neolignans. The mechanisms of lignin-carbohydrate bond formation are also discussed, and a concise review of lignin-carbohydrate linkages proposed in the literature concludes Chapter 1. Chapter 2 presents the methods used in investigating covalent linkages in wood, which include methods of isolating lignin-carbohydrate complexes, chemical cleavage methods, DDQ oxidation and model compound/NMR methods. The synthesis of plant cell wall model compounds and neolignans are reviewed in Chapter 3. The experimental work performed for the completion of this thesis is described in Chapters 4-8. A method which provides β-𝘖-4 lignin model dimers with complete threo stereospecificity is described in Chapter 4. This method is complementary to the current method for the preparation of erythro lignin model dimers. Chapter 5 presents a practical synthesis of methyl 4-𝘖-methy] α-D-glucopyranosiduronic acid. Methyl 4-𝘖-methyl-α-D-glucopyranosiduronic acid was prepared from methyl α-D-glucopyranoside in 4 steps (74% overall yield). Previous preparations of this compound were much lengthier, and had very low overall yields. Chapter 6 deals with the synthesis and rearrangement reactions of ester-linked lignin-carbohydrate model compounds. A series of ester-linked lignin-carbohydrate model compounds were synthesized, and migration of the uronosy] group between the primary (γ) and benzyl (α) position of lignin side chain is discussed. Several approaches to synthetic neolignans are described in Chapter 7. Chapter 8 presents a novel approach for the preparation of chiral aryl alkylethers. The successful application of this novel approach to synthesis of several optically active 8-𝘖-4 neolignans and a 1,4- benzodioxane neolignan is described, as is the introduction of an alkyl] aryl ether bond in carbohydrate molecules. Some of the material of this dissertation has been reported in the following papers: 1. Li, K. and Helm, R. F. Approaches to Synthetic Neolignans. J. Chem. Soc. Perkin Trans 1. Accepted. 6. Li, K. and Helm, R. F. Use of Carbohydrates as Building Blocks to Synthesize Neolignans. 211th ACS National ACS Meeting, New Orleans, March 24-28, 1996. CELL-079. 2. Li, K. and Helm, R. F. A Practical Synthesis of Methyl 4-𝘖-Methylα-D-Glucopyranosiduronic Acid. Carbohydr. Res. 273(1995), 249-253. 3. Li, K. and Helm, R. F. Synthesis and Rearrangement Reactions of Ester-Linked Lignin-Carbohydrate Model Compounds. J. Agric. Food Chem. 48(1995), 2098-2103. 4. Helm, R. F. and Li, K. Complete threo Stereospecificity for the Preparation of β-𝘖-4 Lignin Model Dimers. Holzforschung. 49(1995), 533-536. 5. Helm, R. F. and Li, K. Synthesis and Rearrangement Reactions of Lignin-uronic Acid Model Compounds Related to Hardwood Cell Wall Structure. The 8th International Symposium on Wood and Pulping Chemistry. Helsinki, Finland, June 1995, vol. 1, pp107-114. 7. Li, K. and Helm, R. F. Approaches to Synthetic Neolignans, 34th National Organic Symposium, Williamsburg, VA. June 11-15, 1995. Poster 281. / Ph. D.

Six white volunteer males less than 35 years old, who ran less than 35 miles per week completed a loading regimen. This consisted of a succession of mixed, high-fat and highCHO diets for at least 72 hours each. Subjects ran to exhaustion after each diet stage. Mean times to exhaustion were 61, 63 and 95 minutes for the mixed, high-fat, and high-CHO diets, respectively. Since questions have been raised concerning a detrimental effect of CHO-loading on heart function, subjects were closely examined for evidence of negative effects associated with this procedure. An incomplete right bundle branch block (RBBB), sinus arrhythmia, and early repolarization after the mixed diet was noted in three of the six subjects. These changes were not observed during the high-fat nor high-CHO diets. Prior to the high-CHO exhaustive run, it was also found that the width of the QRS complex was significantly more narrow than the mixed and high-fat diets. The above electrocardiographic (ECG) changes were noted as not being clinically significant by an internal medicine physician. No changes were noted in blood pressures, serum free fatty acids (FFA) and post exhaustive run body weights for diets not pre/post exercise bouts. Serum glucose was significantly higher for the pre-run high-CHO diet when compared to the mixed and high-fat diets pre-run values, yet it remained within normal limits. Body weight following the high-CHO diet was significantly greater than during the high-fat and mixed periods. This may be due to water retention occurring with increased glycogen storage. This probably explains the longer time to exhaustion for the high-CHO diet as compared to the mixed and high-fat trials which both yielded similar times to exhaustion. Although research indicates that a mixed diet prolongs the onset of exhaustion more than a high-fat diet, the similar endurance capacity for the high-fat and mixed diets could be related to a learning effect on the treadmill and/or psychological considerations of consuming a high-fat diet. Also since heart rate and blood pressure were not significantly different for the first 30 minutes of exercise while rate of perceived exertion (RPE) indicated the mixed diet trial most demanding and the high-CHO trial least, one might suspect that some other factor besides physiological values, may cause fatigue. In summary, CHO-loading appeared to enhance endurance of the novice runner with no apparent detrimental effects on cardiac function in these six subjects. / Master of Science

Yes / The polysialyltransferases are biologically important glycosyltransferase enzymes responsible for the biosynthesis of polysialic acid, a carbohydrate polymer that plays a critical role in the progression of several diseases, notably cancer. Having improved the chemical synthesis and purification of the fluorescently-labelled DMB-DP3 acceptor, we report optimisation and validation of a highly sensitive cell-free high-throughput HPLC-based assay for assessment of human polysialyltransferase activity.

La dégradation des fibres alimentaires est une fonction essentielle des écosystèmes digestifs microbiens. Chez le ruminant, elle est assurée par des bactéries, champignons et protozoaires capables de produire de nombreuses enzymes nécessaires à l’hydrolyse des polysaccharides de paroi végétale. Parmi les facteurs susceptibles d’influencer l’efficacité de dégradation des fibres, qui est une composante importante de la productivité et de la santé animales, des additifs tels que des levures probiotiques apparaissent comme un levier intéressant. Afin d’approfondir les connaissances sur les facteurs de modulation de l’activité fibrolytique, une biopuce ADN fonctionnelle, outil moléculaire haut-débit, ciblant les gènes codant les enzymes clés de la dégradation de la cellulose et des xylanes dans les écosystèmes digestifs a été développée. Aussi, une méthode efficace dédiée à des échantillons ruminaux pour la soustraction des ARNr à partir des ARN totaux a été mise au point afin d’accroitre la sensibilité de l’outil. La biopuce fonctionnelle a été validée sur échantillons de complexité croissante et démontre d’excellents caractères de spécificité et de sensibilité tout en étant exploratoire et quantitative. Des régulations différentielles de l’arsenal des gènes de la fibrolyse de la bactérie du rumen Fibrobacter succinogenes ont pu être montrées. De même, les résultats sur échantillons de rumen suggèrent un rôle des microorganismes eucaryotes dans la fibrolyse pouvant être plus important qu’initialement envisagé. Cette approche métatranscriptomique dirigée pourra in fine continuer d’être appliquée dans l’étude de l’impact de facteurs biotiques et abiotiques sur la fonction fibrolytique microbienne chez les animaux d’élevage. / Dietary fibre degradation is an essential function of microbial digestive ecosystems. In ruminants, this function is ensured by bacteria, fungi and protozoa, producing a large array of enzymes able to degrade plant cell wall polysaccharides. Among factors likely to influence the efficiency of fibre degradation, which is an important component in animal productivity and health, dietary additives such as probiotic yeasts appear as an interesting tool. To provide more insight on factors modulating fibrolytic activity, we designed a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and xylan degradation by digestive microbiota. Also, an efficient method dedicated to rumen samples for removing microorganisms’ rRNA from total RNA samples was developed to increase the sensitivity of the tool. The DNA microarray was validated using targets of increasing complexity and demonstrated sensitivity and specificity as well as explorative and quantitative potential. Differential expression of genes involved in fibrolysis was evidenced in the rumen bacterium Fibrobacter succinogenes. Moreover, results on rumen samples suggest a more important role of eucaryotes in fibre degradation than previously thought. This targeted metatranscriptomic approach will be further applied to the study of the impact of biotic and abiotic factors on the microbial mechanisms of fibre degradation in livestock.

The genome of Escherichia coli harbors several catabolic operons involved in the utilization of a wide variety of natural compounds as carbon sources. The chitobiose (chu) operons of E.coli Is involved in the utilization of chitobiose(disaccharide of N-acety1-D-glucosamine) and cellbiose (disaccharide of glucose) derived from the two most abundant naturally occurring carbon sources on earth, chitin and cellulose respectively. The operon consists of the chbBCARFG genes coding for transport, regulation and hydrolysis functions required to utilize these compounds; the chuyBCA genes code for a multi-subuni PTS transporter ; the chuR codes for a dual function repressor/activator of the operon; the chbF codes for a phospho-glucosidase and the chbG codes for a protein of unknown function. The chu operon Is regulated by three transcription factors; NagC, a key regulator of the nag genes involved in amino sugar metabolism; ChbR, a dual function operon-specific regulator; and CRP_cAMP. The operon is repressed by NagC and ChbR in the absence of catabolic substrate. In the presence of chitobiose, expression is induced by the abrogation of NagC-mediated repression by GlcNAc-6-P generated by the hydrolysis of chitobiose-6-P and subsequent activation of transcription by ChbR and CPR-cAMP. Wild type E.coli connot utilize cellbiose due to the inability of cellbiose to induce expression from the operon. The simultaneous presence of a loss of function mutation in nagC and a gain –of-function mutation in chbR is necessary and sufficient to allow cellbiose to induce expression and confer on E.coli the ability to utilize cellbiose. The activation step by ChbR and CPR-cAMP requires an inducer that is recognized by ChbR. The chemical identity of the inducer and the mechanism of transcriptional activation by ChbR and CPR-cAMP are not understood. The studies described in the chapter 2 shows that chbG is essential for the utilization of the acetylated sugars chitobiose and chitotriose while it is dispensable for the sugars lacking the acety1group such as cellobiose and chitosan dimer, a disaccharide of N-glucosamine. ChbG is produced as a cytosolic protein and removes one acety1 group from chitobiose and chitotriose thus shows a mono-decetylase activity. Taken together, the observing suggest that ChbG deacetylates chitobiose-6-P and chitotriose-6-P producing the mono-decetylated from of the sugars. The deacetylateion is necessary for their recognition both as inducers by ChbR to activate transcription along with CRP-cAMP and as substractes by phosop-glucosidase ChbF. Cellobiose positive(Cel+) mutants carrying nagC delection and different gain-of-function mutations in chbR are independent of chbG for induction by chitobiose suggesting that the mutations in ChbR can allow it to recognize the acetylated form of chitobiose-6-P. Despite normal induction, the mutants to grow on chitobiose without chbG are consistant with the requirement of deacetylation for hydrolysis by ChbF. The prediction active site of chbG was validated by demonstrating the loss of chbG function upon alanine substitution of the putative metal binding residues. Vibro cholerace ChbG can complement the function of E.coli ChbG indicating that ChbG is conserved in both the organisms. The studies presented in chapter 3 address the mechanism of transcriptional activation of the chb operon by ChbR and CPR-cAMP. ChbR and CPR-cAMP function in a synergistic manner in response to the induction signal. The synergy is not because of their cooperative binding to the DNA. The role of CRP as a class I activator via the known mechanism involving interaction between the Activation region1 (AR1) and the C-terminal domain of the alpha subunit of RNA polymerase (CTD) was not crucial for the chb operon. A direct interaction between the two activators in virto was observed. Based on these results and the close spacing of the synergy is due to interaction between the two regulators bound to DNA that is enhanced in the presence of the inducer, binding about an optimal confirmation in ChbR required to interact with RNA polymerase. ChbR contacts different residues in the subunit in response to cellbiose and chitobiose; whereas it utilizes the known residues in the presence cellbiose, it appears to require different and unknown residues for induction in the presence of chitobiose. In conclusion, the studies reported in chapter 2 and 3 provide an understanding of the regulation of the chitin oligosaccharides utilization in E.coli at different levels. The broad implications of these studies and possible future directions are discussed in chapter 4. ChbG is an evolutionary conserved protein found in both prokaryotes and enkayotes including humans. ChbG homologs have been implicated in inflammatory bowel disorders in humans and development in metazoans. Therefore, the studies on chbG described in this thesis have been broader significance.

The purpose of this study was to determine attitudes toward low carbohydrate diets among consumers and the attributes that influence their purchase decision. A growing number of diet trends are spreading across the nation in an effort to improve health and lose weight such as the Atkins diet and the South Beach diet.