Factores genéticos y ambientales son decisivos en la etiología de la diabetes tipo 1 (DT1). Los virus se han propuesto como un factor ambiental en el desarrollo de esta enfermedad y se han publicado algunas evidencias: incidencia estacional de la enfermedad, siendo más frecuente en otoño e invierno; la presencia de IgM específicas para algunos virus en el suero de pacientes de nuevo diagnóstico; la detección de IFNs de tipo 1 (citocinas antivirales) en páncreas de pacientes diabéticos, la presencia de RNA de enterovirus en el suero de pacientes diabéticos y datos de estudios en modelos animales experimentales.Nuestra hipótesis es que la expresión de la citocina antiviral IFN-beta en los islotes pancreáticos podría inducir una situación inflamatoria local que favorecería la pérdida de tolerancia inmunológica hacia las células beta y daría lugar a una diabetes autoinmune.El objetivo general del trabajo es estudiar el papel del IFN-beta en los fenómenos que conducen a la destrucción de las células beta pancreáticas en la DT1.Para determinar el papel del IFN-beta en el desarrollo de diabetes se han estudiado ratones transgénicos que expresan IFN-beta humano en las células beta pancreáticas.?Se han observado signos de autoinmunidad: hiperexpresión de MHC de clase I en los islotes, infiltración de estos por linfocitos T y B y transferencia de la enfermedad mediante linfocitos. La expresión de beta-2-microglobulina y algunas hormonas pancreáticas en el páncreas en el timo no se ven afectadas por la presencia de HuIFN-beta lo que sugiere que la enfermedad está causada por un efecto local del IFN-beta suficiente para romper la tolerancia periférica hacia las células beta. Hemos generado por primera vez ratones NOD (nonobese diabetic, modelo espontáneo de diabetes autoinmune) y NOR (nonobese resistant, homólogo resistente) que expresan IFN de tipo I en los islotes: estos animales desarrollan una diabetes autoinmune acelerada con una elevada incidencia de la enfermedad. El infiltrado de estos animales diabéticos presenta un elevado porcentaje de células NK que no se observa a nivel periférico en ganglios regionales pancreáticos o bazo. La depleción de este tipo celular implica una prevención de la diabetes acelerada, lo que indica un papel determinante de las células NK en el desarrollo de la enfermedad en este modelo transgénico. Los resultados obtenidos durante la realización de este estudio indican que la citocina anti-viral IFN-beta rompe la tolerancia periférica a las células beta, influencia la progresión de la insulitis y contribuye al desarrollo de autoinmunidad en ratones susceptibles y resistentes a la enfermedad, el background genético determina el momento de inicio clínico y la incidencia de la enfermedad. Estos datos, apoyan indirectamente el posible papel de los virus en la etiología de la DT1, como causantes de una situación inflamatoria mediada inicialmente por IFNs de tipo 1 en la que las células del sistema inmune reconocen 'señales de peligro' de un tejido atacado, lo que puede desembocar en un proceso autoinmune que varia en función del fondo genético del individuo. / Genetic and environmental factors are decisive in the etiology of type 1 diabetes. Viruses have been proposed as a triggering environmental event and some evidences have been reported: seasonality of the disease with peaks in fall and winter; detection of specific IgMs for some viruses in new diagnosed patients; finding of Type 1 IFNs (antiviral cytokines) in pancreases of diabetic patients, detection of enteroviruses RNA in the sera of diabetic patients and data derived from experimental animal models. Our hypothesis is that the expression of the antiviral cytokine IFN-beta in the pancreatic islets could induce a local inflammation favouring the lost of immunological tolerance to pancreatic beta cells and leading to an autoimmune diabetes. The main aim of the study is to elucidate the role of IFN-beta in the phenomena leading to the destruction of pancreatic beta cells in T1D. To determine the role of IFN-beta in diabetes, we studied transgenic mice expressing human IFN-beta in beta cells. Autoimmune features were found: MHC class I islet hyperexpression, T and B cells infiltrating the islets and transfer of the disease by lymphocytes. Moreover, the expression of beta2-microglobulin and some pancreatic hormones in the thymus was not altered by IFN-beta, thus suggesting that the disease is caused by a local effect of IFN-beta strong enough to break the peripheral tolerance to beta cells. This is the first report of the generation of NOD (a model of spontaneous autoimmune diabetes) and nonobese-resistant (its homologous resistant) transgenic mice expressing a type I IFN in the islets: transgenic NOD and nonobese-resistant mice developed accelerated autoimmune diabetes with a high incidence of the disease. The insulitis of these diabetic animals shows an increased percentage of NK cells which is not detected at the periphery in pancreatic regional lymph nodes or spleen. The depletion of his cellular subset implies a prevention of accelerated diabetes thus suggesting and important role of NK cells in the development o the disease in this transgenic model. The results of this study show that antiviral cytokine IFN-beta breaks peripheral tolerance to pancreatic beta cells, influence the insulitis progression and contributes to the development of autoimmunity in prone and non-prone mice. The genetic background influences the onset and the incidence of the disease. Indirectly, these data support the role of viruses in the aetiology of T1D: the viral infection leads to an inflammatory status induced by Type 1 IFNs. Immune system cells recognize 'danger signals' in the infected tissue leading to a different autoimmune process depending on the genetic background of the individual.

<p>Seven transmembrane spanning receptors (7TMRs), or G-protein coupled receptors (GPCRs), represent the largest and most ubiquitous of the several families of plasma membrane receptors and regulate virtually all known physiological processes in humans. The classical paradigm of signal transduction in response to 7TMR stimulation involves an agonist-induced conformational change of the receptor which leads to interaction with and dissociation of the heterotrimeric G-protein into independent Galpha and Gbeta;gamma signaling subunits. Following their activation, 7TMRs are phosphorylated by G-protein coupled receptor kinases (GRKs) and subsequently recruit beta-arrestins. beta-arrestins are multifunctional adaptor proteins which not only desensitize G-protein signals, but also facilitate receptor internalization and mediate numerous signaling pathways on their own. As beta-arrestins universally interact with members of the 7TMR superfamily, we (1) developed an in vitro model system to assess conformational changes that occur in beta-arrestins in response to phosphorylation and (2) to map the sites of phosphorylation on the beta2 adrenergic receptor by different GRKs which would determine the conformation(s) assumed by beta-arrestin and thereby, in turn, instruct its functional capabilities. </p><p>We determined conformational changes in beta-arrestin1 in vitro using limited tryptic proteolysis and MALDI-TOF MS analysis in the presence of a phosphopeptides derived from the C-terminus of the V2 vasopressin receptor (V2Rpp or V2R4p) or the corresponding unphosphorylated peptide (V2Rnp). Upon V2Rpp binding, we show that the previously shielded R393 becomes accessible, which indicates release of the C-terminus. Moreover, we have shown that R285 becomes more accessible and this residue is located in a region of &beta;-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1. We also show a functional consequence of the release of beta-arrestin1's C-terminus by enhanced clathrin binding. In addition, we have shown marked protection of beta-arrestin1's N-domain in the presence of V2Rpp; consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. Using a differentially phsophorylated V2R peptide (V2R4p), we show that beta-arrestin1 is able to adopt distinct conformations in response to different phosphorylation patterns. Futhermore, a striking difference is observed in the conformation of V2Rpp-bound beta-arrestin1 when compared to beta-arrestin2, namely the flexibility of the inter-domain hinge region. These data represent the first direct evidence that the beta-arrestin1 conformation is differentially instructed by phosphorylation patterns and that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.</p><p>Phosphorylation of 7TMRs by GRKs plays essential roles in regulation of receptor function by promoting interactions of the receptors with beta-arrestins. We hypothesized that different GRKs phosphorylate distinct sets of sites thereby establishing a "bar code." In order to test this hypothesis, we monitored the phosphorylation events of the beta2AR upon stimulation with a classical full agonist, isoproterenol, or a beta-arrestin "biased" agonist, carvedilol, in the presence of a full complement of GRKs or when individual GRKs (2 or 6) were depleted by siRNA. We demonstrate that at least thirteen sites on the beta2AR show changes in phosphorylation in response to the agonist isoproterenol. Of these, phosphorylation increased 10 to more than 300 fold in 12 (S261, S262, S345, S346, S355, S356, T360, S364, S396, S401, S407 AND S411) and decreased 50% in one (S246). Depletion of GRK2 or 6 by siRNA indicates that S355, 356 are GRK6 sites whereas the remainder are GRK2 sites. Phosphorylation of GRK2 sites inhibits that of GRK6 sites. Carvedilol, a beta-arrestin biased agonist, promotes phosphorylation of only the GRK6 sites S355, 356. In HEK293 cells, GRK2 phosphorylation is found to be the major positive regulator of receptor internalization; to contribute to receptor desensitization; and to inhibit beta-arrestin mediated ERK activation. Phosphorylation of the two GRK6 sites contributes to receptor desensitization and internalization and is required for beta-arrestin mediated ERK activation. These data indicate that different ligands promote distinct patterns of receptor phosphorylation which dictate different patterns of beta-arrestin mediated function.</p> / Dissertation

Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

Beta-carotene presence or absence in muskmelon is controlled by two genes, green flesh gf and white flesh wf. In its dominant form the wf gene is responsible for orange flesh color; however, the epistatic interactions of gf and wf can create three flesh colors: orange, white and green. Two F2 populations, consisting of 77 greenhouse grown and 117 field grown plants, from the cross of ‘Sunrise’ (white fleshed) by ‘TAM Uvalde’ (orange fleshed), were used to examine the relationships of beta-carotene content, flesh color, and flesh color intensity. Bulk segregent analysis was used with RAPD markers to identify molecular markers associated with high beta-carotene content. Flesh color and flesh color intensity both had significant relationships with beta-carotene content. A significant correlation between total soluble solids and beta-carotene content was also found. Molecular markers were identified in both F2 populations and all significant, associated markers from ‘TAM Uvalde’ were linked with WF. A single QTL was also found to be linked with the WF locus. The identified QTL can be used to screen potential breeding lines for high beta-carotene. It was also confirmed that the visual ratings of flesh color intensity can be reliably used to select high beta-carotene content melons.

Mechanical unloading and food restriction (FR) are leading causes of bone loss, which increase the risk of fracture later in life. Leptin, a 16kDa cytokine like hormone principally produced by white adipocytes, may be involved in bone metabolism with physiological or mechanical changes causing bone loss. The hypotheses of the first study were aimed at determining if serum leptin is reduced by unloading or FR. The serum leptin level reduced by unloading or by global FR, is associated with the decline in bone formation rate. It was conjectured that decreased serum leptin may be due to reduced adipocyte number/size and/or sympathetic nervous system (SNS) activation of betaadrenoreceptors with unloading or FR, inhibiting the release of leptin from adipocytes. In the second experiment, we tested whether leptin or beta-adrenoreceptor blockade attenuates bone loss during unloading and whether such an effect due to beta blockade is associated with changes in serum leptin level. Beta-blockade mitigated unloading induced reduction in serum leptin and also beta blockade was as effective as leptin administration in mitigating a reduction in cancellous bone mineral density with unloading through both stimulation of bone formation and suppression of resorption. It was previously demonstrated that energy restriction (ER) is a major contributor to the bone loss during global FR. In the third study, we tested whether beta- blockade attenuates bone loss during ER and whether such an effect is associated with changes in serum leptin level and leptin localization in bone tissues. Beta blockade attenuated the ER induced reduction in serum leptin level, cancellous bone mineral density and bone formation rate, and also abolished the ER induced increase in bone resorption. Reduction in leptin expression in bone marrow adipocytes observed with ER was attenuated by beta-blockade. Reduction in the number of cells (bone lining cells, osteocytes and chondrocytes in cartilage) which are stained positive for leptin was also attenuated by beta-blockade. Collectively, these data identify circulating leptin effects on preventing bone loss during mechanical unloading or energy restriction. Also beta blockade is associated with mitigating reduction in serum leptin and subsequently with mitigating reduction in bone mass with unloading or ER.

Expanding the uses of the NCAL and finding the spectrum of substrates best suited for such a transformation has been the main effort of my research. Previous studies had focused on aldedydes as the requisite functionality that would provide the needed electrophilicity in order to complete the aldol; however, recent advancements have introduced ketones as a viable carbonyl. With an established protocol in hand, I set out to explore various substrates that could yield Beta-lactones in good to moderate yields such as amino acid derivatives, diones, and large cyclic formations as well as simple, straight chain acids with varying groups Alpha to the ketone. In general, I was able to establish a basic framework of substrates that are highly and/or moderately susceptible towards the NCAL and current studies continue to further expand the scope. In addition to making Beta-lactones, I investigated alkyl cuprates as soft nucleophiles to afford addition at the Beta carbon yielding a variety of acids. Substrates for cuprate additions have been expanded to bulkier and multi-cyclic Beta-lactones and applied to the synthesis of a Merck IND intermediate. Additions to bi- and tri-chloro Beta-lactones due to the presence of the resulting moity in natural products are currently being studied.

Infectious agents such as bacteria or viruses can grow rapidly. If a microorganism invades a host, it must be recognized rapidly and destroyed before it overwhelms the immune system. Limiting infection to a minimum in the early stage is critical for the outcome and the recovery from infection. The innate immune system has evolved to recognize a few highly conserved, constitutive structures present only in microorganisms, such as bacterial lipopolysaccharide (LPS), called pathogen-associated molecular patterns (PAMP). Toll-like receptors are the host receptors that recognize PAMP, ultimately activating a variety of transcription factors to induce expression of a wide spectrum of immune related genes, e.g. defensins. Defensins are antimicrobial peptides that play an important role in innate defense against microorganisms in plants and animals. Beta-defensins are the largest family of antimicrobial peptides, which can directly kill microorganisms and have regulatory effects on the immune system. Thirteen beta-defensins have been identified; however, the regulation of these genes has not been well-investigated in the chicken. The objective of this research was to understand constitutive and inducible gene expression of beta-defensins in chicken white blood cells. Real-time RT-PCR was used to quantify gene expression level before and after LPS stimulation. Transcription factor binding sites in the genes were identified to understand the gene expression regulation. From the expression profile results, most chicken beta-defensins had induced gene expression by LPS stimulation in the early phase (0- to 3-hour) and reduced gene expression in the late phase (3- to 8-hour). As for the level of gene expression, the results show that the induced gene expression in the early phase corresponded to the higher levels of expression at 3-hours after LPS stimulation, and the reduced gene expression in the late phase corresponded to the lower levels of gene expression at 8-hours after LPS stimulation.

Contamination of seafood by marine toxins has been a consistent public health problem. Gymnodimine (GYM) is a member of a family of spirocyclic imine containing marine natural products which was shown to be highly toxic (LD50 96 mg/kg, intraperitoneal injection); thus ensuring public safety requires stringent monitoring of gymnodimine. Current detection methods for GYM and spirolides include the mouse bioassay and LC-MS-based detection techniques which, however, have significant limitations. Therefore, more efficient and convenient detection methods are required. Building on our recently completed total synthesis of (-)-gymnodimine, the synthesis of two haptens were targeted for eventual production of monoclonal antibodies (mAb) to be used in an eventual Enzyme-Linked Immunosorbent Assay (ELISA) for gymnodimine. As an extension of the intramolecular nucleophilic catalyzed aldol lactonization (NCAL) process from aldehyde acid to keto acid substrates, carbocyclic and nitrogen heterocyclic B-lactones were synthesized. Demonstration of the utility of the NCAL process for keto acids was applied to the synthesis of dihydroplakevulin A and the core of tussilagine. In addition, although initial attempts to develop guanidine catalysts for the asymmetric NCAL process were unsuccessful, homobenzotetramisole (HBTM) was found to be a suitable asymmetric catalyst for keto acid substrates. Finally, synthetic studies toward the proposed structure of thiolyngbyan are described. Thiolyngbyan was isolated from a blue-green algae and it exhibited antifungal activity.

The recent finding that the FDA-approved antiobesity agent orlistat (tetrahydrolipstatin, Xenical) is a potent inhibitor of the thioesterase domain of fatty acid synthase (FAS) led us to develop a concise and practical asymmetric route to pseudosymmetric 3,4-dialkyl-cis-beta-lactones. The well-documented upregulation of FAS in cancer cells makes this enzyme complex an interesting therapeutic target for cancer. The described route to 3,4-dialkyl- beta -lactones is based on a two-step process involving Calter's catalytic, asymmetric ketene dimerization of acid chlorides followed by a facialselective hydrogenation leading to cis-substituted- beta -lactones. Importantly, the ketene dimer intermediates were found to be stable to flash chromatography, enabling opportunities for subsequent transformations of these optically active, reactive intermediates. Subsequent R-epimerization and R-alkylation or acylation led to trans- beta - lactones and beta -lactones bearing alpha-quaternary carbons, respectively. Several of the ketene dimers and beta-lactones displayed antagonistic activity (apparent Ki in the low micromolar range) in competition with a fluorogenic substrate toward a recombinant form of the thioesterase domain of fatty acid synthase. The best antagonist, a simple phenyl-substituted cis- beta -lactone, displayed an apparent Ki (2.5 ( 0.5 muM) of only 10- fold lower than that of orlistat (0.28 ( 0.06 muM). In addition, mechanistic studies of the ketene dimerization process by Reaction View infrared spectroscopy support previous findings that ketene formation is rate determining. A highly diastereoselective, nucleophile-promoted bis-cyclization process, employing readily available and tractable keto-acid substrates, is described. This methodology provides concise access to bicyclic- and tricyclic-beta-lactones bearing tertiary carbinol centers and quaternary carbons, greatly extending the scope of previous routes to bicyclic-beta-lactones from aldehyde acid substrates. This and related processes may be revealing a subtle interplay between [2 plus 2] cycloaddition and nucleophilecatalyzed aldol lactonization (NCAL) reaction manifolds. An early induction period in the bis-cyclization of keto-acids is confirmed via isolation of the complex between 4- pyrrolopyridine and Modified Mukaiyama reagent N-propyl-2-bromo pyridinium triflate. Dyotropic rearrangements of tricyclic keto beta-lactones derived in high yields and >19:1 diastereoselectivity from readily available 1, 3-dione acids is described. Zn (II) salts were found to be most efficient for affecting dyotropic 1, 2-acyl migrations where as sub stoichiometric TMSOTf was found to execute a delta-lactone migration providing bis gamma-lactone in modest yields. Enantioselective desymmetrization with inexpensive (S) - tetramisole has been demonstrated to provide direct evidence of Lewis base involvement in the Nucleophile Promoted Bis-cyclization of keto-acids. Further studies using TsCl as the carboxylate activating agent instead of modified Mukaiyama reagent and catalytic tetramisole are described for achieving practical, catalytic, enantioselective synthesis of beta-lactones from keto-acids. Preliminary studies toward conjugate addition- lactonization pathway provided a hint as to the complexity involved to affect this transformation under the bis-cyclization conditions. An alternate hypotheses concerning the possibility of isomerization-dienolate formation - lactonization is experimentally proven. Additionally, applications of these and related findings in the intramolecular Morita-Baylis-Hillman reaction with cyclic ketones have been investigated which provide new avenues of synthetic methodology development.

Les ?-(1,3)-glucanes se rencontrent chez diverses espèces végétales et ont été reconnus pour leurs activités immunostimulantes chez les mammifères, ainsi que leur rôle dans le système de défense des plantes. Le premier chapitre de ce manuscrit est un rappel bibliographique sur les glycoside-hydrolases et, particulièrement sur les glycosynthases, et présente également une introduction aux "glucan-binding proteins", enzymes impliquées dans la reconnaissance des ?-glucanes, ainsi qu'un état des lieux des connaissances acquises sur les ?-(1,3)-glucanes et ?-(1,3)-gluco-oligosaccharides. Le deuxième chapitre décrit le travail réalisé au CERMAV (Grenoble), afin de disposer des outils nécessaires à l'étude des mécanismes d'action des ?-(1,3)-glucanes et des ?-(1,3)-gluco-oligosaccharides. Une méthode de synthèse de ?-(1,3)-gluco-oligosaccharides linéaires, de DP contrôlé, à l'aide de la ?-(1,3)-glucosynthase GII E231G, ainsi que la synthèse d'un substrat fluorescent destiné au dosage d'activité endo-?-(1,3)-glucanase, ont été mises au point. Le troisième chapitre traite des ?-glucan-binding proteins, et décrit le travail effectué à l'institut botanique de la LMU (Munich), dans le cadre du projet de recherche européen SACC-SIG-NET (n°HPRN-CT-2202-00251). Cette recherche concerne d'une part, l'étude de la ?-glucan-binding protein de Glycine max, et a permis de déterminer pour la première fois le mécanisme moléculaire d'hydrolyse d'une glycoside-hydrolase appartenant à la famille GH-81. D'autre part, un travail de clonage de la famille de gènes Gbps de Medicago truncatula, plante modèle pour les Fabacées, a été réalisé.