Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.

Conformation-specific crosslinking of mitochondrial complex I

Complex I is the only component of the eukaryotic respiratory chain of which no high-resolution structure is yet available. A notable feature of mitochondrial complex I is the so-called active/de-active conformational transition of the idle enzyme from the active (A) to the de-active, (D) form. Using an amine- and sulfhydryl-reactive crosslinker of 6.8 �� length (SPDP) we found that in the D-form of complex I the ND3 subunit crosslinked to the 39 kDa (NDUFA9) subunit. These proteins could not be crosslinked in the A-form. Most likely, both subunits are closely located in the critical junction region connecting the peripheral hydrophilic domain to the membrane arm of the enzyme where the entrance path for substrate ubiquinone is and where energy transduction takes place.

Characterization, crystallization and preliminary X���ray diffraction analysis of acutohaemolysin, a haemolytic toxin from Agkistrodon acutus venom

Acutohaemolysin, a phospholipase A2 (PLA2) from the venom of the snake Agkistrodon acutus, has been isolated and purified to homogeneity by anion-exchange chromatography on a DEAE-Sepharose column followed by cation-exchange chromatography on a CM-Sepharose column. It is an alkaline protein with an isoelectric point of 10.5 and is comprised of a single polypeptide chain of 13 938 Da. Its N-terminal amino-acid sequence shows very high similarity to Lys49-type PLA2 proteins from other snake venoms. Although its PLA2 enzymatic activity is very low, acutohaemolysin has a strong indirect haemolytic activity and anticoagulant activity. Acutohaemolysin crystals with a diffraction limit of 1.60 �� were obtained by the hanging-drop vapour-diffusion method. The crystals belong to the space group C2, with unit-cell parameters a = 45.30, b = 59.55, c = 46.13 ��, [beta] = 117.69��. The asymmetric unit contains one molecule

Type Synthesis of 3-DOF Multi-Mode Translational/Spherical Parallel Mechanisms with Lockable Joints

A 3-DOF (degrees-of-freedom) multi-mode translational/spherical PM (parallel mechanism) with lockable joints is a novel reconfigurable PM. It has both 3-DOF spatial translational operation mode and 3-DOF spherical operation mode. This paper presents an approach to the type synthesis of translational/spherical PMs with lockable joints. Using the proposed approach, several 3-DOF translational/spherical PMs are obtained. It is found that these translational/spherical PMs do not encounter constraint singular configurations and self-motion of sub-chain of a leg during reconfiguration. The approach can also be used for synthesizing other classes of multi-mode PMs with lockable joints, multi-mode PMs with variable kinematic joints, partially decoupled PMs, and reconfigurable PMs with a reconfigurable platform.

Vulnerability and robustness of SME supply chains: An empirical study of risk and disturbance management of fresh food processors in a developing market

The aim of this paper is to analyse vulnerability and robustness of small and medium size enterprises (SMEs) supply chains and to consider contextual factors that might influence the success of their disturbance management: Risky product and business environment. By using an exploratory case study it is shown how these contextual factors attribute vulnerability sources, contribute to the robustness of a company���s performance and supply chain vulnerability, as well as how a company seeks to manage internal and external vulnerability sources. The exploratory case is based on a fresh food supply chain of a manufacturing SME operating in a developing market.<br/>Case findings suggest that fresh food supply chains of a manufacturing SME in developing markets are prone to disruptions of their logistics and production processes due to ���riskiness��� of fresh food products, the ���riskiness��� of developing markets, as well as ���riskiness��� of SMEs themselves. However, this does not necessarily indicate the vulnerability of an SME and its entire supply chain. Findings indicate that SMEs can be very successful in disturbance management by selective use of redesign strategies that aim to prevent or reduce the impact of disturbances. More precise, it is likely that an SME can achieve robust performance by employing preventive redesign strategies in managing disturbances that result from internal, company related vulnerability sources, while impact reduction strategies are likely to contribute to robust performance of an SME if used to manage disturbances that result from internal, supply chain related vulnerability sources, as well as external vulnerability sources.<br/>

Thermal transport in out-of-equilibrium quantum harmonic chains

<p>We address the problem of heat transport in a chain of coupled quantum harmonic oscillators, exposed to the influences of local environments of various nature, stressing the effects that the specific nature of the environment has on the phenomenology of the transport process. We study in detail the behavior of thermodynamically relevant quantities such as heat currents and mean energies of the oscillators, establishing rigorous analytical conditions for the existence of a steady state, whose features we analyze carefully. In particular, we assess the conditions that should be faced to recover trends reminiscent of the classical Fourier law of heat conduction and highlight how such a possibility depends on the environment linked to our system.</p>

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

Helicobacter pylori is a bacterial pathogen that affects more than half of the world���s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a ���6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The ���-fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind ���4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to ���4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of ���4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.

Antioxidant properties of hydroxycinnamic acids: a review of structure- activity relationships

Hydroxycinnamic acids (HCAs) are important phytochemicals possessing significant biological properties. Several investigators have studied in vitro antioxidant activity of HCAs in detail. In this review, we have gathered the studies focused on the structure-activity relationships (SARs) of these compounds that have used medicinal chemistry to generate more potent antioxidant molecules. Most of the reports indicated that the presence of an unsaturated bond on the side chain of HCAs is vital to their activity. The structural features that were reported to be of importance to the antioxidant activity were categorized as follows: modifications of the aromatic ring, which include alterations in the number and position of hydroxy groups and insertion of electron donating or withdrawing moieties as well as modifications of the carboxylic function that include esterification and amidation process. Furthermore, reports that have addressed the influence of physicochemical properties including redox potential, lipid solubility and dissociation constant on the antioxidant activity were also summarized. Finally, the pro-oxidant effect of HCAs in some test systems was addressed. Most of the investigations concluded that the presence of ortho-dihydroxy phenyl group (catechol moiety) is of significant importance to the antioxidant activity, while, the presence of three hydroxy groups does not necessarily improve the activity. Optimization of the structure of molecular leads is an important task of modern medicinal chemistry and its accomplishment relies on the careful assessment of SARs. SAR studies on HCAs can identify the most successful antioxidants that could be useful for management of oxidative stress-related diseases.

Spectroscopic characterization of a novel 2 /[4Fe /4S] ferredoxin isolated from Desulfovibrio desulfuricans ATCC 27774

Inorganica Chimica Acta 356 (2003) 215-221

Factors influencing the seasonal changes in primary productivity of the photosynthetic bacteria of Crawford Lake, Ontario

A naturally occurring population of photosynthetic bacteria, located in the meromictic Crawford Lake, was examined during two field seasons (1979-1981). Primary production, biomass, light intensity, lake transparency, pH and bicarbonate concentration were all monitored during this period at selected time intervals. Analysis of the data indicated that (l4C) bacterial photosynthesis was potentially limited by the ambient bicarbonate concentration. Once a threshold value (of 270 mg/l) was reached a dramatic (2 to 10 fold) increase in the primary productivity of the bacteria was observed. Light intensity appeared to have very little effect on the primary productivity of the bacteria, even at times when analyses by Parkin and Brock (1980a) suggested that light intensity could be limiting (i.e., 3.0-5.0 ft. candles). Shifts in the absorption maxima at 430 nrn of the .bacteriochlorophyll spectrum suggested that changes in the species or strain composition of the photosynthetic bacteria had occurred during the summer months. It was speculated that these changes might reflect seasonal variation in the wavelength of light reaching the bacteria. Chemocline erosion did not have the same effect on the population size (biomass) of the photosynthetic bacteria in Crawford Lake (this thesis) as it did in Pink Lake (Dickman, 1979). In Crawford Lake the depth of the chemocline was lowered with no apparent loss in biomass (according to bacteriochlorophyll data). A reverse current was. proposed to explain the observation. The photosynthetic bacteria contributed a significant proportion (10-60%) of the lake1s primary productivitya Direct evidence was obtained with (14C) labelling of the photosynthetic bacteria, indica.ting that the zooplankton were grazing the photosynthetic bacteria. This indicated that some of the photosynthetic bacterial productivity was assimilated into the food chain of the lake. Therefore, it was concluded that the photosynthetic bacteria made a significant contribution to the total productivity of Crawford Lake.

Synthetic studies on prostaglandins: "synthesis of a new thia-PGEI analog"

A PGE1 analog, namely (��)-trans-2-(6'-carbomethoxyhexyl)-3- (E-3"-thia-1 "-octene)-4-hydroxycyclopentanone 71, has been prepared for the first time. Towards the synthesis of this compound, several synthetic approaches aimed at the preparation of the required acetylenic and E-halovinylic sulfides as building blocks were investigated. Among all the methods examined, it appeared evident that the best route to ethynyl n.pentyl sulfide 81 is via a double dehydrohalogenation of the corresponding 1,2-dibromoethyl sulfide with sodium amide in liquid ammonia. In addition, the isomerically pure E-2-iodoethenyl n.pentyl sulfide 85 is conveniently prepared in high yield and stereoselectivity by hydrozirconation-iodination of the terminal ethynyl sulfide 81. The classical hydroalumination and hydroboration reactions for the preparation of vinyl halides from alkynes gave only small yields when applied as methods towards the synthesis of 85 . The building block 2-(6'-carbomethoxyhexyl)-4-hydroxy-2- cyclopentenone (��)-1 carrying the upper side-chain of prostaglandin E 1 was prepared by a step-wise synthesis involving transformations of compounds possessing the required carbocyclic framework (see scheme 27). The synthesis proved to be convenient and gave a good overall yield of (��)-1 which was protected as the TH P-derivative 37 or the siloxy derivative 38. With the required building blocks 81 and 37 in hand, the target 1S-thia-PGE1 analog (��)-71 was prepared via the in situ higher cuprate formation-conjugate addition reaction. This method proved to be convenient and stereospecific. The standard cuprate method, involving an organocuprate reagent generated from an isolated vinyl iodide, did not work well in our case and gave a complicated mixture of products. The target compound will be submitted for assessment of bio log ical activity.

The nature of avidy�� in Buddhism and Ved�� nta

This thesis deals with the nature of ignorance as it was interpreted in the Upani~adic tradition, specifically in Advaita Vedanta, and in early and Mahayana Buddhism , e specially in the Madhyamika school of Buddhism. The approach i s a historical and comparative one. It examines the early thoughts of both the upanis.a ds and Buddhism abou t avidya (ignorance), shows how the notion was treated by the more speculative and philosphically oriented schools which base d themselves on the e arly works, and sees how their views differ. The thesis will show that the Vedinta tended to treat avidya as a topic for metaphysical s peculation as t he s chool developed, drifting from its initial e xistential concerns, while the Madhyamika remained in contact with the e xistential concerns evident in the first discourses of the Buddha. The word "notion" has been chosen for use in referring t o avidya, even though it may have non-intellectual and emotional connotations, to avoid more popular a lternatives such as "concept" or "idea". In neither the Upani,ads, Advaita Vedanta, or Buddhism is ignorance merely a concept or an idea. Only in a secondary sense, in texts and speech , does it become one. Avidya has more to do with the lived situation in which man finds himself, with the subjectobject separation in which he f eels he exists, than with i i i intel lect ual constr ucts . Western thought has begun to r ealize the same with concerns such as being in modern ontology, and has chosen to speak about i t i n terms of the question of being . Avidya, however, i s not a 'question' . If q ue stions we r e to be put regarding the nature of a vidya , they would be more of t he sort "What is not avidya?", though e ven here l anguage bestows a status t o i t which avidya does not have. In considering a work of the Eastern tradition, we f ace t he danger of imposing Western concepts on it. Granted t hat avidya is customari ly r endered i n English as ignorance, the ways i n which the East and West view i gno rance di f f er. Pedagogically , the European cultures, grounded in the ancient Greek culture, view ignorance as a l ack or an emptiness. A child is i gnorant o f certain t hings and the purpose o f f ormal education , in f act if not in theory, is to fill him with enough knowledge so that he can cope wit h t he complexities and the e xpectations of s ociety. On another level, we feel t hat study and research will l ead t o the discovery o f solutions, which we now lack , for problems now defying solut i on . The East, on the o t her hand, sees avidya in a d i fferent light.Ignorance isn't a lack, but a presence. Religious and philosophical l iterature directs its efforts not towards acquiring something new, but at removing t.he ideas and opinions that individuals have formed about themselves and the world. When that is fully accomplished, say the sages , t hen Wisdom, which has been obscured by those opinions, will present itself. Nothing new has to be learned, t hough we do have t o 'learn' that much. The growing interest in t he West with Eastern religions and philosophies may, in time, influence our theoretical and practical approaches to education and learning, not only in the established educati onal institutions, but in religious , p sychological, and spiritual activities as well. However, the requirements o f this thesis do no t permit a formulation of revolutionary method or a call to action. It focuses instead on the textual arguments which attempt to convince readers that t he world in which they take themselves to exist is not, in essence, real, on the ways i n which the l imitations of language are disclosed, and on the provisional and limited schemes that are built up to help students see through their ignorance. The metaphysic s are provisional because they act only as spurs and guides. Both the Upanisadic and Buddhist traditions that will be dealt with here stress that language constantly fails to encompass the Real. So even terms s uch as 'the Real', 'Absolute', etc., serve only to lead to a transcendent experience . The sections dealing with the Upanisads and Advaita Vedanta show some of the historical evolution of the notion of avidya, how it was dealt with as maya , and the q uestions that arose as t o its locus. With Gau?apada we see the beginnings of a more abstract treatment of the topic, and , the influence of Buddhism. Though Sankhara' S interest was primarily directed towards constructing a philosophy to help others attain mok~a ( l iberation), he too introduced t echnica l t e rminology not found in the works of his predecessors. His work is impressive , but areas of it are incomplete. Numbers of his followers tried to complete the systematic presentation of his insi ghts . Their work focuses on expl anat i ons of adhyasa (superimposition ) , t he locus and object of ignorance , and the means by which Brahman takes itself to be the jiva and the world. The section on early Buddhism examines avidya in the context o f the four truths, together with dubkha (suffering), the r ole it p l ays in t he chain of dependent c ausation , a nd t he p r oblems that arise with t he doctrine of anatman. With t he doct rines of e arly Buddhism as a base, the Madhyamika elaborated questions that the Buddha had said t e nded not t o edi f ication. One of these had to do with own - being or svabhava. Thi s serves a s a centr e around which a discussion o f i gnorance unfolds, both i ndividual and coll ective ignorance. There follows a treatment of the cessation of ignorance as it is discussed within this school . The final secti on tries to present t he similarities and differences i n the natures o f ignorance i n t he two traditions and discusses the factors responsible for t hem . ACKNOWLEDGEMENTS I would like to thank Dr. Sinha for the time spent II and suggestions made on the section dealing with Sankara and the Advait.a Vedanta oommentators, and Dr. Sprung, who supervised, direoted, corrected and encouraged the thesis as a whole, but especially the section on Madhyamika, and the final comparison.

ORGANOSILICON BIOTECHNOLOGY: A BIO-INSPIRED APPROACH TO THE HYDROLYSIS OF ALKOXYSILANES and THE LIPASE-CATALYZED SYNTHESIS OF SILOXANE-CONTAINING POLYESTERS AND POLYAMIDES

The first part of this thesis studied the capacity of amino acids and enzymes to catalyze the hydrolysis and condensation of tetraethoxysilane and phenyltrimethoxysilane. Selected amino acids were shown to accelerate the hydrolysis and condensation of tetraethoxysilane under ambient temperature, pressure and at neutral pH (pH 7��0.02). The nature of the side chain of the amino acid was important in promoting hydrolysis and condensation. Several proteases were shown to have a capacity to hydrolyze tri- and tet-ra- alkoxysilanes under the same mild reaction conditions. The second part of this thesis employed an immobilized Candida antarctica lipase B (Novozym-435, N435) to produce siloxane-containing polyesters, polyamides, and polyester amides under solvent-free conditions. Enzymatic activity was shown to be temperature dependent, increasing until enzyme denaturation became the dominant pro-cess, which typically occurred between 120-130���C. The residual activity of N435 was, on average, greater than 90%, when used in the synthesis of disiloxane-containing polyesters, regardless of the polymerization temperature except at the very highest temperatures, 140-150���C. A study of the thermal tolerance of N435 determined that, over ten reaction cycles, there was a decrease in the initial rate of polymerization with each consecutive use of the catalyst. No change in the degree of monomer conversion after a 24 hour reaction cycle was found.

Semisynthetic aminoglycoside antibiotics : toward biomimetic synthesis, evasion of bacterial resistance and reduced toxicity

Les antibiotiques aminoglycosidiques sont des agents bact��ricides de grande valeur et d���efficacit�� �� large spectre contre les pathog��nes Gram-positifs et Gram-n��gatifs, dont plusieurs membres naturels et semisynth��tiques sont importants dans l���histoire clinique depuis 1950. Des travaux crystallographiques sur le ribosome, r��compens��s par le prix Nobel, ont d��montr�� comment leurs diverses structures polyamin��es sont adapt��es pour cibler une h��lice d���ARN dans le centre de codage de la sous-unit�� 30S du ribosome bact��rien. Leur interf��rence avec l���affinit�� et la cin��tique des ��tapes de s��lection et v��rification des tARN induit la synth��se de prot��ines �� basse fid��lit��, et l���inhibition de la translocation, ��tablissant un cercle vicieux d���accumulation d���antibiotique et de stress sur la membrane. En r��ponse �� ces pressions, les pathog��nes bact��riens ont ��volu�� et diss��min�� une panoplie de m��canismes de r��sistance enzymatiques et d���expulsion : tels que les N ac��tyltransf��rases, les O phosphotransf��rases et les O nucleotidyltransf��rases qui ciblent les groupements hydroxyle et amino sur le coeur des aminoglycosides; des m��thyl-transf��rases, qui ciblent le site de liaison ribosomale; et des pompes d���expulsion actives pour l�����limination s��lective des aminoglycosides, qui sont utilis��s par les souches Gram-n��gatives. Les pathog��nes les plus probl��matiques, qui pr��sentent aujourd���hui une forte r��silience envers la majorit�� des classes d���antibiotiques sur le bord de la pan-r��sistance ont ��t�� nomm��s des bact��ries ESKAPE, une mn��monique pour Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa et Enterobacteriaceae. La distribution globale des souches avec des m��canismes de r��sistance envers les standards cliniques aminoglycosides, tels que la tobramycine, l���amikacine et la gentamicine, est comprise entre 20 et 60% des isol��es cliniques. Ainsi, les aminoglycosides du type 4,6-disubstitu��s-2-deoxystreptamine sont inad��quats comme th��rapies anti-infectieuses �� large spectre. Cependant, la famille des aminoglycosides 4,5-disubstitu��s, incluant la butirosine, la neomycine et la paromomycine, dont la structure plus complexe, pourrait constituter une alternative. Des coll��gues dans le groupe Hanessian et collaborateurs d���Achaogen Inc. ont d��montr�� que certains analogues de la paraomomycine et neomycine, modifi��s par d��soxyg��nation sur les positions 3��� et 4���, et par substitution avec la cha��ne N1-��-hydroxy-��-aminobutyramide (HABA) provenant de la butirosine, pourrait produire des antibiotiques tr��s prometteurs. Le Chapitre 4 de cette dissertation pr��sente la conception et le d��veloppement d���une strat��gie semi-synth��tique pour produire des nouveaux aminoglycosides am��lior��s du type 4,5 disubstitu��s, inspir�� par des modifications biosynth��tiques de la sisomicine, qui frustrent les m��canismes de r��sistance bact��rienne distribu��es globalement. Cette voie de synth��se d��pend d���une r��action d���hydrog��nolyse de type Tsuji catalys��e par palladium, d���abord d��velopp��e sur des mod��les monosaccharides puis subs��quemment appliqu��e pour g��n��rer un ensemble d���aminoglycosides hybrides entre la neomycine et la sisomicine. Les ��tudes structure-activit�� des divers analogues de cette nouvelle classe ont ��t�� ��valu��es sur une gamme de 26 souches bact��riennes exprimant des m��canismes de r��sistance enzymatique et d���expulsion qui englobe l���ensemble des pathog��nes ESKAPE. Deux des antibiotiques hybrides ont une couverture antibacterienne excellente, et cette ��tude a mis en ��vidence des candidats prometteurs pour le d��veloppement pr��clinique. La th��rapie avec les antibiotiques aminoglycosidiques est toujours associ��e �� une probabilit�� de complications n��phrotoxiques. Le potentiel de toxicit�� de chaque aminoglycoside peut ��tre largement corr��l�� avec le nombre de groupements amino et de d��soxyg��nations. Une hypoth��se de longue date dans le domaine indique que les interactions principales sont effectu��es par des sels des groupements ammonium, donc l���ajustement des param��tres de pKa pourrait provoquer une dissociation plus rapide avec leurs cibles, une clairance plus efficace et globalement des analogues moins n��phrotoxiques. Le Chapitre 5 de cette dissertation pr��sente la conception et la synth��se asym��trique de cha��nes N1 HABA �� substitut��es par mono- et bis-fluoration. Des cha��nes qui poss��dent des ��-N pKa dans l���intervalle entre 10 et 7.5 ont ��t�� appliqu��es sur une neomycine t��tra-d��soxyg��n��e pour produire des antibiotiques avanc��s. Malgr�� la r��duction consid��rable du �� N pKa, le large spectre bact��ricide n���a pas ��t�� significativement affect�� pour les analogues fluor��s isosteriques. De plus, des ��tudes structure-toxicit�� ��valu��es avec une analyse d���apoptose propri��taire d���Achaogen ont d��montr�� que la nouvelle cha��ne ��,�� difluoro-N1-HABA est moins nocive sur un mod��le de cellules de rein humain HK2 et elle est prometteuse pour le d��veloppement d���antibiotiques du type neomycine avec des propri��t��s th��rapeutiques am��lior��es. Le chapitre final de cette dissertation pr��sente la proposition et validation d���une synth��se biomim��tique par assemblage spontan�� du aminoglycoside 66-40C, un dim��re C2 sym��trique bis-imine macrocyclique �� 16 membres. La structure propos��e du macrocycle a ��t�� affin��e par spectroscopie nucl��aire �� un syst��me trans,trans-bis-azadi��ne anti-parall��le. Des calculs indiquent que l���effet anom��rique de la liaison �� glycosidique entre les anneaux A et B fournit la pr��-organisation pour le monom��re 6��� ald��hydo sisomicine et favorise le produit macrocyclique observ��. L���assemblage spontan�� dans l���eau a ��t�� ��tudi�� par la dim��risation de trois divers analogues et par des exp��riences d���entre croisement qui ont d��montr�� la g��n��ralit�� et la stabilit�� du motif macrocyclique de l'aminoglycoside 66-40C.

Large scale identification of protein SUMOylation by mass spectrometry in HEK293 cells

Les fichiers qui accompagnent mon document sont des tableaux suppl��mentaires r��alis��s avec Excel (Microsoft Office), dans la version papier du m��moire ces fichiers sont sur un CD-ROM.