No description available.

The first section of this dissertation (Chapter I-III) describes the development of new methodologies for the rhodium catalyzed C-N bond formation between sp3 hybridized carbon atom of phenyl substituted alpha-lactams and the nitrogen atom of indole derivatives. Phenyl substituted alpha-lactams generated in situ from the corresponding alpha-bromoamides reacted with indoles in the presence of rhodium catalyst to afford the ring opening products of alpha-lactams. The scope of this methodology was extended to various types of indole derivatives including electron donating and withdrawing substituents. Furthermore, a series of functionalized phenyl substituted alpha-lactams generated in situ reacted with indole to assess the viability of this methodology. The developed method provides an atom-economical approach for the formation of substituted alpha-amino amides in good to excellent yields. The main goal of the research described in the second section (Chapter IV-VII) is the synthesis of the corannulene-based molecular receptors with polar tethers and their immobilization on silica gel. First, we have considered a preparation of bis-corannulenoanthracene, formally possessing the pentacene core as a potential precursor for a series of barrelene based bis-corannulene receptors with polar groups. Bis-corannulenoanthracene was synthesized by the double Diels-Alder cycloaddition of isocorannulenofuran with bis-benzyne precursor, followed by deoxygenation of the endoxide adducts. While bis-corannulenoanthracene is stable enough to be isolated and stored, its pentacene core undergoes facile cycloaddition with maleic anhydride to afford bis-corannulene molecular receptor with the barrelene tether adorned with the anhydride moiety. The 1H NMR titration experiments carried out in chlorobenzene-d5 proved the high binding affinity of the receptor toward C60. In addition, the presence of polar anchors on its tether allowed for its deposition on silica gel through the (3-aminopropyl)triethoxysilane linker.

Yes / Fused cyclobutanes are found in a range of natural products and formation of these motifs in a straightforward and easy manner represents an interesting synthetic challenge. To this end we investigated an intramolecular variant of the thermal enamine [2 + 2] cyclisation, developing a diastereoselective intramolecular enamine [2 + 2] cyclisation furnishing δ lactone and lactam fused cyclobutenes in good yield and excellent diastereoselectivity. / The work was funded by Yorkshire Cancer Research

Polyketides are a class of natural products that exhibit remarkable structural and functionally diversity and are highly sought after due to their medicinally important activities. For many decades now, polyketide synthases (PKSs), the mega-enzymes responsible for biosynthesis of polyketides have been the focus of extensive investigation to make new polyketides by polyketide engineering strategies. While there are many established methods to investigate polyketide enzymes and biosynthesis mechanisms, they have substantial shortcomings that have limited the extent of success with polyketide engineering efforts. This thesis focuses on developing simple, flexible yet powerful tools for examining polyketide biosynthesis by overcoming some deficiencies in currently used techniques. Reactive heterocylces have been designed for direct labeling of key polyketide synthase enzymes to provide a direct insight into its functions and mechanisms. First β-lactones and then β-lactams have been used as small molecule probes to perform site-specific labeling of acyl carrier proteins and further used for mechanistic interrogation of key steps in polyketide biosynthesis. The utility of these probes has been demonstrated by comparison to traditional probes and has been successfully applied to examine substrate selectivity of keto synthases, key enzymes in polyketide biosynthesis. The applications of the tools described in this manuscript only scratch the surface of their capabilities and are expected to significantly aid in the study of new and existing PKS systems leading to improved understanding of how these extraordinary biosynthetic machines function.

No description available.

Tesis por compendio / [EN] About 10 % of all adverse drug reactions are due to allergies, with ß-lactam antibiotics causing the majority of the episodes. Although the actual incidence remains unknown, individuals suspected of being allergic to a drug end up being prescribed with other medications that are less effective, more expensive or harmful. Consequently, a correct diagnosis is key to reduce the derived economic costs and proceed to an adequate 'delabeling' of the population. At present, clinical approaches to diagnose allergies to ß-lactam antibiotics are based on in vivo and in vitro tests. These tests present limited clinical performances since they are invasive, dangerous, and provide false positives and/or negatives. Moreover, the diagnostic sensitivity is far from what is expected, possibly because the epitopes that cause the allergic episodes are still not well detected. In this respect, the preparation of antigens has commonly been determined by the direct attachment of antibiotics to carrier molecules through the formation of an amide bond between amino lysine groups of the carrier molecule and the carboxylate group of the antibiotic. Even so, specific IgE are barely detected with such antigens. This dissertation addresses the synthesis of haptens and the generation of antigens to ß-lactam antibiotics, which develop a more reliable in vitro diagnosis of allergies to these drugs. The evaluation of the antigens has been carried out by means of multiplex in vitro tests based on compact disc technology. This research begins by focusing on the synthesis and preparation of penicillin antigens. To this end, first, the effect of the incorporation of aliphatic spacer arms in the chemical structure of penicillin has been approached, considering the possibility that a better molecular recognition is obtained by moving the hapten away from the carrier protein. Thirteen haptens derived from benzylpenicillin and amoxicillin were synthesized in order to prepare antigens with human serum albumin. The evaluation of the antigens revealed that even though they were immunogenic and were detected by the raised IgG antibodies, they were not detected by specific IgE from allergic patients. Additionally, the next approach considered the cationization of the carrier proteins, human serum albumin and histone. The modification of carboxylate groups of the protein to amino groups allow for an increase of the molar hapten/protein ratio. This strategy led to the generation of five antigens, four of which (only those histone-based antigens), did increase the sensitivity of the assay. Concretely, specific IgE has been determined in sera from allergic patients at low concentrations (LOD = 0.07 IU/mL) with a diagnostic specificity of 100 % and a sensitivity of 60 and 31 % for benzylpenicillin and amoxicillin, respectively. That means a 60 % improvement in the diagnostic sensitivity when compared to the in vitro reference test. Subsequently, the idea of preparing minor antigens based on penicillin metabolites was approached. Penicilloic, penilloic, penicillic, and 6-aminopenicillanic acids, together with penicillamine, were therefore conjugated to the carrier proteins human serum albumin and histone. Except penilloic acid, the rest of antigens were selectively detected when testing a set of serum samples from allergic patients. The diagnostic specificity obtained was 100 %, 94 % in the case of penicillic acid, and the sensitivity was between 67 and 100 %. Another approach was focused on the production of antigens for other families of ß-lactam antibiotics. The generation of antigens for cephalosporins, carbapenems, monobactams or ß-lactam inhibitors is essential, since in vitro tests for the detection of allergies to these antibiotics are not commercially available. Therefore, the results obtained after the preparation of major and minor antigens for the antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem, and aztreonam were evaluated. / [ES] Alrededor del 10 % de las reacciones adversas a medicamentos son debidas a alergias, siendo los antibióticos ß-lactámicos los que más episodios alérgicos ocasionan. Aunque la incidencia real sigue siendo desconocida, los individuos sospechosos de presentar alergia a algún medicamento acaban siendo prescritos con otros medicamentos, menos efectivos, más caros o perjudiciales. Así pues, un correcto diagnóstico resulta clave para disminuir los costes económicos derivados y proceder a un adecuado 'desetiquetado' de la población. En la actualidad, las pruebas de diagnóstico de alergias a antibióticos ß-lactámicos se basan en ensayos in vivo e in vitro. Estos ensayos muestran bajas prestaciones, ya que son invasivos y peligrosos y proporcionan falsos positivos y/o negativos. Además, la sensibilidad diagnóstica está lejos de ser la esperada, posiblemente porque aún no se ha conseguido reconocer todos los epítopos causantes de los episodios alérgicos. En este sentido, la preparación de antígenos se ha basado hasta el momento, en mayor medida, en la unión directa de los antibióticos a las moléculas portadoras mediante la formación de un enlace amida entre los grupos amino de las lisinas de la molécula portadora y el grupo carboxilato del antibiótico. Aun así, las IgE específicas son vagamente detectadas con estos antígenos. En esta tesis se ha abordado la síntesis de haptenos y la generación de determinantes antigénicos a antibióticos ß-lactámicos con los que poder realizar un diagnóstico in vitro más fiable de alergias a estos fármacos. La evaluación de los mismos se ha llevado a cabo mediante ensayos in vitro multiplex basados en tecnología de disco compacto. Esta investigación comienza centrándose en la síntesis y preparación de antígenos de penicilina. Para ello, en una primera fase se ha estudiado el efecto de la incorporación de brazos espaciadores alifáticos en la generación de antígenos, considerando la posibilidad de que se obtenga un mejor reconocimiento molecular al alejar el hapteno de la proteína portadora. Se sintetizaron trece haptenos derivados de bencilpenicilina y amoxicilina con los que se prepararon antígenos con la proteína albumina de suero humano. La evaluación de los antígenos reveló que a pesar de ser suficientemente inmunogénicos y ser reconocidos por anticuerpos IgG de conejo, éstos no fueron reconocidos por IgE específicas de muestras de pacientes alérgicos. Así bien, por otro lado, la estrategia de cationización de las proteínas albumina de suero humano e histona fue abordada teniendo en cuenta que la modificación de grupos carboxilatos de la proteína a grupos amino aumenta la relación molar hapteno/proteína. Esta estrategia permitió la generación de 5 antígenos, 4 de los cuales (los antígenos de histona), esta vez sí, incrementaron la especificidad de la respuesta inmunológica obtenida, reconociendo IgE específicas. Concretamente, se han determinado IgE específicas en suero de pacientes alérgicos a bajas concentraciones (LOD = 0.07 IU/mL) con una especificidad diagnóstica del 100 % y una sensibilidad del 60 y 31 % para bencilpenicilina y amoxicilina, respectivamente, mejorando la sensibilidad un 60 % en comparación con el ensayo in vitro de referencia. A pesar de las mejoras obtenidas con las estrategias llevadas a cabo, se estudiaron otras vías no clásicas para la síntesis de nuevos haptenos con mayor diversidad química. Este enfoque se basa en la generación de antígenos en librerías químicas de compuestos con diversidad estructural para encontrar nuevos haptenos biológicamente activos. Dichas estrategias, hasta el momento, no han sido empleadas para la generación de antígenos y el análisis de muestras de suero de pacientes alérgicos. Con el fin de incorporar diversidad estructural, se sintetizaron, mediante la técnica combinatoria diversity-oriented synthesis, 22 compuestos de los precursores de las penicilinas y cefalosporinas, ácido 6-aminopenicilánico y ácido 7-amino-desacetoxicefalosporánico, respectivamente, y de los antibióticos amoxicilina y ampicilina. Su evaluación con el inmunoensayo in vitro basado en disco compacto ha demostrado que la incorporación de diversidad permite el reconocimiento de epítopos causantes de episodios alérgicos. Concretamente, se observó que estos antígenos eran capaces de detectar anticuerpos tipo IgG e IgE específicos procedentes de suero de conejos inmunizados y de suero humano de pacientes alérgicos, siendo especialmente selectivos los determinantes de amoxicilina y ampicilina. Concretamente, se obtuvo una sensibilidad diagnóstica del 79 % y una especificidad diagnóstica del 100 % / [CA] Al voltant del 10% de les reaccions adverses a medicaments són degudes a al·lèrgies, sent els antibiòtics ß-lactàmics aquells que més episodis al·lèrgics ocasionen. Encara que la incidència real continua sent desconeguda, els individus sospitosos de presentar al·lèrgia a algun medicament acaben sent prescrits amb altres medicaments, menys efectius, més cars o perjudicials. Així doncs, un correcte diagnòstic resulta clau per a disminuir els costos econòmics derivats i procedir a un adequat 'desetiquetatge' de la població. En l'actualitat, les proves de diagnòstic d'al·lèrgies a antibiòtics ß-lactàmics es basen en assaigs in vivo i in vitro. Aquests assaigs mostren baixes prestacions, ja que són invasius i perillosos i proporcionen falsos positius i/o negatius. A més a més, la sensibilitat diagnòstica està lluny de ser l'esperada, possiblement perquè encara no s'ha aconseguit reconéixer tots els epítopes causants dels episodis al·lèrgics. En aquest sentit, la preparació d'antígens s'ha basat fins al moment, en major mesura, en la unió directa dels antibiòtics a les molècules portadores mitjançant la formació d'un enllaç amida entre els grups amino de les lisines de la molècula portadora i el grup carboxilat de l'antibiòtic. Així i tot, les IgE específiques són vagament detectades amb aquests antígens. En aquesta tesi s'ha abordat la síntesi d'haptens i la generació de determinants antigènics a antibiòtics ß-lactàmics amb els quals poder realitzar un diagnòstic in vitro més fiable d'al·lèrgies a aquests fàrmacs. La seua avaluació s'ha dut a terme mitjançant assaigs in vitro multiplex basats en tecnologia de disc compacte. Aquesta investigació comença centrant-se en la síntesi i preparació d'antígens de penicil·lina. Per a això, en una primera fase s'ha estudiat l'efecte de la incorporació de braços espaiadors alifàtics en la generació d'antígens, considerant la possibilitat que s'obtinga un millor reconeixement molecular en allunyar l'haptè de la proteïna portadora. Es van sintetitzar tretze haptens derivats de bencilpenicil·lina i amoxicil·lina amb els quals es van preparar antígens amb la proteïna albúmina de sèrum humà. L'avaluació dels antígens va revelar que malgrat ser prou immunogènics i ser reconeguts per anticossos IgG de conill, aquests no van ser reconeguts per IgE específiques de mostres de pacients al·lèrgics. Així bé, d'altra banda, l'estratègia de cationització de les proteïnes albúmina de sèrum humà i histona va ser abordada tenint en compte que la modificació dels grups carboxilats de la proteïna a grups amino augmenta la relació molar hapten/proteïna. Aquesta estratègia va permetre la generació de 5 antígens, 4 dels quals (els antígens d'histona), aquesta vegada sí, van incrementar l'especificitat de la resposta immunològica obtinguda, reconeixent IgE específiques. Concretament, s'han determinat IgE específiques en sèrum de pacients al·lèrgics a baixes concentracions (LOD = 0.07 IU/mL) amb una especificitat diagnòstica del 100 % i una sensibilitat del 60 i 31 % per a bencilpenicil·lina i amoxicil·lina, respectivament, millorant la sensibilitat un 60 % en comparació amb l'assaig in vitro de referència. Malgrat les millores obtingudes amb les estratègies dutes a terme, es van estudiar altres vies no clàssiques per a la síntesi de nous haptens amb major diversitat química. Aquest enfocament es basa en la generació d'antígens en llibreries químiques de compostos amb diversitat estructural per a trobar nous haptens biològicament actius. Aquestes estratègies, fins al moment, no han sigut emprades per a la generació d'antígens i l'anàlisi de mostres de sèrum de pacients al·lèrgics. Amb la finalitat d'incorporar diversitat estructural, es van sintetitzar, mitjançant la tècnica combinatòria diversity- oriented synthesis, 22 compostos dels precursors de les penicil·lines i cefalosporines, àcid 6-aminopenicilànic i àcid 7-amino-desacetoxicefalosporànic, respectivament, i dels antibiòtics amoxicil·ina i ampicil·lina. La seua avaluació amb l'immunoassaig in vitro basat en disc compacte ha demostrat que la incorporació de diversitat permet el reconeixement d’epítops causants d'episodis al·lèrgics. Concretament, es va observar que aquests antígens eren capaços de detectar anticossos tipus IgG i IgE específics procedents de sèrum de conills immunitzats i de sèrum humà de pacients al·lèrgics, sent especialment selectius els determinants d’amoxicil·lina i ampicil·lina. Concretament, es va obtindre una sensibilitat diagnòstica del 79 % i una especificitat diagnòstica del 100 %. / This work was supported by the H2020 program (project COBIOPHAD, grant agreement No. 688448 awarded to A.M.), being an initiative of the Photonics Public Private Partnership; Agencia Estatal de Investigación Agencia Estatal de Investigación (CTQ2016-75749-R, FEDER) (PID2019-110713RB-I00, FEDER) awarded to S.M.; Generalitat Valenciana (PROMETEO/2020/094 awarded to A.M. & S.M.); program UPV-La FE 2019 (P105 VALBIOAL awarded to S.M & E. I-E.); and the National Institute of General Medical Sciences (GM-1R35GM127045 awarded to S.L.S.). E.P.M. was supported by a FPU fellowship from the Ministerio de Educación, Cultura y Deporte (FPU15/01738 and EST18/00360). B.K.H. was supported by a fellowship from the National Science Foundation (DGE1144152 and DGE1745303). The authors acknowledge the Instituto de Investigación Sanitaria La Fe, Valencia, Spain, which provided the samples from both allergic patients and controls. / Peña Mendizabal, E. (2022). Synthesis and Evaluation of Antigenic Determinants for ß-lactam Allergy Diagnosis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182979 / TESIS / Compendio

The synthesis of p-lactams has been of foremost importance since the discovery of penicillin by Sir Alexander Fleming, in 1928, and its susequent structure elucidation in 1945. Ethyl N-2-bromo-alkylcarbamates show considerable potential as precursors to p- lactams. In the past, p-lactams have been prepared by many methods, none of which have involved 2-3 bond formation. The proposed ring closure using ethyl N-2-bromoalkylcarbamate involves 2-3 bond formation, making this method of synthesis novel. This work describes two attempted methods of cyclisation. The first using a Grignard reagent, and the second, using abstraction of an acidic proton a to a phosphonate group. These methods of intramolecular cyclisation were based on analogous intermolecular additions, which are also described. The second method was also used to determine the general potential of ethyl N-bromo- alkylcarbamtes as precursors to other heterocyclic systems. / Thesis (M.Sc.)-University of Natal, Durban, 2001. / NRF & NRF/DEA & T.

IIntrodução A utilização excessiva de antimicrobianos na medicina humana, veterinária e agricultura resultou no aparecimento da resistência bacteriana. Este fenômeno gera problemas de saúde pública que podem resultar na reemergência de doenças infecciosas. O uso de antibióticos, principalmente de forma profilática, tornou-se prática na aquicultura, como ocorre no Brasil, onde a regulamentação para o uso de medicamentos veterinários é ineficiente. Além disso, há evidências da transferência de organismos resistentes para humanos por meio do consumo de produtos de origem animal, quando, durante a fase de criação e produção destes foram administrados antibióticos. Objetivo Pesquisar a ocorrência de genes de resistência a antibióticos em filés de tilápia comercializados em supermercados do município de São Paulo SP. Material e Métodos Foram coletadas 10 amostras de filé de tilápia e realizada a pesquisa de coliformes termotolerantes como indicadores das condições higiênico-sanitárias do alimento. Em seguida, as amostras foram inoculadas em Caldo Lúria 0,5 por cento e o DNA total das bactérias cultivadas nesse meio foi extraído por meio de choque térmico para pesquisa de genes de resistência aos antibióticos -lactâmicos e tetraciclinas pela PCR. Os genes identificados pela PCR foram confirmados pelo sequenciamento. Resultados Em 100 por cento das amostras analisadas o resultado para coliformes termotolerantes foi < 3 NMP.g-1. Na pesquisa de genes de resistência a -lactâmicos, o gene blaOXY-5 foi detectado em 90 por cento das amostras, blaTEM-1b em 20 por cento , blaLEN-16 em 10 por cento , blaSHV-28 em 10 por cento , blaKPC-2 em 20 por cento , blaMOX-6 em 10 por cento e blaCphA em 60 por cento . Os genes de resistência a tetraciclinas identificados foram tetB em 10 por cento das amostras, tetC em 20 por cento , tetD em 80 por cento , tetE em 50 por cento , tetG em 60 por cento , tetO e tetS em 10 por cento cada e tetW em 20 por cento . Conclusões Em 90 por cento das amostras foi identificada a presença de genes de resistência aos antibióticos -lactâmicos e tetraciclinas, demonstrando uma grande circulação de resistência bacteriana na aquicultura e verificando a necessidade de legislação e fiscalização mais atuantes no controle do uso de antibióticos na aquicultura / IIntroduction The excessive use of antimicrobials in human medicine, veterinary medicine and agriculture has resulted in the emergence of bacterial resistance. This phenomenon creates public health problems that may result in the re-emergence of infectious diseases. The use of antibiotics, mainly prophylactically, has become a practice in aquaculture, including Brazil, where the legislation for the use of veterinary drugs is inefficient. Furthermore, there is evidence of transmission of resistant organisms to humans through consumption of animal products, especially when the antibiotics have been administered during the raising and production of these animals. Objective To search for the occurrence of antibiotics resistance genes in tilapia fillets commercialized in supermarkets in São Paulo city - SP. Material and Methods 10 tilapia fillet samples were collected and submitted to fecal coliform search as an indicator of the sanitary conditions of the food. Then, the samples were inoculated into Luria broth 0,5 per cent and the total DNA was extracted from growing bacteria by thermal shock for the search of resistance genes to -lactam and tetracyclines antibiotics by PCR. The genes identified by PCR were confirmed by sequencing. Results In 100 per cent of samples the result was < 3 NMP.g-1 for fecal coliform organisms. In the study of -lactam resistance genes, blaOXY-5 gene was detected in 90 per cent of samples, blaTEM-1b in 20 per cent , blaLEN-16 in 10 per cent , blaSHV-28 in 10 per cent , blaKPC-2 in 20 per cent , blaMOX-6 in 10 per cent and blaCphA in 60 per cent . The tetracyclines resistance genes identified were tetB in 10 per cent of samples, tetC in 20 per cent , tetD in 80 per cent , tetE in 50 per cent , tetG in 60 per cent , tetO and tetS in 10 per cent each and tetW in 20 per cent . Conclusions 90 per cent of the samples showed the presence of -lactam and tetracyclines resistance genes, demonstrating a high circulation of bacterial resistance in aquaculture and verifying the need for more active law and surveillance in controlling the use of antibiotics in aquaculture.

IIntrodução A utilização excessiva de antimicrobianos na medicina humana, veterinária e agricultura resultou no aparecimento da resistência bacteriana. Este fenômeno gera problemas de saúde pública que podem resultar na reemergência de doenças infecciosas. O uso de antibióticos, principalmente de forma profilática, tornou-se prática na aquicultura, como ocorre no Brasil, onde a regulamentação para o uso de medicamentos veterinários é ineficiente. Além disso, há evidências da transferência de organismos resistentes para humanos por meio do consumo de produtos de origem animal, quando, durante a fase de criação e produção destes foram administrados antibióticos. Objetivo Pesquisar a ocorrência de genes de resistência a antibióticos em filés de tilápia comercializados em supermercados do município de São Paulo SP. Material e Métodos Foram coletadas 10 amostras de filé de tilápia e realizada a pesquisa de coliformes termotolerantes como indicadores das condições higiênico-sanitárias do alimento. Em seguida, as amostras foram inoculadas em Caldo Lúria 0,5 por cento e o DNA total das bactérias cultivadas nesse meio foi extraído por meio de choque térmico para pesquisa de genes de resistência aos antibióticos -lactâmicos e tetraciclinas pela PCR. Os genes identificados pela PCR foram confirmados pelo sequenciamento. Resultados Em 100 por cento das amostras analisadas o resultado para coliformes termotolerantes foi < 3 NMP.g-1. Na pesquisa de genes de resistência a -lactâmicos, o gene blaOXY-5 foi detectado em 90 por cento das amostras, blaTEM-1b em 20 por cento , blaLEN-16 em 10 por cento , blaSHV-28 em 10 por cento , blaKPC-2 em 20 por cento , blaMOX-6 em 10 por cento e blaCphA em 60 por cento . Os genes de resistência a tetraciclinas identificados foram tetB em 10 por cento das amostras, tetC em 20 por cento , tetD em 80 por cento , tetE em 50 por cento , tetG em 60 por cento , tetO e tetS em 10 por cento cada e tetW em 20 por cento . Conclusões Em 90 por cento das amostras foi identificada a presença de genes de resistência aos antibióticos -lactâmicos e tetraciclinas, demonstrando uma grande circulação de resistência bacteriana na aquicultura e verificando a necessidade de legislação e fiscalização mais atuantes no controle do uso de antibióticos na aquicultura / IIntroduction The excessive use of antimicrobials in human medicine, veterinary medicine and agriculture has resulted in the emergence of bacterial resistance. This phenomenon creates public health problems that may result in the re-emergence of infectious diseases. The use of antibiotics, mainly prophylactically, has become a practice in aquaculture, including Brazil, where the legislation for the use of veterinary drugs is inefficient. Furthermore, there is evidence of transmission of resistant organisms to humans through consumption of animal products, especially when the antibiotics have been administered during the raising and production of these animals. Objective To search for the occurrence of antibiotics resistance genes in tilapia fillets commercialized in supermarkets in São Paulo city - SP. Material and Methods 10 tilapia fillet samples were collected and submitted to fecal coliform search as an indicator of the sanitary conditions of the food. Then, the samples were inoculated into Luria broth 0,5 per cent and the total DNA was extracted from growing bacteria by thermal shock for the search of resistance genes to -lactam and tetracyclines antibiotics by PCR. The genes identified by PCR were confirmed by sequencing. Results In 100 per cent of samples the result was < 3 NMP.g-1 for fecal coliform organisms. In the study of -lactam resistance genes, blaOXY-5 gene was detected in 90 per cent of samples, blaTEM-1b in 20 per cent , blaLEN-16 in 10 per cent , blaSHV-28 in 10 per cent , blaKPC-2 in 20 per cent , blaMOX-6 in 10 per cent and blaCphA in 60 per cent . The tetracyclines resistance genes identified were tetB in 10 per cent of samples, tetC in 20 per cent , tetD in 80 per cent , tetE in 50 per cent , tetG in 60 per cent , tetO and tetS in 10 per cent each and tetW in 20 per cent . Conclusions 90 per cent of the samples showed the presence of -lactam and tetracyclines resistance genes, demonstrating a high circulation of bacterial resistance in aquaculture and verifying the need for more active law and surveillance in controlling the use of antibiotics in aquaculture.

The invention of catalytic processes that convert feedstock chemicals into pharmacologically-privileged amines is a landmark challenge in organic synthesis. This thesis describes the development of three novel transition-metal catalyzed processes for the synthesis of alkylamines that attempts to meet this challenge. The first Pd-catalyzed methylene β-C−H carbonylation of alkylamines to form substituted β-lactams is reported. Through the synergistic use of a Pd-catalyst and Xanpthos ligand, secondary amines underwent exclusive methylene β-C−H activation in high yields and diastereoselectivities. Subsequently, the development of a remarkably selective methylene β-C−H carbonylation of α-tertiary amines (ATAs), is detailed. This methodology enables the C−H carbonylation of methylene C−H bonds over traditionally more reactive methyl and C(sp2)−H bonds. Importantly, a range of functional groups previously incompatible with C−H technologies were tolerated in good yields. Finally, the development of a novel multicomponent synthesis of tertiary amines is described. The novel photocatalytic single-electron reduction of alkyl iminium ions furnishes -amino radicals that engage alkenes forming a new C-C bond. The reaction exhibits broad functional group tolerance and enables the synthesis of amines not readily accessible by existing methods.