Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Galactooligosaccharides are produced by the transglycosylation activity of β-galactosidases (β-gal, EC 3.2.1.23) when utilising lactose as a substrate. They have emerged as important constituents used in the food and pharmaceutical industries owing to their prebiotic properties. Although transglycosylation was discovered in 1951 (Wallenfels 1951), and a number of β-gals have had their transglycosylation activity characterised, the activities of these enzymes are not optimal for industrial use. Their tendency to favour the hydrolytic reaction over the transglycosylation reaction, coupled with the production of shorter chain oligosaccharides has driven scientists to investigate altering protein structure both to increase chain lengths and the amount of oligosaccharide produced at lower substrate concentrations. In an attempt to alter the amount of oligosaccharide produced by a metagenomically derived β-gal belonging to the glycosyl hydrolase 2 family, random and site-directed mutagenesis were used. A randomly mutagenised library was screened on SOB agar plates containing 5% (w/v) lactose which should select for clones that synthesise oligosaccharides at relatively low concentrations. No such activity was detected. Site-directed mutagenesis was also utilised to alter protein structure. It was confirmed that the β-gal utilised in this study belonged to the glycosyl hydrolase 2 family through mutation of the predicted catalytic acid/base glutamic acid to a non-catalytic residue, thus removing activity. Another mutation was utilised to investigate if it was possible to increase the degree of polymerisation of oligosaccharides produced by the β-gal. This mutation was successful in increasing the degree of polymerisation. Biochemical characterisation of the β-gal revealed that it exhibited optimal activity at pH 8.0, with a temperature optimum of 30°C. The β-gal exhibited a Km and Vmax of 54.23 mM and 2.26 μmol/minute-1/mg protein-1 respectively, similar to kinetic parameters that have been determined for a number of previously characterised enzymes. / AFRIKAANSE OPSOMMING: Galaktooligosakkariede word geproduseer deur die transglikosileering aktiwiteit van β-galaktosidase (β-gal, EG 3.2.1.23) wanneer hulle laktose as 'n substraat gebruik. Hierdie oligosakkariede het na vore gekom as 'n belangrike bestandeel vir gebruik in die voedsel en farmaseutiese bedryf as gevolg van hulle prebiotiese eienskappe. Alhoewel transglycosylation al in 1951 ontdek is (Wallenfels 1951) en 'n aantal β-gals se transglycosylation aktiwiteit gekenmerk is, is hierdie ensieme nie ideaal vir industriële toepassings nie. Die geneigdheid om die hidrolitiese reaksie oor die transglycosylation reaksie bevoordeel, tesame met die produksie van korter oligosakkariede het wetenskaplikes ondersoek genoop om die proteïenstruktuur te verander om ketting-lengte en die kwantiteit van oligosakkaried geproduseer teen laer substraat konsentrasies te verhoog. In 'n poging om die opbrengs van die oligosakkaried wat deur 'n metagenomiese β-gal wat aan die glycosyl hidrolase 2 familie behoort te verander, is lukraak en terrein gerigte-mutagenese gebruik. Die mutagenese biblioteek is op SOB agarplate met 5% (w/v) lactose gekeur, om klone wat die fenotipe wat verband hou met die produksie oligosakkaried teen relatiewe lae konsentrasies te selekteer. Geen aktiwiteit is opgemerk nie. Terrein gerigte-mutagenese is ook gebruik om die proteïenstruktuur te verander. Deur ‘n bioinformatiese voorspelling, is dit bevestig dat die β-gal wat in hiedie studie gebruik word tot die glycosyl hidrolase 2 familie behoort. Dit is gedoen deur mutasie van die voorspelde katalitiese suur/basis glutamiensuur na 'n nie-katalitiese oorskot, dus die verwydering van aktiwiteit. Nog ‘n mutasie is gebruik om te ondersoek of dit moontlik was om die ketting-lengte van die oligosakkaried wat deur die β-gal geproduseer is te verhoog. Die mutasie was suksesvol in die verhoging van die oligosakkaried wat geproduseer was. Biochemiese karakterisering van die β-gal het getoon dat hierdie β-gal optimale aktiwiteit het by pH 8.0, met 'n optimum temperatuur van 30°C. Die β-gal het 'n Km en Vmax van 54.23 mM en 2.26 μmol/minute-1/mg proteïen-1 onderskeidelik, soortgelyk aan kinetiese parameters wat bepaal word vir ensieme wat voorheen gekenmerk is.

No description available.

γ-aminobutyric acid is the major inhibitory neurotransmitter in the adult mammalian central nervous system (CNS) and may also play a neurotrophic role during CNS development. Diversification of GABA_A receptor mediated responses are in part a result ofvariation in subunit composition in the receptor complex. This variation arises both from the number of different subtypes of GABA_A receptor subunits (α1-6, β1-4, γ1-3, δ1, ρ1-3, ε, ρ), as well as from post-transcriptional processes such as RNA splicing. In this thesis, I have investigated the developmental onset of GABA_A receptor gene expression and the distribution and temporal expression of GABA_A receptor subunit mRNAs and 12 splice variants within the developing and adult murine CNS. Preliminary studies using S 1 nuclease protection analysis demonstrated that α1, β3 and γ2 were the predominant subtypes of GABA_A receptor subunits expressed at embryonic day 14 and in the adult murine CNS. In situ hybridisation analysis demonstrated overlapping but distinct spatial and temporal patterns of GABA_A subunit mRNA expression during postnatal development and in the adult murine CNS. Analysis of γ2 mRNA splice variants demonstrated that the γ2S transcript is the predominant γ2 mRNA expressed during latter stages of embryo genesis, while the γ2L transcript is the predominant γ2 isoform present inthe adult CNS. Since there is a 29 to 47 percent amino acid identity among the various GABA_A receptor subunits, I have also demonstrated through site-directed mutagenesis studies, that changes in a conserved amino acid in the cysteine loop of the bovine a 1 GABA_A receptor subunit resulted in a loss of agonist and antagonist binding (DI49N), while a change in a conserved amino acid in the M1 transmembrane domain of the bovine α1 GABA_A receptor subunit resulted in loss of agonist binding and reduction in the B_max and K_d for antagonist binding (P243A). 'These results are in contrast to the effect of identical mutations in the bovine β1 subunit and suggest that if the pentameric GABA_A receptor assembly is composed of (α1)2(β1)1(γ2)2, then changes in highly conserved amino acids in the α1 receptor subunit would have a greater distortion on the structure of the receptor complex.

AN ABSTRACT OF THE THESIS OF JOSHUA GUNTHER, for the Masters of Science degree in Plant, Soil and Agricultural Systems, presented on November 2nd, 2015, at Southern Illinois University Carbondale. TITLE: DEVELOPING CHEMICALLY MUTAGENIZED EMS FORREST SOYBEAN POPULATION FOR HIGH OIL PROFILE. MAJOR PROFESSOR: Dr. Khalid Meksem SUPERVISOR: Dr. Naoufal Lakhssassi Soybeans (Glycine max (L.) Merr.) are the most important crop that provides a sustainable source of oil and protein worldwide. Five major fatty acids are known, Palmetic, Stearic, Oleic, Linoleic and Linolenic acid, and each is essential for both nutrition and biodiesel. Oil demand for biodiesel production is constantly on the rise both because of high crude oil prices and because of the search for a sustainable fuel source. In biodiesel production a high level of Oleic Acid is ideal. Commodity soybean oil usually contains around 20% oleic acid. The objective of this research is to increase the Oleic acid content to increase the quantity of biodiesel that can be produced from one bushel of soybeans. With the process of chemical mutagenesis using Ethyl Methanesulfonate (EMS), soybean populations can be produced with varying fatty acid levels. Once these populations are isolated and tested they can be bred into a new cultivar with higher percentages of Oleic acid and grown on a large scale for biodiesel production. From the ‘Forrest’ cultivar that was used in the chemical mutagenesis process, there was one mutant line (FM3 2014-2031) that produced 43.26% Oleic Acid. This was almost twice the concentration of the Forrest wild type that was used as a control which had an Oleic Acid concentration of 23.17%.

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-27Bitstream added on 2014-06-13T19:53:56Z : No. of bitstreams: 1 porto_mp_me_botib.pdf: 2132236 bytes, checksum: b1dd297e7ebc3769eafbc4dbcaa5705a (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Devido a propriedades terapêuticas, várias plantas e seus constituintes químicos vêm sendo muitas vezes utilizados como o primeiro recurso para o tratamento de diversas doenças. Nesse contexto, compostos isolados de plantas têm sido alvos de inúmeros estudos que avaliam, além da atividade, seus possíveis mecanismos de ação. Dentre os compostos com potencial quimioprotetor, o citral e o eugenol merecem atenção devido suas estruturas químicas de monoterpeno e de composto fenólico, respectivamente, e por seus potenciais anti-inflamatório, antiparasitário e antioxidante. Considerando que mutação no DNA pode ser a primeira etapa de várias doenças, e que lesões induzidas nessa molécula podem ser prevenidas ou moduladas por compostos naturais, este estudo objetivou avaliar, pelo teste do cometa, o potencial genotóxico do citral (25, 50 e 100 Tg/mL) e do eugenol (0,31, 0,62, 1,24 e 2,48 Tg/mL), após diferentes tempos de tratamento (6, 10, 24 e 30 h) e, também, seus possíveis efeitos moduladores sobre danos induzidos no DNA pela doxorrubicina (DOX), em diferentes protocolos de tratamento (pré, pós e simultaneamente à DOX) e momentos de análise (12, 24 e 36 h), em macrófagos peritoneais de camundongos. Além disso, foi avaliado o potencial toxicogenômico do citral e do eugenol por meio da modulação da expressão dos genes NF-kB1, COX-2 e TNF-α (relacionados a processos inflamatórios) em macrófagos ativados ou não por lipopolissacarideo de bactéria (LPS). Os resultados mostraram que ambos os compostos apresentaram potencial genotóxico. No caso do citral, a genotoxicidade foi observada para as duas maiores concentrações, mas apenas no tempo de 6h; para o eugenol, o aumento de danos no DNA foi detectado para todas as concentrações, em pelo menos um momento de análise. Com relação ao potencial... / Because of the therapeutic properties, several plants and their chemical constituents have been used for treatment of various diseases. Therefore, isolated compounds from plants have been targets of numerous studies that evaluate their activity and mechanisms of action. Among compounds with chemopreventive potential, citral and eugenol have gain attention because of their chemical structures, monoterpene and phenol,respectively, and for their anti-inflammatory, antioxidant and antiparasitic potentials. Since DNA mutation is the first step for some diseases, and since the lesions induced in this molecule can be prevented or modulated by natural compounds, aim of the present study was first to evaluate the genotoxic potential of citral (25, 50 and 100 Tg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 Tg/mL) at different times after exposure (6, 10, 24 and 30 h), and then, their ability to modulate DNA damage induced by doxorubicin (DOX) at different treatment protocols (pre, post and simultaneous with DOX) and times (12, 24 and 36 h) in mice peritoneal macrophages. In addition, the toxicogenomic potential of citral and eugenol by modulating the expression of NF-KB1, COX-2 and TNF-α (related to inflammatory processes) genes in macrophages activated or not by bacterial lipopolysaccharide (LPS) was also investigated. The results showed that both compounds have genotoxic potential. In the case of citral, genotoxicity was observed for the two major concentrations, but only 6h after the exposure. For eugenol, increased DNA damage was detected for all concentrations, in at least one moment of analysis. Related to the antigenotoxicity, both citral and eugenol presented protective effects at different concentrations and treatment protocols, and the more effective activities were detected at simultaneous and pre-treatment... (Complete abstract click electronic access below)

Made available in DSpace on 2015-04-09T12:28:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-25Bitstream added on 2015-04-09T12:47:59Z : No. of bitstreams: 1 000817146.pdf: 479870 bytes, checksum: d3427b349dc719a504332ddf82064866 (MD5) / O estudo da interação planta-patógeno é de grande importância para o entendimento do cancro cítrico, justificando assim a busca por genes que estejam ligados a patogenicidade e virulência em Xanthomonas citri subsp. citri (Xac), agente causal dessa doença. Neste estudo, foi realizada mutagênese aleatória por inserção do transposon EZ-Tn5 in vitro no genoma da Xac. Obteve-se 8000 mutantes onde 292 foram conduzidos em ensaio experimental in planta. Cinco mutantes expressaram sintomatologia alterada, dois com ausência total de sintomas e três com leve hiperplasia. A análise da sequência dos genes onde se inseriu o transposon indicam mutações nos genes purF, yapH, oar, um gene que codifica uma proteína hipotética (XAC 0196), e na região entre os genes pobB (XAC0362) e glpR (XAC0361). As análises de curvas de crescimento bacteriano in planta demonstraram que, exceto o gene purF, todos os demais podem ser genes envolvidos na patogenicidade de Xac. Dois destes, yapH e oar são descritos como relacionados à adesividade bacteriana, evidenciando que a interferência nesse processo exerce influência direta no sucesso da infecção de Xac. Destaca-se também a importância da identificação de uma proteína hipotética, já que essa apresentou sintomatologia atenuada quando ocorreu a inserção do transposon / The study of plant-pathogen interaction is very important to citrus canker understanding, justifying the search for virulence and pathogenicity related genes in Xanthomonas citri subsp. citri (Xac), causal agent of this disease. In this study, a random mutagenesis by Tn5 transposon insertion into Xac’s genome was performed. Eight thousand mutants were produced and 292 mutants were tested in planta. From those, five mutants expressed altered symptomatology, two showed complete absence of symptoms and three reduced hyperplasia. Gene sequences analysis where transposon was inserted, indicated mutations in purF, yapH and oar genes, in a region that codes for a hypothetical protein (XAC0196), and in a region between pobB (XAC0362) and glpR (XAC0361) genes. Analysis of bacteria growth curve in planta showed that, except for purF gene, all the others genes may be involved in Xac pathogenicity. Two of these genes, yapH and oar, are described as bacterial adhesion related genes, highlighting that interference in this process has direct influence in the Xac infection success. The importance of hypothetical protein identification is emphasized, since it presented attenuated symptomatology when mutated

Orientador: Daisy Maria Fávero Salvadori / Coorientador: Glenda Nicioli da Silva / Banca: Luís Fernando Barbisan / Banca: Denise Crispim Tavares / Resumo: Devido a propriedades terapêuticas, várias plantas e seus constituintes químicos vêm sendo muitas vezes utilizados como o primeiro recurso para o tratamento de diversas doenças. Nesse contexto, compostos isolados de plantas têm sido alvos de inúmeros estudos que avaliam, além da atividade, seus possíveis mecanismos de ação. Dentre os compostos com potencial quimioprotetor, o citral e o eugenol merecem atenção devido suas estruturas químicas de monoterpeno e de composto fenólico, respectivamente, e por seus potenciais anti-inflamatório, antiparasitário e antioxidante. Considerando que mutação no DNA pode ser a primeira etapa de várias doenças, e que lesões induzidas nessa molécula podem ser prevenidas ou moduladas por compostos naturais, este estudo objetivou avaliar, pelo teste do cometa, o potencial genotóxico do citral (25, 50 e 100 Tg/mL) e do eugenol (0,31, 0,62, 1,24 e 2,48 Tg/mL), após diferentes tempos de tratamento (6, 10, 24 e 30 h) e, também, seus possíveis efeitos moduladores sobre danos induzidos no DNA pela doxorrubicina (DOX), em diferentes protocolos de tratamento (pré, pós e simultaneamente à DOX) e momentos de análise (12, 24 e 36 h), em macrófagos peritoneais de camundongos. Além disso, foi avaliado o potencial toxicogenômico do citral e do eugenol por meio da modulação da expressão dos genes NF-kB1, COX-2 e TNF-α (relacionados a processos inflamatórios) em macrófagos ativados ou não por lipopolissacarideo de bactéria (LPS). Os resultados mostraram que ambos os compostos apresentaram potencial genotóxico. No caso do citral, a genotoxicidade foi observada para as duas maiores concentrações, mas apenas no tempo de 6h; para o eugenol, o aumento de danos no DNA foi detectado para todas as concentrações, em pelo menos um momento de análise. Com relação ao potencial... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Because of the therapeutic properties, several plants and their chemical constituents have been used for treatment of various diseases. Therefore, isolated compounds from plants have been targets of numerous studies that evaluate their activity and mechanisms of action. Among compounds with chemopreventive potential, citral and eugenol have gain attention because of their chemical structures, monoterpene and phenol,respectively, and for their anti-inflammatory, antioxidant and antiparasitic potentials. Since DNA mutation is the first step for some diseases, and since the lesions induced in this molecule can be prevented or modulated by natural compounds, aim of the present study was first to evaluate the genotoxic potential of citral (25, 50 and 100 Tg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 Tg/mL) at different times after exposure (6, 10, 24 and 30 h), and then, their ability to modulate DNA damage induced by doxorubicin (DOX) at different treatment protocols (pre, post and simultaneous with DOX) and times (12, 24 and 36 h) in mice peritoneal macrophages. In addition, the toxicogenomic potential of citral and eugenol by modulating the expression of NF-KB1, COX-2 and TNF-α (related to inflammatory processes) genes in macrophages activated or not by bacterial lipopolysaccharide (LPS) was also investigated. The results showed that both compounds have genotoxic potential. In the case of citral, genotoxicity was observed for the two major concentrations, but only 6h after the exposure. For eugenol, increased DNA damage was detected for all concentrations, in at least one moment of analysis. Related to the antigenotoxicity, both citral and eugenol presented protective effects at different concentrations and treatment protocols, and the more effective activities were detected at simultaneous and pre-treatment... (Complete abstract click electronic access below) / Mestre

Orientador: Maria Inês Tiraboschi Ferro / Coorientador: Jesus Aparecido Ferro / Banca: Flavia Maria de Souza Carvalho / Banca: Fabrício José Jaciani / Resumo: O estudo da interação planta-patógeno é de grande importância para o entendimento do cancro cítrico, justificando assim a busca por genes que estejam ligados a patogenicidade e virulência em Xanthomonas citri subsp. citri (Xac), agente causal dessa doença. Neste estudo, foi realizada mutagênese aleatória por inserção do transposon EZ-Tn5 in vitro no genoma da Xac. Obteve-se 8000 mutantes onde 292 foram conduzidos em ensaio experimental in planta. Cinco mutantes expressaram sintomatologia alterada, dois com ausência total de sintomas e três com leve hiperplasia. A análise da sequência dos genes onde se inseriu o transposon indicam mutações nos genes purF, yapH, oar, um gene que codifica uma proteína hipotética (XAC 0196), e na região entre os genes pobB (XAC0362) e glpR (XAC0361). As análises de curvas de crescimento bacteriano in planta demonstraram que, exceto o gene purF, todos os demais podem ser genes envolvidos na patogenicidade de Xac. Dois destes, yapH e oar são descritos como relacionados à adesividade bacteriana, evidenciando que a interferência nesse processo exerce influência direta no sucesso da infecção de Xac. Destaca-se também a importância da identificação de uma proteína hipotética, já que essa apresentou sintomatologia atenuada quando ocorreu a inserção do transposon / Abstract: The study of plant-pathogen interaction is very important to citrus canker understanding, justifying the search for virulence and pathogenicity related genes in Xanthomonas citri subsp. citri (Xac), causal agent of this disease. In this study, a random mutagenesis by Tn5 transposon insertion into Xac's genome was performed. Eight thousand mutants were produced and 292 mutants were tested in planta. From those, five mutants expressed altered symptomatology, two showed complete absence of symptoms and three reduced hyperplasia. Gene sequences analysis where transposon was inserted, indicated mutations in purF, yapH and oar genes, in a region that codes for a hypothetical protein (XAC0196), and in a region between pobB (XAC0362) and glpR (XAC0361) genes. Analysis of bacteria growth curve in planta showed that, except for purF gene, all the others genes may be involved in Xac pathogenicity. Two of these genes, yapH and oar, are described as bacterial adhesion related genes, highlighting that interference in this process has direct influence in the Xac infection success. The importance of hypothetical protein identification is emphasized, since it presented attenuated symptomatology when mutated / Mestre

Insight into the biochemical mechanisms utilized by retaining and inverting glycosyltransferase enzymes is an important stepping stone to the directed design of stereospecific inhibitor based drugs. The suitability of proposed mechanisms was probed using site directed mutagenesis of catalytically relevant residues as well as the use of catalytically inactive substrate analogs UMP-PO2-CH2-D-Gal and α-L-Fuc-(1→2)-β-D-(3-deoxy)-Gal-O(CH2)5CH3 with the retaining human enzyme a specific α-1,3-N-acetylglucosaminyltransferase (GTA) in conjunction with kinetic and structural approaches including two dozen high resolution X-ray structures and a 2.5 Å resolution neutron structure. The neutron structure depicts a remarkably non-polar active site which lacks suitably positioned hydrogen atoms to support a dissociative mechanism. Site directed mutagenesis of residues which should be essential to initiate and stabilize a dissociative oxocarbenium ion do not abolish enzyme activity. The catalytically inactive substrate analogs depict the acceptor nucleophile to lay very close to the anomeric carbon (2.5 Å), which is considerably closer than the closest observed enzymatic dipoles (4.8 Å). This is an indication that the active site architecture is more suited to facilitate a mechanism initiating with nucleophilic attack than dissociation. To ensure that these observations are applicable to other glycosyltransferases, in depth geometric analysis of all published liganded structures of GT-A fold glycosyltransferase enzymes are reported that display conserved architectures in which the acceptor nucleophile approach is closer than enzymatic dipoles required for dissociation for both inverting and retaining enzymes. Inverting and retaining enzymes present the donor sugar through different conserved geometries about the divalent cation cofactor: all inverting enzymes position the donor for inline nucleophilic attack by the acceptor, the retaining enzymes position the sugar to be attacked from an orthogonal angle. Such an orthogonal associative mechanism is the most direct proposed approach, and seems supported by all available evidence. / Graduate