Investigations detailed in this thesis constitue a part of continuing programme of research work undertaken in this laboratory on vitamin binding proteins. Avidin from the chicken egg white, streptavidin &om the bacterium Streptromyces avidin and biotin binding proteins (BBP-I and BBP-11) from chicken egg yolk constitute a family of proteins that bind the vitamin biotin with extremely high affinities. The yolk BBPs are involved in the deposition of the vitamin in the developing oocyte in chicks whereas an antimicrobial function has been attributkl to avidin.. The fact that all these proteins bind the vitamin in the same manner, unlike biotin-dependent enzymes, indicates that the structural features involved in ligand binding could be similar, if not identical in these proteins. To delineate the basis of putative structural similarity among these proteins, studies were carried out using antibodies as the immunological probes. Avidin, a homotetremer glycoprotein, with a subunit Mr of 17,000 has been purified to homogeneity from chicken egg white using a novel procedure involving ammonium sulphate fractionation, ethanol precipitation and S-Sepharose column chromatography. Despite their lesser abundance in chicken egg yolk associated with a large amount of interfering lipids during the purification, both BBP-I (monomer and shown to be precursor for BBP-11) and BBP-I1 (tetramer) have been purified to homogeneity by employing a common method using butanol extraction to remove the lipids, DEAE-Sephacel column chromatography, biotin-AH-Sepharose affinity chromatography and fast performance liquid chrometography (FPLC) system. The purity of all these proteins was confirmed by SDS-PAGE analysis.

<p>Autism is a pervasive developmental disorder (PDD) that&rsquo / s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman&reg / SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman&reg / study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.</p>

Background: Hepatitis E (HEV) is a small, non-enveloped virus that belongs to the viral genus Hepevirus. HEV is a positive sense single-stranded RNA virus and there is insufficient information regarding its replication. This is mainly because the virus has low capacity to grow in normally used cell cultures. Many specific strand assay detection studies have been done in order to understand more about HEV replication. Unfortunately, these assays have the disadvantage of giving false positive results. Aim: The aim of this project was to improve the positive strand assay to increase specificity and eliminate false positivity which is due to high sensitivity of the polymerase chain reaction (PCR). False positivity occurs as remains of transfected material in the cell are amplified. Method: The samples used in this project were swine samples from Sweden and a human sample (plasmid clone of genotype 1) from India. Negative samples, extracted positive samples and transcribed RNA positive sense samples were used. The methods applied were cDNA synthesis, exonuclease I and RNase treatments, DNA purification kits followed by first and nested PCR. Result: The results of this study indicated great improvement of the detection assay especially for the transcribed RNA samples. Best results were obtained at a final concentration of 1.5mM MgCl2 in the mastermix.  Conclusion: Changing the concentration of MgCl2 appeared to have a great effect on PCR specificity. Improving detection assays is very essential as they can be applied in the research field and in public health centers either for diagnosis or tracking disease outbreaks.

Strawberry (Fragaria ananassa) is an economically important soft fruit that is highly valued as a fresh product and flavouring. During ripening, strawberry fruits undergo a number of physiological changes affecting colour, texture and flavour. An understanding of these changes at the biochemical and molecular level will be important in developing strategies for enhancing the quality attributes of this fruit. A cDNA encoding a ripening- enhanced acyl carrier protein (RE-ACP) was previously isolated from strawberry fruit. AC? is an essential component of fatty acid synthesis in both plants and animals. The aims of this thesis were to isolate and characterise this and other members of the ACP multigene family expressed in strawberry fruit. Six closely related putative AC? cDNA isoforms were identified from strawberry. Two of these were obtained by screening a cDNA library from ripe fruit and three were obtained by a technique known as candidate fragment length polymorphism (CFLP) that utilised ACP gene-specific primers for AFLP-cDNA display. Northern analysis was not able to differentiate their expression but ACP was highly up-regulated in ripening fruit whereas low levels of expression were detected in other strawberry tissues, including achenes (seeds), expanding leaves and flowers. The RE-ACP was over-expressed in E. coli and the recombinant protein partially purified. The over-expressed protein had a M(_r) of 20kDa on SDS-PAGE and appeared to form a dimer. A genomic library was constructed from F. ananassa from which two different genomic clones closely related to RE-ACP were obtained. Promoter analysis indicated the presence of regulatory elements. The characterization of putative ACP cDNA and genomic clones, including the 5' upstream regions, is described and their possible role in strawberry fruit is discussed. Key words: Strawberry, fruit, ripening, gene expression, genomic, cDNA, fatty acid, acyl carrier protein, aroma, promoter.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-06T11:29:59Z No. of bitstreams: 2 ariadne_marques.pdf: 1829125 bytes, checksum: 01fca719846b682abc79cca9f78f836a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-06T11:30:33Z (GMT) No. of bitstreams: 2 ariadne_marques.pdf: 1829125 bytes, checksum: 01fca719846b682abc79cca9f78f836a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-06T11:31:10Z (GMT) No. of bitstreams: 2 ariadne_marques.pdf: 1829125 bytes, checksum: 01fca719846b682abc79cca9f78f836a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2015-01-06T11:31:10Z (GMT). No. of bitstreams: 2 ariadne_marques.pdf: 1829125 bytes, checksum: 01fca719846b682abc79cca9f78f836a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2013 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O Ceratocystis fimbriata Ellis & Halsted ? um fitopat?geno vascular de ampla distribui??o geogr?fica e gama de hospedeiros. A murcha de Ceratocystis em Eucalyptus ? considerada de dif?cil controle devido ao seu car?ter sist?mico e a variabilidade gen?tica dos isolados. O plantio de material resistente ? a principal forma de controle desta doen?a. No entanto, a obten??o de material gen?tico resistente pode demandar longos per?odos em um programa de melhoramento. A identifica??o de gen?tipos resistentes dentre os clones utilizados comercialmente pode ajudar a abreviar o tempo necess?rio ao melhoramento. Assim, objetivou-se avaliar a resist?ncia de clones de eucalipto ? murcha de Ceratocystis, analisar as respostas ecofisiol?gicas desses clones sob infec??o e fornecer suporte ao processo de melhoramento gen?tico para tal caracter?stica. Foram avaliados 27 clones de resist?ncia desconhecida e 2 clones caracterizados como resistente e suscet?vel ? murcha de Ceratocystis, todos inoculados artificialmente com isolados de C. fimbriata. Os 27 clones apresentaram, aos 50 dias ap?s inocula??o, varia??o quanto ? resist?ncia ? doen?a. Os dois clones de resist?ncia conhecida tiveram crescimento, ?ndice de clorofila e efici?ncia qu?ntica do fotossistema II (Fv/Fm) avaliados em quatro diferentes tempos. As vari?veis analisadas apresentaram potencial para uso na avalia??o da resist?ncia/suscetibilidade de gen?tipos de Eucalyptus spp. ? murcha de Ceratocystis. O clone resistente (R) e o suscet?vel (S) foram utilizados para a constru??o de uma biblioteca subtrativa supressiva. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013. / ABSTRACT The Ceratocystis fimbriata Ellis & Halsted is a vascular phytopathogen of wide geographic distribution and host range. Ceratocystis wilt in Eucalyptus is considered difficult to control due to its systemic character and the genetic variability of the isolated. Planting resistant material is the main way to control this disease. However, the acquisition of genetic material resistant may require long periods in a breeding program. The identification of resistant genotypes among clones used commercially can help shorten the time needed to improve. The aim was to evaluate the resistance of eucalyptus clones to Ceratocystis wilt, analyze the ecophysiological responses of these clones under infection and provide support to the process of breeding for this trait. We evaluated 27 clones of unknown resistance and 2 clones characterized as resistant and susceptible to Ceratocystis wilt, all artificially inoculated with isolates of C. fimbriata. The 27 clones showed, 50 days after inoculation, variation in disease resistance. The two clones of known resistance grew, chlorophyll content and quantum efficiency of photosystem II (Fv / Fm) evaluated at four different times. The variables analyzed showed potential for use in the evaluation of the resistance/susceptibility of genotypes of Eucalyptus spp. to Ceratocystis wilt. The clone resistant (R) and susceptible (S) were used for the construction of a suppressive subtractive library.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T10:54:29Z No. of bitstreams: 2 marcele_santos_ferreira.pdf: 1547534 bytes, checksum: a0861513c3025ada252d9e4fa8f0c494 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T10:55:14Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) marcele_santos_ferreira.pdf: 1547534 bytes, checksum: a0861513c3025ada252d9e4fa8f0c494 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T10:55:10Z (GMT) No. of bitstreams: 2 marcele_santos_ferreira.pdf: 1547534 bytes, checksum: a0861513c3025ada252d9e4fa8f0c494 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-07T10:55:34Z (GMT) No. of bitstreams: 2 marcele_santos_ferreira.pdf: 1547534 bytes, checksum: a0861513c3025ada252d9e4fa8f0c494 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2015-01-07T10:55:34Z (GMT). No. of bitstreams: 2 marcele_santos_ferreira.pdf: 1547534 bytes, checksum: a0861513c3025ada252d9e4fa8f0c494 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2013 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / O g?nero Eucalyptus engloba centenas de esp?cies conhecidas e de elevado interesse comercial. Este g?nero florestal ? altamente respons?vel pelo abastecimento da cadeia produtiva da madeira no Brasil. Diversos aspectos podem afetar negativamente a produtividade dos plantios, como, por exemplo, a ocorr?ncia de pragas e doen?as. O percevejo bronzeado Carpintero e Dellap?, 2006 (Hemiptera: Thaumastocoridae) ? uma praga ex?tica, nova no Brasil. Uma estrat?gia para diminuir os efeitos negativos dos insetos-praga ? selecionar materiais gen?ticos resistentes. O isolamento e a identifica??o de genes envolvidos no processo de resist?ncia podem ser usados na obten??o de indiv?duos com caracter?sticas desej?veis. O presente trabalho teve por objetivos a avalia??o de materiais gen?ticos de Eucalyptus spp. quanto ? inj?ria ocasionada pelo percevejo bronzeado e a constru??o, a partir de materiais contrastantes, de bibliotecas subtrativas de cDNA. A escolha dos gen?tipos baseou-se em metodologias distintas, conduzidas em laborat?rio, sala climatizada e em casa de vegeta??o. Foram estudados 27 clones h?bridos. Os gen?tipos C03 e C17 foram escolhidos e, juntamente com mudas de E. camaldulensis, submetidos ao ataque de T. peregrinus. A partir das amostras de RNA mensageiro foi poss?vel construir duas bibliotecas subtrativas: uma com genes diferencialmente expressos entre o clone C03 e o E. camaldulensis e outra a partir do clone C17 e o E. camaldulensis. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013. / ABSTRACT The genus Eucalyptus encompasses hundreds of known species with high commercial interest. This forest genus is largely responsible for the supply of the wood production chain in Brazil. Several aspects can negatively affect the plantation productivity, for example the occurrence of pests and diseases. The bronze bug Carpintero e Dellap?, 2006 (Hemiptera:Thaumastocoridae) is a new exotic pest in Brazil. A strategy to reduce the negative effects of the insect pests is to select genetic material with resistance. The isolation and identification of resistance genes can be used in order to obtain individuals with desirable characteristics. The present study aimed to evaluate genetic materials of Eucalyptus spp. under the attack of Thaumastocoris peregrinus and make cDNA libraries from contrasting materials. The choice of genotypes relied on different methodologies conducted in laboratory, climate-controlled room and greenhouse. There were studied 27 hybrid clones. The genotypes C03 and C17, were selected along with E. camaldulensis seedlings and subjected to the attack of T. peregrinus. Two genomic subtractive libraries were made from the total RNA samples, one containing the differentially expressed genes between C03 and E. camaldulensis, and another from C17 and E. camaldulensis.

Made available in DSpace on 2016-06-02T20:21:25Z (GMT). No. of bitstreams: 1 3272.pdf: 1459537 bytes, checksum: 78eeb30fb7d9022c1a6c97b55c565d47 (MD5) Previous issue date: 2010-08-02 / Financiadora de Estudos e Projetos / The genus Anastrepha is the largest in the family Tephritidae (Trypetinae, Toxotrypanini). The construction of cDNA libraries is important for identifying new genes and establishing genetic markers on non-models organisms such as the genus Anastrepha. Among the proteins involved in reproduction, we can cite those belonging cascade sexual and those responsible for forming the eggshell. The purpose of this study was to build a cDNA library from a pool of female reproductive tissues Anastrepha obliqua to breed ESTs, to search for genes with a high evolutionary rate, focusing on chorionic and vitelline proteins. We were sequenced 2304 clones obtained from the reproductive tissues of female flies Anastrepha obliqua. In total, 310 contigs and 506 singlets were generated wich were classified into different proteins classes. The chorionic proteins and Sxl revealed to be under selection positive as well as the vitelline Vm 26Aa ', which was possibly generated by a gene duplication of Vm 26aa, while Tra2 seems under purifying selection. The inference of the secondary structure of proteins studied revealed that the sites under positive selection were present on outside membrane. The population analysis of genes chorionic, vitelline, Sxl and Tra2 revealed no separation between the 3 species, although in some situations have occurred separation between some of the haplotypes for a single specie. With these results, it was possible to identify new genes, make the full sequencing for some vitelline and chorionic genes on pools of other species of Anastrepha, and we also identified that some of these genes are under positive selection pressure. / O gênero Anastrepha é o maior dentro da família Tephritidae (Trypetinae, Toxotrypanini). A construção de bibliotecas de cDNA é importante para a identificação de novos genes e no estabelecimento de marcadores genéticos em organismos não-modelos como do gênero Anastrepha. Dentre as proteínas envolvidas na reprodução podemos citar as pertencentes da cascata sexual e as que são responsáveis por formar a parede do ovo. Assim, este trabalho teve como objetivo construir uma biblioteca de cDNA a partir de um pool de tecidos reprodutivos femininos de Anastrepha obliqua para gerar ESTs, afim de buscar genes com alta taxa evolutiva, focando em proteínas coriônicas e vitelínicas. No Total foram seqüenciados 2304 clones obtidos a partir dos órgãos reprodutivos de fêmeas de moscas de Anastrepha obliqua. Ao todo foram gerados 310 contigs e 506 singlets que foram classificados em diferentes classes de proteínas. As proteínas coriônicas e o Sxl revelaram estar sob seleção positiva, assim como a vitelínica Vm 26Aa´ que possivelmente foi gerada por uma duplicação gênica do Vm 26Aa, enquanto que o Tra2 parece estar sob seleção purificadora. A inferência da estrutura secundária das proteínas estudadas revelou que os sítios sob seleção positiva estavam presentes do lado externo da membrana. As análises populacionais dos genes coriônicos, vitelínicos, Sxl e Tra2 não revelaram uma separação entre as 3 espécies, apesar de em algumas situações ter ocorrido uma separação entre alguns dos haplótipos para uma determinada espécie. Com resultados obtidos, foi possível identificar novos genes, fazer o seqüenciamento completo para alguns genes coriônicos e vitelínicos em pools de outras espécies de Anastrepha, e também foi possível identificar que alguns destes genes estão sob pressão de seleção positiva.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Em etapa anterior a este trabalho, uma proteÃna purificada a partir do lÃtex da planta Calotropis procera foi purificada e sua caracterizaÃÃo bioquÃmica revelou ser esta uma proteÃna similar a osmotinas e que a mesma, denominada de CpOsm, exibia forte atividade contra fungos fitopatogÃnicos. Neste trabalho foram investigados sistemas de expressÃo heterÃlogos, procarionte e eucarionte, com o objetivo de estabelecer sistemas de expressÃo que pudessem produzir CpOsm recombinante e avaliar se sua expressÃo produzia a proteÃna ativa, com aÃÃo antifÃngica. A partir do sequenciamento do cDNA da CpOsm e ensaios de cristalizaÃÃo desenvolvidos com a proteÃna purificada do lÃtex foi possÃvel estudar suas caracterÃsticas moleculares. Para a expressÃo em E. coli foi utilizado o vetor pET303&#61487;CT-His e P. pastoris o vetor pPICZ&#945;A. A proteÃna recombinante (rCpOsm) expressa no sistema procarionte nÃo foi secretada para o meio externo, acumulando-se no espaÃo intracelular, formando corpos de inclusÃo, nos quais a proteÃna estava insolÃvel. Embora a insolubilidade represente um passo limitante, este sistema de expressÃo pode ser muito interessante para a produÃÃo quantitativa de rCpOsm para outros fins como produÃÃo de anticorpos ou estudos de folding/refolding proteico, considerado suas caracterÃsticas moleculares peculiares, observadas nos estudos cristalogrÃficos, tais como o conjunto de pontes dissulfeto intracadeia. rCpOsm foi tambÃm expressa em cÃlulas de P. pastoris, entretanto o sistema de expressÃo deverÃ, necessariamente, sofrer melhorias para maximizar o rendimento. rCpOsm de P. pastoris foi inicialmente detectada por sequenciamento de novo por espectrometria de massas a partir da digestÃo trÃptica. A identificaÃÃo de peptÃdeos internos confirmou sua presenÃa no meio extracelular. A limitaÃÃo deu-se pela baixa taxa de expressÃo, o que, por conseguinte, nÃo permitiu uma caracterizaÃÃo mais ampla da rCpOsm deste sistema. rCpOsm expressa em P. postoris estava em sua forma ativa. Em uma anÃlise comparativa, rCpOsm e CpOsm, de modo e intensidade similares, foram capazes de alterar drasticamente a morfologia de esporos de F. solani e reduzir seu volume, comparados a esporos nÃo tratados, revelado atravÃs de microscopia de forÃa atÃmica. CpOsm foi cristalizada pelo mÃtodo de gota pendente e os cristais obtidos difrataram a uma resoluÃÃo de 1,61 à com caracterÃsticas morfolÃgicas do espaÃo cristalogrÃfico P6122. Os dados coletados sugerem que a proteÃna mantÃm uma estrutura em monÃmero, correspondendo a sequÃncia de aminoÃcidos do cDNA, com 203 resÃduos. A estrutura pode ser vista como trÃs regiÃes distintas, presentes em outras osmotinas jà descritas, formando um domÃnio central onde hà um conjunto de folhas beta, sendo este o mais longo, e dois outros menores, formados de estrutura predominantemente desordenadas com pequenos segmentos em alfa-hÃlice (domÃnio II) e longas alÃas (domÃnio III). Oito pontes dissulfeto estabilizam a estrutura e envolvem todos os resÃduos de cisteÃna da estrutura primÃria. NÃo hà evidencias cristalogrÃficas para formaÃÃo de oligÃmeros. Este estudo conclui que a expressÃo heterÃloga em sistema eucarionte produz rCpOsm ativa e isto representa uma etapa a mais cumprida para a sua possÃvel expressÃo em plantas com o objetivo de proteÃÃo contra fitopatÃgenos. / In a previous step to this work, a latex protein belonging to Calotropis procera was described. The protein, named CpOsm, exhibited biochemical characteristics closely related to pathogenesis related proteins joined into PR-5 group. The new protein with osmotin characteristics displayed activity against phytopatogenic fungi. Here, attempts to obtain a suitable heterologous system to express functional recombinant CpOsm were performed in Prokaryote and Eukaryote expression systems. Further, cDNA sequencing and crystallographic assays were performed using CpOsm and the molecular and structural properties of the functional protein. The vector pET303&#61487;CT-His and PICZ&#945;A were used to express CpOsm in E. coli and P. pastoris, respectively. The ecombinant protein (rCpOsm) produced in the Prokaryote system was retained into E. oli cells and deposited as inclusion bodies. rCpOsm was insoluble. Although insoluble roteins into inclusion bodies represent an adverse phase for obtaining the active CpOsm, this system of expression can be interesting to other goals as quantitative roduction of rCpOsm for producing antibodies or to study protein folding/refolding, ince CpOsm possesses peculiar structural characteristics such as occurrence of an xtended network or intra chain disulfide bonds stabilizing the overall structure. rCpOsm as also successfully expressed in P. pastoris. However, this protocol must to undergo improvement in order to maximize yield. rCpOsm was initially detected in the P. pastoris expression system by MS/MS de novo sequencing after tryptic digestion. Identification of internal peptides present in the extracellular media confirmed the production and excretion of rCpOsm in P. pastoris cells. The very low yield of recombinant protein avoided enough amount of purified rCpOsm to perform a broad characterization. On a comparative basis, native CpOsm, purified of the latex, and rCpOsm, purified from P. pastoris cultures, at similar mode and intensity were capable of drastically alter the morphological architecture of spores of F.solani and reduced their olume as compared to non-treated spores, as revealed by atomic force microscopy measurements. CpOsm was crystallized by the pendant drop method and the crystals grown diffracted at 1.61 Ã. They fit on the P6122 space. The data collecting, supported by the cDNA deduced amino acid sequence suggested that CpOsm occurs as an monomeric structure composed of a unique chain of 203 amino acid residues and no evidences for quaternary association was seen. The overall structure can separated in three structural domains, which have been reported in other osmotins. The central region preserves the set of beta-sheets and is the largest. The others exhibit short segments on alpha-helix interconnected by randomized sequences (domain II). In domain III predominates randomized sequences and long loops. Eight disulfide bonds stabilize the structure and involve all cysteine residues of the primary sequence. The heterologous expression of CpOsm on Eukaryote system produces rCpOsm active and this support the hypothesis that rCpOsm is a suitable candidate for heterologous expression in plants in order to obtain improved crops against selected phytopatogens.

Magister Scientiae - MSc / Autism is a pervasive developmental disorder (PDD) that's incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman® SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman® study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples. / South Africa

A pipeline was developed for the quantitative screening and sequence management of clones from suppression subtractive hybridization (SSH) cDNA libraries. The pipeline is particularly useful for gene discovery in non-sequenced organisms, and was illustrated with SSH library data from pearl millet (Pennisetum glaucum) and cowpea (Vigna unguiculata) and Arabidopsis (Arabidopsis thaliana) ecotype Kil-0. The objective of each library was to identify stress-response genes. cDNA microarrays provide a high-throughput screening method. Accordingly, these SSH libraries were amplified by PCR and spotted onto glass microarray slides. Subtracted and un-subtracted cDNA samples, that were used to construct the SSH libraries were prepared as Cy3- and Cy5-labeled targets and hybridized to the microarrays. The R package SSHscreen version 2.0.0, available from http://microarray.up.ac.za/SSHscreen/, was developed to analyze the resulting microarray data using limma (linear models for microarray data) functions. Commonly, loess normalization is used for within-slide normalization, however this is based on the assumption that most of the genes on the array are not differentially expressed. This is legitimate for most whole genome microarray experiments, however it is not appropriate when the array is constructed from an SSH library which is enriched for differentially expressed genes. Therefore, control spot-based normalization was used in the SSHscreen analysis. Empirical Bayes methods were employed to calculate the moderated t-statistic using functions from the limma package. This procedure in effect borrows information from the ensemble of genes to aid with inference about individual genes, taking advantage of the parallel structure whereby the same model is fitted to the data for each gene. In the Arabidopsis, pearl millet and cowpea forward libraries, 18%, 58% and 58% of the clones were identified as significantly up-regulated (adjusted p-value < 0.05) and in the reverse libraries, 18%, 30% and 28% significantly down-regulated, respectively. SSHscreen analysis was used to assist in selection of clones for sequencing. The SSHscreen data output (ranked gene lists in terms of differential expression), as well as the selected sequences in FASTA format were uploaded to SSHdb. For the Arabidopsis library, 114 out of the 262 sequenced clones (55%) were identified as unique/non-redundant; and for the pearl millet and cowpea libraries respectively, 37% and 33% of the sequenced clones were unique. SSHdb was developed as a web-based tool for sequence management and annotation of clones in SSH libraries and can freely be accessed at http://sshdb.bi.up.ac.za. BLAST analysis that was carried out when sequences were uploaded to SSHdb was used to combine clones with the same sequence into redundant partner groups, as well as identify putative annotations for each group. Individual clones from the abovementioned SSH libraries were selected and an independent technique, quantitative PCR, was used to validate the microarray/SSHscreen results. The pipeline was applied successfully to Arabidopsis, pearl millet and cowpea SSH cDNA libraries. Interesting genes in each case were identified for further study. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted