Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 106-119).

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is the only endemic species of commercial value. Aquaculture remains the only avenue for expanding the industry, since the closure of the fishery. The current focus is on implementing a molecular breeding programme; thus the development of molecular markers for linkage mapping and QTL analysis is a priority. Various markers, mainly anonymous, have been developed for H. midae; however emphasis is being placed on the development of gene-linked type I molecular markers. The present study investigates and demonstrates the use of public sequence collections to develop type I markers for a species with limited genomic resources, via three strategies: Surveying anonymous H. midae microsatellite markers’ flanking regions to find homology to gene sequences in public databases, cross-species marker transfer of anonymous markers from H. rubra and H. discus hannai demonstrating putative gene associations and lastly EST marker mining (SNP and microsatellites) from various Haliotids and testing transfer to the target species. Approximately 17% of H. midae anonymous markers showed significant similarity to genes. The current study also reports higher cross-species transferability from both H. rubra and H. discus hannai to H. midae (39% and 20.5%, respectively) than previously demonstrated and 15 EST-microsatellites and 16 EST-SNPs were successfully mined. Furthermore, the non-random distribution of microsatellites and high nucleotide diversity in the H. midae genome was confirmed. This is a low cost and time effective method for marker development and presents a continuous and dynamic resource that could be used for future marker development and characterisation as sequence information in public databases grow exponentially. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is die enigste van vyf inheemse spesies van kommersiële waarde. Na die noodgedwonge sluiting van die vissery, is akwakultuur die mees praktiese oplossing om die perlemoen industrie uit te brei. Die huidige fokus is gerig op die implementering van ‘n molekulêre teel-program en dus is die ontwikkeling van molekulêre merkers vir genetiese kartering en kwantitatiewe kenmerk lokus analise, van uiterste belang. Tipe II merkers is voorheen vir die perlemoen ontwikkel, maar huidige tendense lê klem op die ontwikkeling van geen-gekoppelde tipe I merkers. Die huidige studie ondersoek die gebruik van publieke databasisse vir die ontwikkeling van tipe I molekulêre merkers vir ‘n spesie met beperkte genomiese bronne. Drie strategieë is geïmplementeer: Eerstens is ‘n opname gemaak van die homologie van perlemoen tipe II merker-vleuelende volgordes met geen volgordes in databasisse. Verder is die oordraagbaarheid van tipe II merkers vanaf H. rubra en H. discus hannai wat assosiasie met gene toon ondersoek. Laastens is ‘n Uitgedrukte Volgorde Merk (UVM) (Expressed Sequence Tag, EST) merker-ontginnings metode vanaf verskeie Haliotis spesies en toetsing van oordraagbaarheid na die teiken spesie uitgevoer. Ongeveer 17% van die tipe II H. midae merkers het geniese assosiasie getoon. ‘n Hoër tussen-spesie oordraagbaarheid vanaf beide H. rubra en H. discus hannai na H. midae (39% en 20.5%, onderskeidelik) word gerapporteer in vergelyking met vorige studies en 15 UVM-mikrosatelliete en 16 UVM-enkel nukleotied polimorfismes (single nucleotide polimorphism, SNP) is ontwikkel. Verder bevestig die studie die nie-lukrake verspreiding van mikrosatelliete en hoë nukleotied diversiteit in die perlemoen genoom. Die gebruik van publieke databasise vir die ontwikkeling en karakterisering van tipe I molekulêre merkers is tyd- en koste-besparend en bied ‘n volgehoue en dinamiese bron vir toekomstige gebruik.

Philosophiae Doctor - PhD / Esophageal cancer (EC) ranks among the ten most frequent cancers worldwide. Mortality rates associated with EC are very similar to the incidence rates due to the relatively late stage of diagnosis and the poor efficacy of treatment. The aim of this study was to enhance our insights of putative transcriptional circuitry of EC genes, thereby potentially positively impacting our knowledge of therapeutic targets, providing indications as to more appropriate lines of treatment, and additionally allowing for the determination of putative candidate diagnostic markers for the early stage detection of EC. This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer. DDEC contains known and novel information for 529 differentially expressed EC genes compiled using scientific publications from PubMed and is freely accessible for academic and non-profit users at http://apps.sanbi.ac.za/ddec/. The novel information provided to users of the DDEC is the lists of putative transcription factors that potentially control the 529 manually curated genes. The value of the information accessible through the database was further refined by providing precompiled text-mined and data-mined reports about each of these genes to allow for easy exploration of information about associations of EC-implicated genes with other human genes and proteins, metabolites and enzymes, toxins, chemicals with pharmacological effects, disease concepts and human anatomy. This feature has the capacity to display potential associations that are rarely reported and thus difficult to identify, and it enables the inspection of potentially new ‘association hypotheses’ generated based on the precompiled reports. This study further illustrates how the biocurated esophageal squamous cell carcinoma (ESCC) genes in the database may represent a reliable starting point for exploring beyond current knowledge of the transcriptional circuitry of estrogen related hormone therapy. The genes were used to develop a method that identified 44 combinations of transcription factors (TFs) that characterize the promoter sequence of estrogen responsive genes implicated in ESCC. These significantly over-represented combinations of TFs were then used to increase confidence in the 47 novel putative estrogen response genes that may be related to ESCC too. Coincidently, two of the novel putative estrogen response genes were verified by current (2009), experimental publications. / South Africa

A cromodomínio-helicase (CHD) e a SET/TAF-Iβ (chaperona de histonas) são proteínas conhecidas por estarem envolvidas em processos de remodelagem de cromatina e controle da expressão gênica. Em organismos como Caenorhabditis elegans, Drosophila melanogaster, camundongo e o homem, estas proteínas estão associadas ao controle de vários processos de desenvolvimento. Em Mesocestoides corti, um modelo de parasito cestódeo, sequências relacionadas à CHD e à SET/TAF-Iβ foram identificadas em uma seleção de genes diferencialmente expressos em larvas e vermes estrobilizados. Visando à identificação de marcadores moleculares de processos e estágios de desenvolvimento da Classe Cestoda, os padrões de expressão de ortólogos das proteínas CHD e SET/TAF-Iβ (McCHD e McSET/TAF) em M. corti foram investigados. Inicialmente as sequências codificadoras da McCHD e McSET/TAF foram amplificadas por RT-PCR, clonadas em vetor de expressão modificado (pGEX-TEV) e expressas em Escherichia coli para produção de proteínas recombinantes. Análises por imunoblot e imuno-histoquímica foram realizadas utilizando anticorpos gerados contra versões recombinantes das proteínas-alvo do estudo. Foram analisados quatro estágios de desenvolvimento de M. corti: larvas (tetratirídeos, TT), TT após 24h de indução ao processo de estrobilização (o qual confere o desenvolvimento da larva em adulto) (24h-Ind), TT após 72h de indução (72h-PI) e vermes estrobilizados (VE). Em imunoblots, a McCHD apresentou altos níveis de expressão em três estágios de desenvolvimento de M. corti: TT, 72h-PI e VE, sendo detectado um menor nível de expressão no estágio 24h-Ind quando comparado aos demais. Já a McSET/TAF apresentou um aumento gradual no seu nível de expressão após a indução (nos estágios de 24h-Ind, 72h-PI e VE), não sendo detectada em TT. Em secções longitudinais foram observados um maior nível de expressão da McCHD em estágios iniciais de desenvolvimento (TT e 24- Ind) enquanto que McSET/TAF foi observado um maior nível de expressão nos estágios intermediários e no final do desenvolvimento de M. corti. Para ambas as proteínas a localização foi citoplasmática e não houve diferenças de distribuição nos tecidos analisados. Para compreender melhor as diferenças encontradas nos níveis de expressão das proteínas, análises por PCR em tempo real (RT-qPCR) foram realizadas para quantificação dos níveis de transcritos dos genes McCHD e McSET/TAF. Foram encontradas diferenças significativas nos níveis de expressão gênica de McCHD e McSET/TAF somente em dois estágios: o inicial (TT) e o adulto (VE), conforme análises pelo teste de Duncan. Níveis maiores de expressão gênica de ambos os genes foram encontrados no último estágio de desenvolvimento (VE) de M. corti. A caracterização de genes e proteínas envolvidas no processo de estrobilização de platelmintos da classe Cestoda contribuirá para o melhor entendimento de rotas do desenvolvimento de cestódeos, podendo auxiliar também na determinação de novos alvos terapêuticos para o tratamento de cestodíases. / Chromodomain-helicase (CHD) and the SET/TAF-Iβ histone chaperone are proteins known to be implicated in processes of chromatin remodeling and regulation of gene expression associated with the control of developmental processes in different organisms, such as Caenorhabditis elegans, Drosophila melanogaster, mouse and human. In Mesocestoides corti, a model cestode parasite, CHD and SET/TAF-Iβ related sequences were isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Aiming at identifying molecular markers of processes and stages of development of Class Cestoda, the expression patterns of M. corti CHD and SET/TAF-Iβ orthologous proteins (McCHD and McSET/TAF) were investigated. Initially, the coding sequences of McCHD and McSET/TAF were amplified by RT-PCR, cloned into a modified expression vector (pGEX-TEV) and expressed in Escherichia coli for recombinant proteins production. Analysis by immunoblotting and immunohistochemistry was performed using antibodies raised against recombinant versions of the proteins target the study. We analyzed four developmental stages of M. corti: bona fide tetrathyridia (TT), tetrathyridia 24 h after strobilation induction (24h-Ind), strobilating worms 72 h after induction (72h-PI), and fully strobilated worms (VE). Immunoblots showed high levels of expression McCHD in three developmental stages of M. corti: TT, 72h-PI e VE, and detected a lower level of expression in stage 24-Ind in comparison to others. The McSET/TAF showed a gradual increase in their level of expression after induction (stages of 24h-Ind, 72h- PI e VE) was not detected in TT. In longitudinal sections were observed an increased level of expression of McCHD in early stages of development (TT and 24-Ind) while McSET/TAF there was a higher level of expression in the intermediate and late stages of development of M. corti. Both McCHD and McSET/TAF showed a cytoplasmic location and a uniform pattern of immunostaining, no differences in distribution between the tissues. For a better understanding the differences in levels of protein expression, analysis by real-time PCR (RT-qPCR) was performed to quantify the transcript levels of genes McCHD and McSET/TAF. There were significant differences in levels of gene expression McCHD and McSET/TAF only in two stages: the initial (TT) and adult (VE), as analysis by Duncan test. Higher levels of gene expression of both genes were found in the last stage of development (VE) of M. corti. The characterization of genes and proteins involved in the process of strobilation flatworms of the class Cestoda contribute to a better understanding of the development of cestodes routes and may also assist in the determination of new therapeutic targets for the treatment of cestodiasis.

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No. of bitstreams: 1 TeseCAS.pdf: 3874357 bytes, checksum: 0bd0fdf47146d9a5d45f62a5203ac011 (MD5) Previous issue date: 2016-11-28 / 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) / The present work had as general objective to perform the genomic annotation of Expressed Sequences (ESTs) of Litopenaeus vannamei shrimp, available in the database of Project ShEST and to evaluate the polymorphism of mined SSR and SNP tags. These markers were located in the main chain of protein genes with function related to performance traits and were validated in SPF (Specific Pathogen Free) shrimp families submitted to selection for rapid growth and survival. In addition to the EST-SSR and EST-SNP loci, obtained by Sanger sequencing, Next Generation Sequencing (NGS) analyzes were included in the initial proposal of work with the objective of expanding the set of SNPs available and verifying the differential gene expression. The new assembly of ESTs was performed and produced a set of 2.984 unigenes with protein products for 41% of them, with 1.983 SSRs and 3.472 SNPs being identified. Among the loci with gene product identified, 231 were enzymes with 127 unique EC numbers inserted in 94 KEGG metabolic pathways. Loci validation showed that the loci of the 60S ribosomal (SSR-EST) and crustacyanin (SNP-EST) proteins were polymorphic in the animals sampled from Genearch. Statistical analyzes were conducted to verify the existence of a possible association between the genotypes and the analyzed weight phenotypes, although no association was observed. In addition, cross-species amplification tests were performed on seven species of marine and two freshwater prawns, demonstrating successful transferability for these species. The RNAseq approach was included in the present work with the purpose of increasing the number of SNPs detected in candidate genes with performance-related function and identifying differentially expressed (DE) genes in animals under experimental conditions. A second transcriptome was assembled from the muscle and hepatopancreas tissues of L. vannamei individuals (i) evaluated for rapid growth and survival and (ii) exposed to the White Spot Syndrome Virus (WSSV). A total of 63.105 transcripts were generated, with an average size of 2.511 bp and N50 of 3.464 bp. More than 15.500 SNPs were identified (frequency > 50%). Functional annotation was also performed on the bases of SwissProt, Gene Ontology (GO) and KEGG. Differential gene expression analyzes were performed on the animal samples evaluated for growth and response to WSSV infection. The data generated showed differences in the expression profile between the genes of (i) high and low growth animals, (ii) the hepatopancreas and muscle and (iii) the uninfected (healthy) and infected (ill) animals by WSSV, considering the effect of the tissue. Two-hundred and seven DE genes were identified for growth, 5.816 for hepatopancreas and muscle and 1.017 for ill and healthy animals. / O presente trabalho teve como objetivo geral realizar a anotação genômica de sequências expressas (ESTs) de Litopenaeus vannamei, disponíveis no banco de dados do Projeto ShEST e avaliar o polimorfismo de marcas SSR e SNP mineradas. Esses marcadores estavam localizados na cadeia principal de genes de proteínas com função relacionada a traços de desempenho e foram validados em famílias de camarões SPF (Specific Pathogen Free) submetidas à seleção para rápido crescimento e sobrevivência. Adicionalmente aos locos SSR-EST e SNP-EST, obtidos por sequenciamento Sanger, análises de Sequenciamento de Próxima Geração (NGS) foram incluídas na proposta inicial de trabalho com o objetivo de ampliar o conjunto de SNPs disponíveis e verificar a expressão gênica diferencial. A montagem de novo das ESTs foi realizada e produziu um conjunto de 2.984 unigenes com produtos proteicos para 41% destes, sendo identificados 1.983 SSRs e 3.472 SNPs. Dentre os locos com produto gênico identificado, 231 eram enzimas com 127 EC numbers únicos inseridos em 94 vias metabólicas do KEGG. A validação dos locos SSR-EST e SNP-EST mostrou que os locos das proteínas 60S ribossomal (SSR-EST) e crustacianina (SNP-EST) apresentaram-se polimórficos nos animais amostrados da Genearch. Análises estatísticas foram conduzidas para verificação da existência de uma possível associação entre os genótipos e os fenótipos de peso analisados, embora não tenha sido observada associação. Além disso, testes de amplificação heteróloga foram realizados em sete espécies de camarões marinhos e duas de água doce, demonstrando sucesso na transferabilidade para estas espécies. A abordagem de RNA-seq foi incluída no presente trabalho com o propósito de ampliar o número de SNPs detectados em genes candidatos com função relacionada a traços de desempenho e identificar genes diferentemente expressos (DE) em animais sob condições experimentais. Foi realizada a montagem de novo de um segundo transcriptoma, dos tecidos músculo e hepatopâncreas de indivíduos de L. vannamei (i) avaliados para rápido crescimento e sobrevivência e (ii) expostos ao vírus da Síndrome da Mancha Branca ou White Spot Syndrome Virus (WSSV). Foram gerados 63.105 transcritos, com tamanho médio de 2.511 pb e N50 de 3.464 pb. Foram identificados mais de 15.500 SNPs (frequência > 50%). Também foi realizada a anotação funcional nas bases do SwissProt, Gene Ontology (GO) e KEGG. Análises de expressão diferencial gênica foram realizadas nas amostras dos animais avaliados para crescimento e resposta a infecção pelo WSSV. Os dados gerados demonstraram diferenças no perfil de expressão entre os genes (i) de animais de alto e baixo crescimento, (ii) do hepatopâncreas e músculo e (iii) dos animais não-infectados (saudáveis) e infectados (doentes) pelo WSSV, considerando-se o efeito do tecido. Foram identificados 207 genes DE para crescimento, 5.816 para a comparação entre os tecidos e 1.017 para os animais doentes e saudáveis. / FAPESP: 2012/13069- 6 / FAPESP: 2012/17322-8