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.

This thesis is focused upon chemistry as a school subject and students' interpretations and use of formally introduced teaching models. To explore students' developing repertoire of chemical models, a longitudinal interview study was undertaken spanning the first year of upper secondary school chemistry. Matter in its different states was selected as the target framework for this study. The results presented are derived from both generalisations of groups of students as well as a case study describing an individual learner's interpretation of formal content. The results obtained demonstrated that the formal teaching models provided to the students included in this study were not sufficient to afford them a coherent framework of matter in its different states or for chemical bonding. Instead, students' expressed models of matter and phase change were to a high degree dependent on electron movement (Paper I), anthropomorphism (Paper II) and, for one student, a mechanistic approach based on small particles and gravitation (Paper III). The results from this study place focus on the importance of learners' prior learning (previous experiences) and the need to develop a coherent framework of formal teaching models for the nature of matter and phase change.

<p>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.</p>

La tesi esta basada en l'adaptació de l'ús de codons a genomes procariotes, especialment l'adaptació de l'ús de codons a una alta expressió. Hi ha un grup de genomes procariotes, els quals estan sota una selecció traduccional, que tenen un grup de gens amb un ús de codons esbiaixat de la resta de gens del genoma i adaptats a l'abundància dels tRNA. Hem desenvolupat un nou algoritme per a avaluar si un genoma esta sota selecció traduccional i predir els gens altament expressat de tots els genomes sota selecció traduccional. Aquestes prediccions són públiques a la base de dades HEG-DB (http://genomes.urv.cat/HEG-DB), la qual s'ha publicat a la revista Nucleic Acids Research. Les prediccions de gens altament expressats s'han fet servir com a filtre en les prediccions de gens adquirits per transferència horitzontal, ja que els gens altament expressats molts cops son predits com a falsos positius en la predicció de gens adquirits. Amb les dades de la predicció de gens altament expressats, també hem desenvolupat una nova eina Bioinformàtica, anomenada OPTIMIZER (http://genomes.urv.cat/OPTIMIZER) i publicada al Nucleic Acids Research, per tal d'optimitzar l'ús de codons d'un gen per a incrementar la seva expressió en experiments d'expressió heteròloga de proteïnes. També hem estudiat un cas particular d'adaptació de l'ús de codons. El cas de l' 'amelioration', que és l'adaptació de l'ús de codons que pateix un gen inserit en un genoma hoste. Aquest cas l'hem estudiat amb els gens mitocondrials que varen saltar al genoma nuclear i varen haver d'adaptar el seu us de codons mitocondrial a l'ús de codons del genoma nuclear. Per tal d'estudiar l''amelioration', hem desenvolupat un nou índex anomenat CAI esperat (eCAI) i una nova eina Bioinformàtica anomenada CAIcal (http://genomes.urv.cat/CAIcal), que està en procés de revisió a la revista BMC Bioinformatics. Analitzant l'anàlisi de l'ús de codons dels genomes completament sequenciats vàrem realitzar una troballa que s'aparta una mica del tema central de la tesi. Vàrem veure que els genomes que estan adaptats a la (hiper)termofília tenen un patró de l'ús de codons i d'aminoàcids diferent a la resta de genomes (mesòfils). Aquest fet ens ha permès descobrir casos de guany i pèrdua (recents i antics) de la capacitat d'adaptació termofílica en genomes procariotes. Aquests resultats han donat lloc a una publicació a la revista Trends in Genetics. Durant la tesi he realitzat una estada de 4 mesos (Febrer - Juny, 2006) en el laboratori de bioinformàtica del departament de biologia de la universitat nacional d'Irlanda a Maynooth sota la supervisió del Dr James McInerney on vaig desenvolupar un nou programa per a la comparació d'arbres filogenètics anomenat TOPD/FMTS (http://genomes.urv.cat/topd) el qual està publicat a la revista Bioinformatics. / This thesis is based in codon usage adaptation in prokaryotic genomes, especially the codon usage adaptation to a high expression. In genomes under translational selection, the group of highly expressed genes has a codon usage adapted to the most abundant tRNA species. We have developed a new iterative algorithm which predicts a group of highly expressed genes in genomes under translational selection by using the Codon Adaptation Index and the group of ribosomal protein genes as a seed. We have developed a new genomic database, called HEG-DB, to store genes that are predicted as highly expressed in prokaryotic complete genomes under strong translational selection. The database is freely available at http://genomes.urv.cat/HEG-DB and it has been published in Nucleic Acids Research. The predicted highly expressed genes are used as an initial filter to reduce the number of false positives of the Horizontal Gene Transfer Database, due to highly expressed genes are usually false positive in predictions of acquired genes. We have developed a new web sever, called OPTIMIZER (http://genomes.urv.cat/OPTIMIZER), which has been published in Nucleic Acids Research, to optimize the codon usage of DNA or RNA sequences. This new web server can be used to predict and optimize the level expression of a gene in heterologous gene expression or to express new genes that confer new metabolic capabilities in a given species. We have also analyzed an especial case of codon usage adaptation, which is called 'amelioration'. The 'amelioration' is the adaptation of foreign genes to a new genome. This is the case of mitochondrial genes encoded in the human nuclear genome and originally encoded in the proto-mitochondria. To test the 'amelioration' process we have developed an expected value of CAI (eCAI) to find out whether the differences in the CAI are statistically significant or whether they are the product of biased nucleotide and/or amino acid composition and a new bioinformatics tool called CAIcal (http://genomes.urv.cat/CAIcal). We have also analyzed the evolution of thermophilic adaptation in prokaryotes and we suggest that the amino acid composition signature in thermophilic organisms is a consequence of or an adaptation to living at high temperatures, not its cause. Our findings suggest that there have been several cases where the capacity for thermophilic adaptation has been gained or lost throughout the evolution of prokaryotes. These results have been published in Trends in Genetics. During my thesis I have worked for four months in the Bioinformatics Laboratory of the Biology Department at the National University of Ireland under the supervision of Dr James O. McInerney where I developed a new software program to compare phylogenetic trees called TOPD/FMTS (http://genomes.urv.cat/topd), that has been published in Bioinformatics.

Expressed sequence tags (ESTs) are derived from the developing tilapia brain was established in our lab. There are 9 transcripts were identified as thermosensitive/Neural development-related gene. The effects of different temperatures on the ontogenetic expression of these thermosensitive/Neural development-related gene during the critical period of brain sexual differentiation were investigated in the present study. The ontogenetic expression of inhibitor of DNA binding/differentiation protein 2 (Id2), thermosensitive/Neural development-related gene, were enhanced by both lower (20¢J) and higher (32¢J) temperatures before 10 days post-hatching. In this study, bioinformatics were searched for Id2, which is a gene with 738 bp of patial cDNA sequence, open reading frame (ORF) is 411bp, and deduced 137 amino acids of protein sequence. The protein of Id2 was expressed in a prokaryotic system, BL21 (Escherichia coli) and purified with Ni-NTA affinity chromatography. Also, the ORF of Id2 was cloned into pEGFP vector, and plasmid (pEGFP-Id2) was transfected into the eukaryotic system, mouse neuroblastoma cell (Neuro-2a cell). The distribution of Id2 expressed in the Neuro-2a cell was identified by fluorescence microscopy.

<p>In this thesis the effect of normalization methods on the identification of differentially expressed genes is investigated. A zebrafish microarray dataset called Swirl was used in this thesis work. First the Swirl dataset was extracted and visualized to view if the robust spline and print tip loess normalization methods are appropriate to normalize this dataset. The dataset was then normalized with the two normalization methods and the differentially expressed genes were identified with the LimmaGUI program. The results were then evaluated by investigating which genes overlap after applying different normalization methods and which ones are identified uniquely after applying the different methods. The results showed that after the normalization methods were applied the differentially expressed genes that were identified by the LimmaGUI program did differ to some extent but the difference was not considered to be major. Thus the main conclusion is that the choice of normalization method does not have a major effect on the resulting list of differentially expressed genes.</p>

<p>This project is an investigation of whether analysing subsets of time series gene expression data can give additional information about putatively co-regulated genes, compared to only using the whole time series. The original gene expression data set was partitioned into subsets and similarity was computed for both the whole timed series and subsets. Pearson correlation was used as similarity measure between gene expression profiles. The results indicate that analysing co-expression in subsets of gene expression data derives true-positive connections, with respect to co-regulation, that are not detected by only using the whole time series data. Unfortunately, with the actual data set, chosen similarity measure and partitioning of the data, randomly generated connections have the same amount of true-positives as the ones derived by the applied analysis. However, it is worth to continue further analysis of the subsets of gene expression data, which is based on the multi-factorial nature of gene regulation. E.g. other similarity measures, data sets and ways of partitioning the data set should be tried.</p>

<p>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.</p>

Mother-to-child transmission (MTCT) of HIV is the most significant source of HIV infection in young children. As the HI virus has been identified in cell-free and cell-associated compartments of breast milk, it is clear that breast milk is one of the ways in which mother-to-child transmission of HIV can take place in addition to in utero and intrapartum transmission. While breastfeeding carries the risk of HIV transmission, not breastfeeding carries significant health risks for infants and young children, such as an increased risk of diarrhoea and pneumonia, morbidity and mortality. When an HIV-positive mother decides to breastfeed her baby, pasteurisation of expressed breast milk (EBM) could be a possible infant-feeding option to limit transmission of the HI virus through breast milk, since this method has shown to effectively inactivate HIV type 1. Three methods of pasteurisation of human milk were investigated in this study: Holder pasteurisation, flash-heating and Pretoria pasteurisation. The systematic review is a helpful method to summarise the best-quality empirical evidence of the benefits and limitations of a specific method, such as heat treatment, and to provide recommendations for future research. Therefore, the aim of this study was to critically synthesise by means of a systematic review the best available existing evidence and to provide a clear overview of the effectiveness of heat treatment of EBM as an in-home procedure to inactivate the HI virus, and in so doing limit mother-to-child transmission of HIV. This study provides the clinical practitioner with accessible information on the effectiveness of heat treatment of EBM as an in-home procedure in terms of (1) safety, inactivation of the HI virus and retaining the protective and nutritional value of the EBM; (2) feasibility as an in-home procedure; and (3) acceptability by the mothers and their communities. This information could be used to improve clinical practitioners‟ knowledge and include it in their health education to contribute to the prevention of mother-to-child transmission. This study is based on the framework of the model for evidence-based clinical decisions of Haynes, Devereaux and Guyatt (2002). The search strategy was conducted in March/April 2009. The initial search resulted in 574 articles. After thorough screening of potentially relevant studies on heat treatment of EBM, the studies that met the inclusion criteria were critically appraised and scored based on their methodological qualities using standardised instruments. After 6 months, the search was updated. The search obtained 1 article. The final sample involved 12 articles. Conclusions were integrated and synthesised as a basis for developing a clear overview of the best available existing evidence. Finally, the findings of the study were synthesised and the research was evaluated, a conclusion was given, limitations were identified and recommendations were formulated for nursing practice, education and research. The bottom-line answer concluded that heat treatment of EBM should be emphasised as a safe alternative for feeding exposed infants (those of an HIV-positive mother, those of uncertain HIV status or during weaning if the mother cannot afford formula or cow‟s milk), but should be supported with appropriate information to the individual mother, her family and the community. Overall it can be concluded that existing evidence of the effectiveness (in terms of safety, feasibility and acceptability) of heat treatment of EBM, particularly Pretoria pasteurisation, used as a simple in-home procedure, is insufficient, and further research is required. / Thesis (M.Cur.)--North-West University, Potchefstroom Campus, 2010

Mother-to-child transmission (MTCT) of HIV is the most significant source of HIV infection in young children. As the HI virus has been identified in cell-free and cell-associated compartments of breast milk, it is clear that breast milk is one of the ways in which mother-to-child transmission of HIV can take place in addition to in utero and intrapartum transmission. While breastfeeding carries the risk of HIV transmission, not breastfeeding carries significant health risks for infants and young children, such as an increased risk of diarrhoea and pneumonia, morbidity and mortality. When an HIV-positive mother decides to breastfeed her baby, pasteurisation of expressed breast milk (EBM) could be a possible infant-feeding option to limit transmission of the HI virus through breast milk, since this method has shown to effectively inactivate HIV type 1. Three methods of pasteurisation of human milk were investigated in this study: Holder pasteurisation, flash-heating and Pretoria pasteurisation. The systematic review is a helpful method to summarise the best-quality empirical evidence of the benefits and limitations of a specific method, such as heat treatment, and to provide recommendations for future research. Therefore, the aim of this study was to critically synthesise by means of a systematic review the best available existing evidence and to provide a clear overview of the effectiveness of heat treatment of EBM as an in-home procedure to inactivate the HI virus, and in so doing limit mother-to-child transmission of HIV. This study provides the clinical practitioner with accessible information on the effectiveness of heat treatment of EBM as an in-home procedure in terms of (1) safety, inactivation of the HI virus and retaining the protective and nutritional value of the EBM; (2) feasibility as an in-home procedure; and (3) acceptability by the mothers and their communities. This information could be used to improve clinical practitioners‟ knowledge and include it in their health education to contribute to the prevention of mother-to-child transmission. This study is based on the framework of the model for evidence-based clinical decisions of Haynes, Devereaux and Guyatt (2002). The search strategy was conducted in March/April 2009. The initial search resulted in 574 articles. After thorough screening of potentially relevant studies on heat treatment of EBM, the studies that met the inclusion criteria were critically appraised and scored based on their methodological qualities using standardised instruments. After 6 months, the search was updated. The search obtained 1 article. The final sample involved 12 articles. Conclusions were integrated and synthesised as a basis for developing a clear overview of the best available existing evidence. Finally, the findings of the study were synthesised and the research was evaluated, a conclusion was given, limitations were identified and recommendations were formulated for nursing practice, education and research. The bottom-line answer concluded that heat treatment of EBM should be emphasised as a safe alternative for feeding exposed infants (those of an HIV-positive mother, those of uncertain HIV status or during weaning if the mother cannot afford formula or cow‟s milk), but should be supported with appropriate information to the individual mother, her family and the community. Overall it can be concluded that existing evidence of the effectiveness (in terms of safety, feasibility and acceptability) of heat treatment of EBM, particularly Pretoria pasteurisation, used as a simple in-home procedure, is insufficient, and further research is required. / Thesis (M.Cur.)--North-West University, Potchefstroom Campus, 2010