<p>The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.</p>

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2001. / Thesis statement in Swedish and English abstract inserted. Based on 4 previously prepared or published papers reprinted here. Includes bibliographical references.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Biochemistry. Title from pdf file main screen (viewed on February 12, 2010). Includes bibliographical references.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Joseph Le Doux; Committee Member: Anthanassios Sambanis; Committee Member: David Archer; Committee Member: Michelle LaPlaca; Committee Member: Steve Stice; Committee Member: Todd McDevitt. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (M. Sc.)--University of Stellenbosch, 2002. / Includes bibliographical references. Also available via the Internet.

The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.

Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry.

Five cultivars, all belonging to the family Cucurbitaceae, have been tested for the ability to regenerate shoots or somatic embryos from cotyledonary explants. The influence of several combinations of growth regulators on regeneration from cotyledonary and other explants was tested. No regeneration was obtained from the two cultivars Cucurbita maxima Duch. cv A-Line and Cucurbitapepo L. cv Rolet. Somatic embryos developed on Cucurbita maxima Duch. cv Chicago Waited, a Hubbard squash. A shoot regeneration response was observed for the cultivar Cucumis sativus L. cv Ashley, but the frequency was low and results could not be repeated in subsequent experiments. A reliable shoot regeneration protocol was developed for Cucumis melo L. cv Hales Best 36. The influence of the antibiotics kanamycin sulphate and cefotaxime on shoot regeneration from cotyledonary explants of Cucumis melo L. cv Hales Best 36 was tested. The plasmid pBI121 was transferred from Escherichia coli strain HB101 into Agrobacterium tumefaciens strain LBA4404 via a triparental mating. The plasmid pBI121, contains the screenable marker gene β-glucuronidase (GUS) and the selectable neomycin phosphotransferase-II gene (NPT-II) that confers kanamycin resistance. Cotyledonary tissue was transformed using this Agrobacterium tumefaciens transformation system. The influence of co-cultivation time, inoculation time and the wound factor acetosyringone on transformation was established. Rooted plantlets were regenerated from transformed cotyledonary tissue placed on kanamycin supplemented regeneration media. Plantlets tested positive for the presence of the GUS gene, using fluorometric and histochemical assays. The developed protocol was used to transform Cucumis melo cv Hales best 36 with the pat gene that provides resistance to the herbicide Ignite®. A selection medium was developed containing phosphinothricin, the active ingredient of the herbicide Transformants were selected on this medium and five lines were recovered. These plants were acclimatized and the herbicide resistance was confirmed in greenhouse spray tests. The ploidy level of these plants was deduced from indirect evidence of micro- and macroscopic characteristics that have been shown to have a correlation with the chromosome number of melon plants. The five lines were subjected to molecular analysis. The polymerase chain reaction was used to give an indication of the transformed nature of the selected plants. Agarose gel electrophoresis confirmed that the correct size band could be obtained from the putative transformants and the presence of pat in the product was verified using a non-radioactive system for nucleic acid analysis. Stable gene insertion into the genome of the plant was verified with a Southern blot of the total genomic DNA. This was achieved by hybridising a radioactively labelled ³²P probe specific for the pat gene to a blot of restriction digested plant DNA. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

Increasing the sucrose yield and the disease resistance of plants are two major objectives of the transgenic sugarcane plant programme in South Africa. The sugarcane culm has thus been identified as one of the main target areas for transgene expression. A shortage of reliable promoter elements as well as patent limitations have necessitated the isolation of promoters that are preferentially expressed in the sugarcane culm. In the present study two different approaches were followed to isolate such promoters, and the bacterial promoter, rolC, was evaluated for tissue-specific expression in sugarcane. Differential display is a non-directed technique that was used to identify genes that are differentially expressed in the mature sugarcane culm. The original method was modified, and four putative culm-preferential fragments were isolated. Sequence and hybridisation analyses revealed that these fragments were false positives, and could therefore not be used to obtain a culm-specific promoter. Activity of the Agrobacterium rolC promoter was evaluated by analysing expression patterns of two reporter genes in the mature culm of transgenic sugarcane plants. Nucleic acid analyses indicated that the foreign DNA was incorporated into the sugarcane genome, and that mRNA transcripts were produced. Histochemical analysis was done to visualise rolC-driven GUS and GFP expression in the mature sugarcane culm. In both cases the reporter gene expression was restricted to the vascular bundles and specifically to the phloem. A directed approach was followed to isolate the gene and subsequently the promoter of the β-subunit of pyrophosphate-dependent phosphofructokinase (PFP-β). An incomplete cDNA clone was obtained from a mature culm cDNA library, and was used for the screening of a sugarcane genomic library. Two clones containing different parts of the PFP-β gene were isolated. A Deletion Factory™ system was used to analyse the clone containing the 5' end of the gene. The first five exons and 1747 bp of the 5' flanking region of the gene were sequenced. Preliminary activity analysis of the promoter region was done by constructing two expression vectors, and analysing transient GUS expression in sugarcane callus. Results indicated that the promoter is capable of driving foreign gene expression in callus. Transient expression levels were lower than that of the maize Ubi-1 promoter. Further analysis of the 5' flanking region will be done to establish whether cis-acting elements outside the analysed area have an influence on the activity of the promoter. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.