No abstract available. / Thesis (Ph.D.)-University of Durban-Westville, 1999.

The transfer and expression of genes in cells is an important technique for basic research and gene therapy of human disease. A model for gene therapy has been investigated making use of a transfection complex consisting of three components, the DNA i.e. the gene to be transferred and expressed; a gene delivery vehicle viz. a cationic liposome and a cell specific targeting ligand, asialoorosomucoid (AOM). Cationic liposomes are positively charged liposomes that have been prepared from synthetic lipids and have been shown to complex or bind to DNA via electrostatic attraction. They have shown potential as an efficient non-viral gene delivery vehicle in human gene therapy. In this investigation, a novel cationic liposome consisting of 3B [N -(N',N'-dimethylaminopropane)carbamoyl] cholesterol (Chol-T), dioleoylphosphatidylethanolamine (DOPE) and biotinylcholesteryl formylhydrazide was prepared and assessed as a mediator of DNA delivery in a mammalian cell culture system viz. the HepG2 cell line. The cationic liposome was synthesised and characterised by electron microscopy. Foreign DNA may be specifically delivered to target cells by a carrier system which makes use of the recognition of the asialoglycoprotein AOM by cognate receptors on the HepG2 cell plasma membrane. The positively charged AOM was biotinylated and due to this biotinylation, binds streptavidin which contains specific binding sites for biotin. The cationic liposome itself contains biotin residues in its bi-Iayer which in turn binds streptavidin resulting in a ternary complex. Further, due to the DNA binding capability of the cationic liposome, a transfection complex is produced consisting of the three components. The experiments were based on the following concepts: (i) Hepatocytes possess a unique receptor that binds to and internalises galactose-terminal asialoglycoproteins by receptor mediated endocytosis. (ii) Due to electrostatic attraction, DNA binds to cationic liposomes forming soluble complexes. (iii) Through the biotin-streptavidin reaction, the biotinylated AOM is attached to the cationic liposome containing biotin forming complexes enabling targeted delivery of the DNA. (iv) DNA containing the pGL3 gene for the luciferase enzyme was used and following transfection experiments, the luciferase assay was performed to ensure successful transfection. The complexes were tested on the hepatocellular carcinoma cell line, HepG2, which possess the asialoglycoprotein receptor. Transfection studies were conducted using a transient expression system, the luciferase assay system. Some degree of success in the transfection of HepG2 cells was observed. Results obtained in this study suggest that transfection using our targeted transfection complex consisting of cationic liposomes and cell specific targeting ligands does in fact transfect cells by receptor mediation. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2006.

The work reported here was done at the Wheeler Orchid Collection and Species Bank and the Department of Biology at Ball State University. We have developed a research teaching program with two applied research goals: genetically transforming and DNA fingerprinting orchid tissue. As part of their molecular biology education, students have investigated the genetic transformation of orchids for mitigating viral symptoms and the identification of unknown orchids by DNA fingerprinting. In a second application of the technology, DNA fingerprinting has been used to determine evolutionary relationships and to quantify genetic diversity among orchids.This dissertation details the background and need for this project and the research that was done to start it. As the early work has, developed and students have added their contributions, the data have developed into two papers formatted for submission to scientific journals. They are included as results.The first is a project designed to insert exognenous DNA into orchid tissue. The soil microbe Agrobacterium tumefaciens causes crown-gall tumors to develop in its plant hosts by inserting DNA into their cells which then controls the biosynthesis of development-controlling hormones. A. tumefaciens which has been disarmed has been routinely used to bioengineer dicotyledonous plants but its use has been rare on monocotyledons. In this paper, we report that A. tumefaciens transformed embryonic orchid tissue and caused alteration in its normal developmental course.The second paper details the DNA fingerprinting of tissue from Aplectrum hymale, a terrestrial orchid native to this climate. Three populations of A. hymale have been sampled and DNA extracted from the tissue samples. RAPD primers were used to prime PCR amplifications of random sequences of the DNA and the amplified DNA was visualized by gel electrophoresis. Loci of the resulting bands were treated as potentially multiallelic gene loci and heterozygosity between and within subpopulations was calculated. We report that the three populations could be partially differentiated by this procedure and that the two populations located nearest to each other yielded the least between -ubpopulation heterozygosity. We report very high levels of genetic diversity between individuals within small subpopulations in spite of the fact that these subpopulations are considered to be primarily clonal in reproductive nature. / Department of Biology

A major goal of our laboratory is to confer resistence specifically to the Odontoglossum ringspot virus [ORSV; sometimes referred to as tobacco mosaic virus strain O (TMV-O)] in orchids. The chosen strategy may also provide cross-protection to other pathogens. The experimental design for the entire project is presented here along with the results obtained in several preliminary experiments performed in this research. Our approach involved RT-PCR amplification of the viral coat protein gene with gene-specific primers and digestion of the cDNAs into oligonucleotides. These fragments were cloned into the selectable vector pG35barB (which confers herbicide resistence) in both sense and antisense orientations. The cloned DNA was coated with tungsten beads and shot into orchid callus tissue using a makeshift biolistic gun. Tranformant callus cells were selected for by herbicide resistance. Unfortunately the potential transformants became contaminated with fungus and could nto be analyzed to determine which oligonucleotide was received and the effect each oligonucleotide had on pathogen resistance. Due to the uncertainty of the relatedness between ORSV and TMV-O, we also sequenced the coat protein gene of TMV-O and compared the amino acid sequence with those of several strains of ORSV: the Japanese strain had the highest percent amino acid similarity (99.4%), the Type strain the second highest (98.7%), and the Korean strain the lowest (96.9%). It was concluded that TMV-O is most likely one strain of ORSV, the Japanese strain. / Department of Biology

Living cells are exquisite systems. They are strongly regulated to perform in highly specific ways, but are at the same time wonderfully robust. This combination arises from the sophistication of their construction and operation: their internal variables are carefully controlled by complex networks of dynamic biochemical interactions, crafted and refined by billions of years of evolution. Using mod- ern DNA engineering technology, scientists have begun to circumvent the long process of evolution by employing a rational design-based approach to construct novel gene networks inside living cells. Currently, these synthetic networks are relatively simple when compared to their natural counter- parts, but future prospects are promising, and synthetic biologists would one day like to be able to control cells using genetic circuits much in the way that electronic devices are controlled using electrical circuits. The importance of precise dynamical behaviour in living organisms suggests that this endeavour would benefit greatly from the insights of control theory. However, the nature of bio- chemical networks can make the implementation of even basic control structures challenging. This thesis focusses specifically on the concept of integral control in this context. Integral control is a fun- damental strategy in control theory that is central to regulation, sensory adaptation, and long-term robustness. Consequently, its implementation in a synthetic gene network is an attractive prospect. Here, the general challenges and important design considerations associated with engineering an in-cell synthetic integral controller are laid out. Specific implementations using transcriptional regu- lation are studied analytically and then in silico using models constructed with commonly available parts from the bacterium Escherichia coli. Finally, using a controller based on post-translational signalling, an on-paper design is proposed for an integral-controlled biosynthesis network intended to allow a population of engineered Saccharomyces cerevisiae cells to actively regulate the extracellular concentration of a small molecule.

Living cells are exquisite systems. They are strongly regulated to perform in highly specific ways, but are at the same time wonderfully robust. This combination arises from the sophistication of their construction and operation: their internal variables are carefully controlled by complex networks of dynamic biochemical interactions, crafted and refined by billions of years of evolution. Using mod- ern DNA engineering technology, scientists have begun to circumvent the long process of evolution by employing a rational design-based approach to construct novel gene networks inside living cells. Currently, these synthetic networks are relatively simple when compared to their natural counter- parts, but future prospects are promising, and synthetic biologists would one day like to be able to control cells using genetic circuits much in the way that electronic devices are controlled using electrical circuits. The importance of precise dynamical behaviour in living organisms suggests that this endeavour would benefit greatly from the insights of control theory. However, the nature of bio- chemical networks can make the implementation of even basic control structures challenging. This thesis focusses specifically on the concept of integral control in this context. Integral control is a fun- damental strategy in control theory that is central to regulation, sensory adaptation, and long-term robustness. Consequently, its implementation in a synthetic gene network is an attractive prospect. Here, the general challenges and important design considerations associated with engineering an in-cell synthetic integral controller are laid out. Specific implementations using transcriptional regu- lation are studied analytically and then in silico using models constructed with commonly available parts from the bacterium Escherichia coli. Finally, using a controller based on post-translational signalling, an on-paper design is proposed for an integral-controlled biosynthesis network intended to allow a population of engineered Saccharomyces cerevisiae cells to actively regulate the extracellular concentration of a small molecule.

In mehrjährigen Feldversuchen wurde der Anbau von Bt-Mais mit unbehandelt angebautem Mais und einer Anbauvariante, die mit Insektiziden gegen den Maiszünsler behandelt wurde, verglichen. Untersucht wurden Maiszünslerbefall, entstandene Ertragsverluste, Futterwert, Futterqualität, Polleneintrag in Bienenvölker und Auskreuzung. Ökonomische Vorteile von Bt-Mais zeigen sich bei Körnermais ab Ertragsverlusten von 3 - 7 dt/ha und bei Silomais ab 5 Prozent Ertragsverlust gegenüber der konventionellen Sorte. Eine Veränderung der Futterqualität bei Bt-Mais oder der behandelten Variante konnte nicht festgestellt werden. Bt-Maispolleneintrag in Honig wurde im Spurenbereich nachgewiesen. Die Kennzeichnungsschwelle von 0,9 Prozent wird dabei deutlich unterschritten. Bt-Maispollenanteile im Honig sind in einer Entfernung von 3 km nachweisbar. In Bodenproben konnte keine Anreicherung mit Cry1Ab-Protein, das im Bt-Mais (MON 810) auf den Maiszünsler toxisch wirkt, nachgewiesen werden.

The research detailed in this dissertation is focused on the use of biomolecules (i.e., peptides and proteins) to form non-biologically produced materials under mild reaction conditions (i.e, neutral pH, aqueous solutions, and room temperature). The peptides utilized in the studies detailed in this dissertation were identified through the screening of single crystal rutile TiO2 substrates or Ge powder with a phage-displayed peptide library. Twenty-one peptides were identified which possessed an affinity for Ge. Those peptides possessing a basic isoelectric point as well as hydroxyl- and imidazole-containing amino acid residues were found to be the most effective in precipitating amorphous germania from an alkoxide precursor. The phage-displayed peptide library screening of TiO2 substrates yielded twenty peptides. The titania formation activity of these peptides was found to correlate with the number of positive charges they carried. The titania materials generated by the library-identified and designed peptides were found to be composed of amorphous titania as well as <10 nm anatase and/or monoclinic TiO2 crystallites. Four recombinant proteins, derived from the amino acid sequences of proteins (silaffins) associated with biosilicification in diatoms, were also investigated for titania precipitation activity. The two most basic of these recombinant silaffins, rSil1L and rSilC, were able to induce the formation of titania. The titania precipitates generated by rSil1L were found to be similar to those produced by the phage-displayed library identified peptides. The second recombinant silaffin, rSilC, was found to produce hollow spheres of titania, which, following dehydration, were observed to transform into larger, solid spheres composed of radially aligned columns of rutile TiO2. The highly repetitive nature of the rSilC s amino acid sequence is believed to be responsible for the differences in TiO2 polymorph generated by the different recombinant silaffins and peptides. This dissertation also details research conducted on the formation of titania utilizing rSilC conjugated to synthetic and biogenic silica surfaces. These silica surfaces were functionalized with a newly developed drendritic growth technique. The dendritic functional-group amplification process was demonstrated to increase the loading of hexahisitidine tagged proteins on silica surfaces by more than 40%, as compared to traditional immobilization procedures.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 104-124). / Microfiche. / xii, 124 leaves, bound photos. 29 cm

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