Understanding complex traits is a fundamental challenge in plant genetics and a prerequisite for molecular breeding. Tools for trait dissection are markers, maps, and quantitative trait locus (QTL) analysis. Marker-assisted selection (MAS) is an application that integrates these tools. In this thesis research, a new sequence-based marker was evaluated, maps were constructed and used, and QTLs were detected using two types of populations. Marker-assisted selection was used to develop a novel class of barley. Restriction-site Associated DNA (RAD), a sequence based-marker technology, allows for simultaneous high-density single nucleotide polymorphism (SNP) discovery and genotyping. We assessed the value of RAD markers for linkage map construction using the Oregon Wolfe Barley (OWB) mapping population. We compared a RAD-based map to a map generated using Illumina GoldenGate Assay (EST-based SNPs). The RAD markers generated a high quality map with complete genome coverage. We then used the RAD map to locate QTL for agronomic fitness traits. A paper describing this research was published (Chutimanitsakun et al., 2011). Marker-assisted selection was used to rapidly develop fall-sown barley germplasm for human food uses. The target traits were high grain β-glucan, vernalization sensitivity (VS) and low temperature tolerance (LTT). The target loci were WX and VRN-H2. Marker-assisted selection was effective in fixing target alleles at both loci and waxy starch led to increase in grain β-glucan. Unexpected segregation at VRN-H1 and VRN-H3, revealed by genome-wide association mapping (GW-AM), led to unanticipated phenotypic variation in VS and LTT. We found that GW-AM is an efficient and powerful method for identifying the genome coordinates of genes determining target traits. Precise information is obtained with perfect markers; additional research may be needed when multiple alleles are segregating at target loci and significant associations are with markers in linkage disequilibrium (LD) with the target loci. A paper describing this research will be submitted for publication. / Graduation date: 2012

Crop productivity is limited by a number of important constraints that need to be addressed urgently in order to avoid an imminent humanitarian crisis. My thesis provides three diverse yet converging examples of biotechnological solutions that can deliver fundamental knowledge, tools and potential products in the form of improved/enhanced crop plants. I conclude my thesis by discussing the potential of biotechnology to address the MDGs. My key conclusion is that although biotechnology can contribute positively and substantially towards many of the MDGs, political expediency and an over-burdening regulatory system threaten to prevent those needing the technology from gaining access, i.e. impoverished subsistence farmers and their families in the developing world.

Med genteknik är det möjligt att ändra gensammansättningen i våra livsmedel och applikationen har väckt stort intresse, inte minst bland allmänheten. Genmodifierade (GM) livsmedel har varit föremål för diskussion sedan 1970-talet. Syftet med denna uppsats är att studera olika uppfattningar och representationer om genmodifierade livsmedel. Enligt teorin om sociala representationer har representationer dubbla funktioner. Den ena är att konventionalisera objekt och den andra innebär att representationerna intar en förutbestämd form. För att analysera uppfattningar och representationer har jag använt mig av en tematisk innehållsanalys samt en analys av kommunikativa strategier av samtal i fyra fokusgrupper. Analysen av fokusgruppsdatan visar att diskussionerna cirkulerar kring tre teman: risker, möjligheter och mervärden med genmodifierade livsmedel. De risker som associeras med GM livsmedel diskuteras främst utifrån begreppsparet naturligt/antropogent och utgår ofta från ett grundläggande antagande om att naturen har ett positivt värde. De möjligheter som associeras med GM livsmedel diskuteras utifrån begreppsparet Nord/Syd och utgår ofta från antagandet att GM livsmedel först och främst gör nytta i utvecklingsländer. Antagandet om naturens positiva värde samt uppfattningen om GM livsmedlens frånvaro av fördelar för konsumenter i industrialiserade länder resulterar i att deltagarna inte ser några eller få konsumentfördelar med GM livsmedel. Representationerna kring GM livsmedel kan genom ett gemensamt meningsskapande ses både ha en konventionaliserande funktion där GM livsmedel förankras och förstås samt en preskriptiv funktion där representationerna leder till ett visst sätt att tänka.

Two computer programs were developed and used to determine the optimum operating parameters of a fedbatch and a continuous two-stage process for fermentation of recombinant bacteria. The study was conducted in three phases: (a) developing two computer programs for simulation and optimization of the above processes, (b) conducting batch culture fermentations to verify the performance of the biokinetic model, and (c) conducting fedbatch and two-stage continuous fermentation experiments to closely examine the simulation and optimization results. The Miao and Kompala (1992) biokinetic model was used for simulation of the bacterial growth and cloned gene expression. The Pattern-Search method, developed by Hooke and Jeeves (1962), was incorporated in the programs to determine the optimum values of the parameters. Extensive studies of the optimization results showed 30-40% higher productivities for the two stage continuous process over the fedbatch process when using the same media in both processes. In addition, increasing the number of stages in the continuous two-stage process resulted in very limited improvement in the productivity of the process (10-12%). The information from the process optimization was then used to design batch, fedbatch nd two stage continuous experiments. Recombinant <i>E. coli </i>(strain BL21DE3) with an inducible gene (sensitive to IPTG, isopropyl-â-D-thiogalactopyranoside) was used throughout the experiments. The experimental results from the fedbatch and two stage continuous processes clearly showed good agreement with the simulation and optimization results $(\cong$15% deviation). The experiments also revealed that the maintenance of plasmid harboring cells over the long-term operation could be an important barrier in achieving the predicted high productivity in the two stage continuous process. Finally, in addition to computer programs for optimization of genetically modified microorganisms, a new computer program with a generic algorithm for optimization of multiple CFSTR fermentation with any kind of biokinetic model was developed. The program was used to optimize multiple CFSTRs with the cybernetic biokinetic model for the first time. Besides finding the optimum residence times for multiple CFSTRs operation, the effect of inaccuracies in different cybernetic model parameters on the overall productivity of the process was investigated. The simulation results illustrated that, a single CFSTR was more sensitive in its operation to inaccuracies in the biokinetic constants as compared to optimized CFSTRs in series (2-8 times more sensitive).

Creating receptors for control of transcription with arbitrary small molecules has widespread applications including gene therapy, biosensors, and enzyme engineering. Using the combination of high throughput docking, codon randomization, and chemical complementation, we have created new receptors to control transcription with small molecules. Chemical complementation, a new method of protein engineering, was used to discover retinoid X receptors (RXR) variants that are activated by compounds that do not activate wild-type RXR. A first library of 32,768 RXR variants was designed for the synthetic retinoid-like compound LG335. The library produced ligand-receptor pairs with LG335 that have a variety of EC50s and efficacies. One engineered variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at 10 and #64979;fold lower LG335 concentration than wild-type RXR with 9cRA in yeast. The activity of this variant in mammalian cells correlates with its activity in yeast. A second library of 262,144 RXR variants was designed for two purposes: (i) to develop a high-throughput chemical complementation method to select variants that have high efficacies and low EC50s; and (ii) to find variants which are activated by small molecules not known to bind RXR variants. Selection conditions were manipulated to find only variants with high efficacies and low EC50s. This library was also selected for variants that activate transcription specifically in response to gamma-oxo-1-pyrenebutyric acid (OPBA), which is different from any known RXR ligand. OPBA was chosen as a potential ligand using high-throughput docking with the software program FlexX. Two variants are activated by OPBA with an EC50 of 5 mM. This is only ten-fold greater than the EC50 of wild type RXR with its ligand 9cRA (500 nM) in yeast. An improved method synthesizing LG335 and a method for quantifying intracellular ligand concentrations were developed. Although the LG335 synthetic method has an additional step, the overall yield was improved to 8% from 4% in the original publication. Liquid chromatography and mass spectrometry was used to quantify the intracellular concentration of LG335, which was found to be within four fold of the LG335 concentration in the media.

Protein engineering can be performed by combinatorial techniques (directed evolution) and data-driven methods using machine-learning algorithms. The main characteristic of directed evolution (DE) is the application of an effective and efficient screen or selection on a diverse mutant library. As it is important to have a diverse mutant library for the success of DE, we compared the performance of DNA-shuffling and recombination PCR on fluorescent proteins using sequence information as well as statistical methods. We found that the diversity of the libraries DNA-shuffling and recombination PCR generates were dependent on type of skew primers used and sensitive to nucleotide identity levels between genes. DNA-shuffling and recombination PCR produced libraries with different crossover tendencies, suggesting that the two protocols could be used in combination to produce better libraries. Data-driven protein engineering uses sequence, structure and function data along with analyzed empirical activity information to guide library design. Boolean Learning Support Vector Machines (BLSVM) to identify interacting residues in fluorescent proteins and the gene templates were modified to preserve interactions post recombination. By site-directed mutagenesis, recombination and expression experiments, we validated that BLSVM can be used to identify interacting residues and increase the fraction of active proteins in the library. As an extension to the above experiments, DE was applied on monomeric Red Fluorescent Proteins to improve its spectral characteristics and structure-guided protein engineering was performed on penicillin G acylase (PGA), an industrially relevant catalyst, to change its substrate specificity.

Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway. In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target. / QC 20110506

La thérapie germinale est une avenue médicale qui est loin de pouvoir être appliquée de manière sécuritaire et responsable car les connaissances médicales actuelles sont insuffisantes. De surcroît, l'encadrement normatif qui l'entoure est unanime et clame la non-acceptabilité de son application humaine. Certains instruments adoptent une approche rigide en la prohibant formellement, d'autres adoptent une approche flexible en demeurant ouverts à une éventuelle application. Il y a donc divergence quant à la légitimité de cette technique. La médecine moderne doit reposer sur des principes directeurs issus de diverses sources, empruntées au droit et à l'éthique. Les principes retenus pour examiner la légitimité de la thérapie germinale sont tirés, d'une part, des droits et libertés fondamentales: ce sont les principes fondamentaux de dignité, de liberté, d'égalité. D'autre part, ils sont issus des règles d'éthique de la recherche: plus particulièrement le principe de bienfaisance (nonmalfaisance) et celui du respect de la personne. La perspective d'une éventuelle application humaine de la thérapie germinale ne porte pas nécessairement atteinte aux principes fondamentaux, dépendamment du genre d'application qui est envisagé. Une application restreinte, appliquée dans des circonstances particulières et en vue de soulager ou d'éliminer certaines formes de détresses et de souffrances, pourrait être conforme aux principes qui soutiennent les droits et libertés fondamentales. La thérapie germinale soulève des questions éthiques difficiles et parfois inédites, notamment l'extension des risques aux générations futures et l'obligation d'un suivi à long terme pour des descendants qui n'auront pas eux-mêmes donné leur consentement à cette «thérapie». La thérapie germinale est présentement non acceptable mais ne devrait pas faire l'objet d'une prohibition totale. / Germ-line therapy is far from being applied in a secure and responsible way because of insufficient medical knowledge. The unanimity against its human application is manifest in the normative frameworks which universally reject it as unacceptable. Certain instruments adopt a rigid approach and formally prohibit it, while others adopt a flexible approach by remaining open to possible applications. There is significant divergence on the legitimacy of this technique. Modem medicine must rest on guiding principles stemming from various sources borrowed from law and from ethics. Framing principles are derived, on one hand, from fundamental rights and freedoms such as the principles of dignity, liberty, and equality, and on the other hand, from the rules of research ethics based on principles such as beneficence and the respect for persons. The prospect of human applications of germ-line therapy does not inevitably infringe on fundamental principles. It depends the application envisaged. A restricted application, used in specific circumstances to relieve or eliminate certain forms of suffering, could respect the principles endorse fundamental rights and freedoms. Germ-line therapy raises difficult and sometimes new ethical questions: notable examples include the extension of the risks to persons other than the treated subject and the obligation of long-term follow-up for the descendants who did not consent to the research. Germ-line therapy is presently unacceptable given the current state ofknowledge; however, it should not be the object of a total prohibition. / "Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maîtrise en droit (LL.M.) Option droit, biotechnologies et société". Ce mémoire a été accepté à l'unanimité et classé parmi les 10% des mémoires de la discipline. Commentaires du jury : "Mémoire de très haute tenue. Recherche exhaustive. Traitement cohérent du sujet. Approche souvent innovatrice".

Today, Africa has more countries with food security problems than any other region on the globe. Two-thirds of all countries suffering food insecurity are in Africa. Present trends would mean that the number of chronically undernourished people in the Southern region of Africa would rise from 180 to 300 million by the year 2010. In this research, I note that in the face of this food or hunger crisis, particularly in Africa, some have argued that genetic engineering biotechnology promises to combat food insecurity. Opponents of the technology argue that, to the contrary, genetic engineering biotechnology undermines food security, food sovereignty and livelihoods on the continent. The technology is designed to block access to food and kill agricultural biodiversity, vest excessive, monopolistic and exclusive power in the hands of a few biotechnologists and giant multinational corporations, and ultimately, create hunger and poverty in Africa and other developing countries by undermining organic and conventional means of farming. The thesis offers a critical theological assessment of the structural, ecological and socioeconomic effects of genetic engineering and biotechnology on agriculture, food production, food security and sovereignty in Africa against some core theological principles. The study, therefore, brings a careful critique to the growing area of science in its relationship to the current issues of food security and sovereignty. The theological framework provides a moral framework for analysis that can be applied in the debate about genetic engineering and biotechnology. In this thesis, I will consistently demonstrate that opponents of the GE technology think that proponents of r-DNA technology are mostly driven by the intent to generate and maximize profits rather than a concern for the common well being, and the intent to control all the stages of agricultural production. The corporate control over essential agricultural resources such as seeds and food entails that multinational companies have control over fundamental human rights of access to healthy, safe and adequate food, nutrition, and ultimately to social and economic development itself. This, then, becomes an issue of justice and hence the concern of the churches and theologians. In this light, then, the study argues that issues of food security and sovereignty cannot be meaningfully and credibly pursued without taking adequate recognition of moral, ethical and theological insights. Such framework would guide scientific and GE technological activities. / Thesis (M.Th.)-University of Natal, Pietermaritzburg, 2002.

This research was an attempt to use a dicot transformation vector to transform a monocot. The initial purpose of this thesis was to transform orchids with the sense and antisense coat protein genes from the Odontoglossum ringspot virus (ORSV) in an effort to mitigate viral symptoms in transgenic plants using the transformation vector, Agrobacterium tumefaciens. However, it soon became apparent that much time would be needed to develop a transformation protocol. The transformation vectors used included the Agrobacterium tumefaciens disarmed strain LBA4404 with the binary plasmid pB1121, the disarmed strain At699 with the binary plasmid pCNL65, and the wild-type strain Chry5. The marker gene on the binary plasmids of both disarmed strains was p-glucuronidase (GUS).Several transformation protocols were used in an effort to determine if this transformation system would work on orchids. Transformation was not achieved even though a number of experimental conditions were varied. These included using two different types of orchid tissue, callus and protocorms; using two different species of orchids, Cattleya Chocolate Drop x Cattleytonia Kieth Roth and Cymbidium maudidum; varying the time the plant tissue was exposed to the bacteria from 1 hour to 96 hours; performing experiments with and without the wound signal molecule acetosyringone; and exposing the tissue to the virulent strains of A. tumefaciens mentioned previously.This research also developed GUS assay conditions necessary to decrease the number of false positives due to bacterial contamination. These conditions included chloramphenicol in the GUS assay buffer. / Department of Biology