Dissertations / Theses on the topic 'Plant cell biotechnology'

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.

A novel technique was developed to immobilize plant cells. The cells are deposited on a surface of man-made fibrous material which provides for strong binding of the plant tissue biomass growing in the submerged culture. It was shown that the plant cells need to be fully viable for the attachment process to occur.
The scale-up of this technique to laboratory size specifically designed bioreactors was performed successfully. The cell immobilizing matrix was formed into a vertical spirally wound configuration to provide for a high immobilizing area-to-volume ratio (0.8-1.2 cm$ sp{-1}$). A modified airlift (riser-to-downcomer area ratio of 0.03 and vessel height-to-diameter (H/D ratio of 3) and a low H/D ($ sim$1.5) mechanically stirred vessel delivered the optimum bioreactor performance characterized by low foaming of the broth and highly efficient plant cell attachment and retention ($ geq$96%).
The growth of Catharantus roseus plant cells was investigated in these bioreactors. This process was found not to be mass transfer limited above minimal mild mixing and aeration levels ensuring sufficient supply of nutrients, especially oxygen (k$ sb{ rm L}$a $ sim$ 10-15 h$ sp{-1}$) to the immobilized biomass.
The gentle surface immobilization technique developed in this work did not hinder the biosynthesis potential of the SIPC. In fact, it appeared to induce a partial secretion of some valuable compounds into the culture medium. The mildness, easiness, efficiency, mass transfer characteristics, scale-up potential and biomass loading capacity (11-13 g d.w./L) of the surface immobilization technique make it superior to all other immobilization techniques used to culture plant cells. In addition, its bioreactor overall biomass concentration compares favourably to suspended plant cell concentrations attainable in bioreactors (15-20 g d.w./L).

Exocyst positive organelle (EXPO) is a newly discovered double membrane organelle involved in exocytosis and likely other vesicle trafficking processes. EXPO is likely generated from the ER, fused with plasma membrane and released a single membrane vesicle to cell exterior. The Arabidopsis protein Exo70E2 was found to be associated with EXPO and therefore is considered as a marker of EXPO and might play a role in EXPO-mediated vesicle trafficking. Understanding the biological function of AtExo70E2 (abbreviated as E2 in this thesis) will be very helpful in unraveling the function of EXPO. The aim of this work was to use various molecular, genetic and physiological approaches to determine the possible role of Arabidopsis Exo70E2 in biological pathways. By using the Exo70E2pro:GUS line, the expression pattern of Exo70E2 was determined. Exo70E2 was expressed mainly in roots, especially in root tips and epidermal cells in the division and elongation zones of roots. Its expression level was induced when the seedlings were treated with Flg22, a peptide derived from bacterial flagillin protein that induces the plant defense response. The tissue subcellular localization of Exo70E2 was also studied using the 35S:Exo70E2-eYFP and Exo70E2pro:Exo70E2-GFP reporter lines. The GFP fusion protein was found primarily in the epidermal cells of roots even in the 35S:Exo70E2-eYFP lines. For phenotypic analysis resulting from mutations of the Exo70E2 gene, I obtained three T-DNA insertion mutant lines and generated its overexpression lines. The two mutant alleles, e2-2 and e2-3 are in the Columbia ecotype background and further characterized. e2-2 which has a T-DNA insertion in an exon is likely a knock out line as Exo70E2 gene transcript could not be detected. e2-3, which carries a T-DNA insertion in its promoter region, was found to accumulate a higher level of the transcript, suggesting that the insertion causes its enhanced expression of Exo70E2. There was no obvious difference between wild type and e2-2 in their phenotypes under different conditions tested in this study. However, e2-3 had a retarded growth phenotype when grown in soil or on MS medium. The seedlings of e2-3 on MS medium also had a yellowish color although such a phenotype was not obvious when they were grown in soil. When supplementing the MS medium with sucrose, glucose or mannitol, the growth of e2-3 was more reduced compared to wild type under these conditions. However, on the medium with NaCl or under phosphate deficiency, the yellowish phenotype of e2-3 was rescued and the mutant seedlings became relatively healthier than the seedlings under the regular MS medium. A proteomics approach was taken to compare protein secreted from the seedlings of wild type and the mutants. Proteins secreted by seedlings to the liquid medium were collected, concentrated and subjected to MS analysis. Comparison of the profiles of secreted proteins between the wild type and the mutants leaded to identification of candidate proteins whose secretion might be affected by the mutation. My study indicates that Exo70E2 and EXPO are involved in transporting proteins (likely also metabolites) to the exterior of cells and the rhizosphere and might play an important role in stress responses.

Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016.
Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities. In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.

Tese de Doutoramento em Ciências Veterinárias
Xyloglucan (XG) is the major plant cell wall hemicellulose. Biochemical and structural studies on a xyloglucanase, Xgh74A, from Clostridium thermocellum are presented here. In addition, the carbohydrate-binding modules (CBMs) from C. thermocellum Cel9D-Cel44A, CtCBM30 and CtCBM44, which recognize XG, are characterized in this work. CtCel9D-Cel44A’s C-terminal module of unknown function is a CBM, constituting the founder member of family 44. Structural studies revealed that both CtCBM30 and CtCBM44 present Type B binding site topologies where tryptophans play an important role in ligand recognition. Cel44A is an endoglucanase domain displaying some xylanase activity, which action is potentiated by CtCBM44 through a targeting effect. Biochemical and structural studies on CtLic26A-Cel5E are also described here. CtLic26A is a mixed b-1,4-b-1,3-glucanase and Cel5E an endo-b-1,4- glucanase. The three-dimensional structure of CtLic26A provides insights in the mechanism of lichenan recognition by family 26 glycoside hydrolases. Finally, novel biotechnological applications for CBMs were investigated. An experiment was conducted where a barley-based diet for broilers was supplemented with CtLic26ACel5 derivatives, with or without CtCBM11, and with a commercial enzyme. The fusion of four antimicrobial peptides with CipA from CtCBM3 originated recombinants with high affinity for crystalline cellulose, indicating CBMs could fix bioactive molecules to cellulose.
RESUMO: O xiloglucano é a principal hemicelulose das paredes celulares vegetais. Neste trabalho apresentam-se estudos bioquímicos e estruturais sobre a xiloglucanase Xgh74A do Clostridium thermocellum. São também estudados módulos de ligação a carbohidratos (CBMs) com afinidade para o xiloglucano, o CtCBM30 e o CtCBM44 da CtCel9DCel44A. O módulo C-terminal revelou-se um CBM, sendo o membro fundador da família 44. As estruturas cristalográficas desses CBMs mostram locais de ligação aos polissacáridos com topologia do Tipo B, nos quais os triptofanos representam um papel importante no reconhecimento dos ligandos. A Cel44A é uma endoglucanase com actividade xilanásica, cuja acção é potenciada pelo CtCBM44. Estudos bioquímicos e estruturais sobre a CtLic26A-Cel5E são também apresentados, sendo a CtLic26A uma b-1,4-b-1,3-glucanase e a Cel5E uma endo-b-1,4-glucanase. A estrutura tridimensional da CtLic26A revelou os mecanismos estruturais que modulam a especificidade da enzima. Neste trabalho são pesquisadas novas aplicações biotecnológicas para os CBMs. Efectuou-se um ensaio com frangos de carne, suplementando dietas à base de cevada com derivados recombinantes da CtLic26A-Cel5, com e sem CtCBM11, e com uma enzima comercial. A fusão de quatro péptidos antimicrobianos com a CipA do CtCBM3 originou recombinantes com elevada afinidade para celulose cristalina, indicando que os CBMs poderão fixar moléculas bioactivas a materiais celulósicos.
This work was funded by Fundação para a Ciência e a Tecnologia, grant SFRH/BD/16731/2004, and co-funded by POCI 2010 and FSE from Ministério da Ciência, Tecnologia e Ensino Superior

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

Plants produce a variety of secondary metabolites, e.g. to defend themselves against herbivores or to attract pollinating insects. Plant cell biotechnology offers excellent opportunities in order to use such secondary plant metabolites to produce goods with consistent quality and quantity throughout the year, and therefore to act independently from biotic and abiotic environmental factors. This article presents results of an extensive study of plant cell in vitro cultivation in a modern shake flask system with non-invasive online respiration activity monitoring unit. Comprehensive screening experiments confirm the successful transfer of a model culture (sunflower suspension) into the shake flask monitoring device and the suitability of this respiration activity monitoring unit as qualified tool for screening of plant in vitro cultures (sunflower and sage suspension). The authors demonstrate deviations between online and offline data due to varying water evaporation from different culture flask types. The influence of evaporation on growth-specific parameters thereby rises with increasing cultivation time. Furthermore, possibilities to minimize the impact of evaporation, either by adjusting the inlet air moisture or by measuring the evaporation in combination with an appropriate correction of the measured growth values, are shown.

Plants produce a variety of secondary metabolites, e.g. to defend themselves against herbivores or to attract pollinating insects. Plant cell biotechnology offers excellent opportunities in order to use such secondary plant metabolites to produce goods with consistent quality and quantity throughout the year, and therefore to act independently from biotic and abiotic environmental factors. This article presents results of an extensive study of plant cell in vitro cultivation in a modern shake flask system with non-invasive online respiration activity monitoring unit. Comprehensive screening experiments confirm the successful transfer of a model culture (sunflower suspension) into the shake flask monitoring device and the suitability of this respiration activity monitoring unit as qualified tool for screening of plant in vitro cultures (sunflower and sage suspension). The authors demonstrate deviations between online and offline data due to varying water evaporation from different culture flask types. The influence of evaporation on growth-specific parameters thereby rises with increasing cultivation time. Furthermore, possibilities to minimize the impact of evaporation, either by adjusting the inlet air moisture or by measuring the evaporation in combination with an appropriate correction of the measured growth values, are shown.

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences. This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities. The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies. The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.

Membrane protein mechanotransduction is the altered function of an integral membrane protein in response to mechanical force. Such mechanosensors are found in all kingdoms of life, and increasing numbers of membrane proteins have been found to exhibit mechanosensitivity. How they mechanotransduce is an active research area and the topic of this thesis. The methodology employed is classical molecular dynamics (MD) simulations. MD systems are complex, and two programs were developed to reduce this apparent complexity in terms of both visual abstraction and statistical analysis. Bendix detects and visualises helices as cylinders that follow the helix axis, and quantifies helix distortion. The functionality of Bendix is demonstrated on the symporter Mhp1, where a state is identified that had hitherto only been proposed. InterQuant tracks, categorises and orders proximity between parts of an MD system. Results from multiple systems are statistically interrogated for reproducibility and significant differences at the resolution of protein chains, residues or atoms. Using these tools, the interaction between membrane and the Escherichia coli mechanosensitive channel of small conductance, MscS, is investigated. Results are presented for crystal structures captured in different states, one of which features electron density proposed to be lipid. MD results supports this hypothesis, and identify differential lipid interaction between closed and open states. It is concluded that propensity for lipid to leave for membrane bulk drives MscS state stability. In a subsequent study, MscS is opened by membrane surface tension for the first time in an MD setup. The gating mechanism of MscS is explored in terms of both membrane and protein deformation in response to membrane stretch. Using novel tension methodology and the longest MD simulations of MscS performed to date, a molecular basis for the Dashpot gating mechanism is proposed. Lipid emerges as an active structural element with the capacity to augment protein structure in the protein structure-function paradigm.

Aquesta tesi doctoral descriu diversos avenços conceptuals per a la comprensió molecular de la senyalització de brassinosteroides en el nínxol de cèl·lules mare de l’arrel d’Arabidopsis thaliana. Els brassinoesteroides són les hormones esteroides de les plantes que juguen un paper important en el creixement i desenvolupament vegetal. En l’arrel primària d’Arabidopsis, els brassinoesteroides estan involucrats en el desenvolupament del meristem i manteniment de les cèl·lules mare. Les cèl·lules mare són les cèl·lules més indiferenciades, aquestes es van dividint i diferenciant per generar els diferents tipus cel·lulars de l’arrel. Aquests processos estan estretament controlats per factors interns i externs. El baix nombre de cada població de cèl·lules mare fa complicat el seu estudi de manera individual, per tant, el desenvolupament de mètodes amb resolució per estudiar tipus cel·lulars i fins i tot cèl·lules individuals representa una oportunitat única per investigar aquesta població cel·lular tan escassa. En aquesta tesi doctoral, utilitzem una estratègia multidisciplinària, que inclou genètica, anàlisi transcriptòmica i models matemàtics, per identificar les característiques moleculars de les cèl·lules mare de l’arrel enfocant-nos en el paper dels brassinoesteroides en aquestes cèl·lules. Defectes en els processos de creixement i desenvolupament vegetal generalment són deguts a defectes en el creixement de l’arrel principal. Com que la quantificació precisa de la longitud de l’arrel requereix molt de temps, en aquesta tesi doctoral es descriu el desenvolupament de l’eina MyROOT per a la mesura d’arrels d’Arabidopsis d’una manera semiautomàtica (Capítol 2). A més a més, els resultats presentats en aquesta tesi revelen quin és el paper dels brassinoesteroides en el nínxol de cèl·lules mare. Mitjançant una estratègia de biologia de sistemes s’ha estudiat el paper del factor de transcripció BRAVO, regulat per brassinoesteroides, juntament amb WOX5 en el creixement i desenvolupament de l’arrel (Capítol 3). Igualment, una aproximació específica per tipus cel·lulars ha revelat la resposta transcripcional mediada per BRAVO al centre quiescent i en les cèl·lules mare vasculars adjacents (Capítol 4). Finalment, l’ús de seqüenciació massiva de l’RNA (RNAseq) amb resolució cel·lular ha estat implementat per generar el primer atles transcriptòmic del nínxol de cèl·lules mare de l’arrel. Aquesta aproximació ha permès identificar les característiques moleculars de les cèl·lules mare i la presència de diferents poblacions d’aquestes en el domini d’expressió de BRAVO (Capítol 5). Aquesta tesi doctoral avança en el coneixement de les cèl·lules mare de les plantes i posa de manifest la necessitat d’estratègies multidisciplinàries per descobrir processos fonamentals de el desenvolupament vegetal.
Esta tesis doctoral reporta avances conceptuales en la respuesta molecular mediada por la ruta de señalización de los brasinoesteroides en el nicho de células madre de Arabidopsis thaliana. Los brasinoesteroides son las hormonas esteroideas de las plantas y juegan un papel importante en el crecimiento y desarrollo vegetal. En la raíz primaria de Arabidopsis, los brasinoesteroides están involucrados en el desarrollo del meristemo y mantenimiento de las células madre. En el nicho de células madre, las células madre son las células más indiferenciadas que se van dividiendo y diferenciando para generar los distintos tipos celulares de la raíz. Estos procesos están estrechamente controlados por factores internos y externos. El bajo número de cada población de células madre hace complicado su estudio individualmente, por lo tanto, el desarrollo de métodos con resolución para estudiar tipos celulares e incluso células individualmente representa una oportunidad única para investigar esta población celular tan escasa. En esta tesis doctoral, utilizamos una estrategia multidisciplinar, que incluye genética, análisis transcriptómicos y modelos matemáticos, para identificar las características moleculares de las células madre de la raíz y enfocándonos al papel de los brasinoesteroides en esas células. Defectos en procesos de crecimiento y desarrollo vegetal se reflejan generalmente en defectos en el crecimiento de la raíz principal. Como la cuantificación precisa de la longitud de la raíz requiere mucho tiempo, en esta tesis doctoral se describe el desarrollo de la herramienta MyROOT para la medida de raíces de Arabidopsis de una forma semiautomática (Capítulo 2). Además, los resultados presentados en esta tesis revelan el papel de los brasinoesteroides en el nicho de células madre. Una estrategia de biología de sistemas revela el papel del factor de transcripción BRAVO, regulado por brasinoesteroides, junto con WOX5 en el crecimiento y desarrollo de la raíz (Capítulo 3). Igualmente, una aproximación específica para tipos celulares revela la respuesta transcripcional mediada por BRAVO en el centro quiescente y en las células madre vasculares adyacentes (Capítulo 4). Por último, el uso de RNAseqs con resolución celular ha sido implementado para generar el que creemos es el primer atlas transcriptómico del nicho de células madre de la raíz. Esta aproximación ha permitido identificar las características moleculares de las células madre y la presencia de diferentes poblaciones de estas células en el dominio de expresión de BRAVO (Capítulo 5). Esta tesis doctoral avanza en el conocimiento de las células madre de las plantas y pone de manifiesto la necesidad de estrategias multidisciplinares para descubrir procesos fundamentales del desarrollo vegetal.
This PhD thesis dissertation reports a number conceptual advances for the molecular understanding of brassinosteroid signaling in the root stem cell niche of Arabidopsis thaliana. Brassisnosteroids are the plant steroid hormones that play important roles in plant growth and development. In the Arabidopsis primary root, brassinosteroids are involved in meristem development and stem cell maintenance. At the root stem cell niche, stem cells are the more undifferentiated cells that divide and differentiate to give rise to the distinct cell types of the root. These processes are tightly controlled by internal and external factors. The low number each stem cell population makes it difficult to study them individually, therefore, the advent of cell-type and single-cell specific approaches represents a unique opportunity to investigate this rare cell population. In this PhD thesis, we used an interdisciplinary approach, including genetics, transcriptomics analysis and mathematical modelling, to identify the molecular signatures of the root stem cells with a focus on the role of brassinosteroid hormones in those cells. Defects in growth and development processes is often reflected in abnormal primary root growth. As the accurate quantification of plant primary root length is time consuming, in this PhD dissertation, we describe the development of MyROOT software for the semi-automatic measurement of Arabidopsis primary roots (Chapter 2). In addition, the results presented in this thesis uncover the role of brassinosteroids in the stem cell niche. A systems biology approach revealed a role of the brassinosteroid-mediated BRAVO transcription factor together with WOX5 in overall root growth and development (Chapter 3). Moreover, cell-type specific transcriptomic analysis uncover the transcriptional response mediated by BRAVO in the QC and adjacent vascular stem cells (Chapter 4). Finally, the use of single-cell RNAseq has been implemented to generate to our knowledge the first transcriptomic atlas of the root stem cell niche. This approach allowed to characterize the molecular signatures of the stem cells and to find novel stem cell populations within the BRAVO expression domain (Chapter 5). Overall, the present PhD thesis advances in the understanding of stem cells in plants and expose the necessity of multidisciplinary approaches to uncover fundamental biological questions in plant development.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegetal

Les fucosyltransférases sont les enzymes responsables du transfert d'un groupement fucose à partir du GDP-fucose sur des accepteurs variés (oligosaccharides, protéines,...). Chez l'homme, les rôles de ces glycosyltransférases sont importants dans un grand nombre de processus biologiques et pathologiques. De nombreuses fucosyltransférases existent chez les végétaux. Notamment FuT1, qui transfert un fucose en 1,2 sur un résidu galactose du xyloglucane : l'une des hémicelluloses majeures de la paroi des dicotylédones. Ce polysaccharide ramifié est très étudié en raison de ses applications actuelles et potentielles dans différents secteurs de l'industrie : textile, alimentation, pharmaceutique, etc. Les objectifs de ce doctorat ont été d'obtenir des informations biochimiques et structurales sur la fucosyltransférase AtFuT1 de la plante modèle Arabidopsis thaliana. Pour cela, une forme recombinante soluble de l'enzyme a été produite avec le système baculovirus /cellules d'insectes. De manière à obtenir suffisamment de protéines pour les études structurales, une méthode de culture en suspension des cellules a été mise en place au laboratoire. Un protocole de purification en deux étapes, impliquant une chromatographie par affinité puis par exclusion de taille, a permis d'obtenir à partir d'un litre de culture cellulaire entre un à deux mg de protéine pure et homogène. Ce résultat a permis de mieux comprendre le comportement de l'enzyme vis-à-vis de ses substrats (GDP-fucose et xyloglucane), d'obtenir des cristaux de la protéine et de résoudre la structure 3D d'AtFuT1 en complexe avec le GDP et un oligosaccharide dérivé du xyloglucane, à une résolution de 2,2 Å. La forme AtFut1 produite se comporte en solution et dans le cristal sous forme d'un dimère non covalent. La protéine adopte un repliement qui est un variant du type GT-B classique. Parallèlement à ces travaux, des tests d'activité glycosyltransférase ont été mis au point permettant le criblage de nombreuses conditions de réactions. L'ensemble des méthodes et techniques développées constitue une base utile, devant faciliter la caractérisation d'autres glycosyltransférases
Fucosyltransferases are enzymes that transfer a fucose residue from GDP-fucose on varied acceptors (oligosaccharides, proteins). In Human, these glycosyltransferases are involved in many biological and pathological processes. Numerous fucosyltransferase exist in the plant kingdom. Among them, FuT1 transfers a fucose linked in 1,2 onto a galactose of xyloglucan: a major hemicellulose of dicots cell wall. This branched polysaccharide is intensively studied because of its current and potential industrial applications in textile, food, pharmaceuticals, etc. The main objective of this PhD program was to obtain biochemical and structural information on the fucosyltransferase AtFuT1 from the model plant Arabidopsis thaliana. A recombinant form of this protein has therefore been produced, using the baculovirus/insect cell system. In order to get sufficient amount of protein for structural studies, a suspension cell culture method has been set-up in the lab. A two-step purification protocol, involving affinity and size exclusion chromatography was established. The active, and highly pure recovered protein was used to determine the biochemical properties of the protein towards its substrates (GDP-fucose and xyloglucan), to get protein crystals and hence to solve its 3D structure in complex with GDP and a xyloglucan derived oligosaccharide (2.2 Å resolution). AtFuT1 behaves in solution and in crystallo as a non-covalent dimer. The protein adopts a variant of the classical GTB fold. In addition, novel glycosyltransferase assay have been designed allowing the screening of numerous reaction conditions. Methods and techniques that were developed during this study should be a useful base for the characterization of other glycosyltransferase

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Necrotrophic fungi infect many economically important crop plants. This results in great losses in the agricultural sector world-wide. Understanding the nature by which plants respond to pathogens is imperative for genetically enhancing disease resistance in plants. Research tools have significantly contributed to our understanding of how the plant responds to pathogen attack, identifying an array of defence mechanisms used by plants upon attack. Many fungal pathogens secrete endopolygalacturonases (endoPGs) when infecting plants. These hydrolytic enzymes are inhibited by polygalacturonase-inhibiting proteins (PGIPs) associated with plant cell walls. PGIPs are well characterised and their current known functions are all linked to endoPG inhibition and the subsequent upregulation of plant defence pathways. Work on grapevine PGIPs have shown that apart from being efficient antifungal proteins, leading to protection of the plant against Botrytis cinerea when overexpressed, PGIPs might also have additional functions linked to cell wall strengthening. This working hypothesis formed the motivation of this study where a cinnamyl alcohol dehydrogenase (CAD) (1.1.1.195) gene was targeted for functional analysis in tobacco (Nicotiana tabacum). Some previous work and genetic resources obtained is relevant to this study, specifically previously characterized transgenic tobacco lines overexpressing the Vitis vinifera pgip1 (Vvpgip1) gene. These lines have confirmed PGIP-specific resistance phenotypes against B. cinerea, as well as increased levels of CAD transcripts in healthy plants. Moreover, preliminary evaluations indicated increased lignin levels as well as differential expression of several other cell wall genes in these overexpressing lines (in the absence of infections). In this study we generated a transgenic tobacco population, overexpressing the native CAD14 gene, via Agrobacterium transformations. The transgene was overexpressed with the Cauliflower Mosaic Virus promoter (CaMV 35Sp). The CAD transgenic population was analyzed for transgene integration and expression and showed active transcription, even from leaves that normally don’t express CAD to high levels. These lines, together with the untransformed control, and a representative transgenic VvPGIP1 tobacco line previously characterized with elevated expression of CAD were used for all further analyses, specifically CAD activity assays of stems and leaves, as well as whole plant infections with B. cinerea. CAD enzyme activity assays were performed on healthy uninfected plant lines, without inducing native CAD expression or resistance phenotypes (i.e. without Botrytis infection). CAD activity was detected in leaves and stems, but a statistically sound separation between the CAD population and the untransformed control was only observed in the stems. The CAD assays also confirmed previous results that indicated that CAD transcription was upregulated in the PGIP line in the absence of infection. Overall, in all plant lines the stems exhibited 10-fold higher levels of CAD activity than the leaves, but the transgenic VvPGIP1 line showed a further 2-3-fold increase in CAD activity in the stems, when compared to the untransformed control and the majority of the CAD overexpressing lines. Disease assessment by whole plant infections with B. cinerea of the CAD transgenic plants revealed reduced disease susceptibility towards this pathogen. A reduction in disease susceptibility of 20 – 40% (based on lesion sizes) was observed for a homologous group of transgenic lines that was statistically clearly separated from the untransformed control plants following infection with Botrytis over an 11-day-period. The VvPGIP1 transgenic line displayed the strongest resistance phenotype, with reduction in susceptibility of 47%. The reduction in plant tissue maceration and lesion expansion was most pronounced in the VvPGIP1 line compared to the CAD transgenic plants, while the CAD transgenic plants showed more reduction than the untransformed control. In combination, the data confirms that CAD upregulation could lead to resistance phenotypes. Relating this data back to the previously observed upregulation of CAD in the VvPGIP1-overexpressing lines, the findings from this study corroborates that increased CAD activity contributes to the observed resistance phenotypes, possibility by strengthening the cell wall. In conclusion, this study yielded a characterized transgenic population overexpressing the CAD14 gene; this overexpression contributed to increased RNA transcription compared to the untransformed control plant, increased CAD activity (most notably in the stems) and a disease resistance phenotype against Botrytis. These findings corroborates the current working hypothesis in our group that PGIPs might have a role in preparing the plant cell for attack by contributing to specific cell wall changes. The exact mechanisms are still currently unknown and under investigation. The transgenic lines generated in this study will be invaluable in the subsequent analyses where these various phenotypes will be subjected to profiling and accurate cell wall analyses.
AFRIKAANSE OPSOMMING: Nekrotrofiese swamme infekteer en beskadig verskeie ekonomies belangrike gewasse. Dit lei wêreldwyd tot groot verliese vir die landbousektor. Dit is noodsaaklik om te verstaan hoe plante reageer teenoor patogene, sodat die siekteweerstand van plante verbeter kan word. Navorsingshulpbronne het beduidend bygedra tot die kennis van plantreaksies tydens patogeniese aanvalle, en het sodoende ‘n reeks van weerstandmeganismes, wat die plant inspan tydens ‘n aanval, geïdentifiseer. Verskeie patogeniese swamme skei endopoligalakturonases (endoPGs) af tydens plantinfeksie. Hierdie hidrolitiese ensieme word geïnhibeer deur poligalakturonase-inhiberende proteïene (PGIPs) wat met die plantselwand geassosieerd is. PGIPs is goed gekarakteriseerd en al hulle huidiglik bekende funksies is gekoppel aan endoPG inhibisie en die daaropvolgende opregulering van plant weerstandspaaie. Navorsing op wingerd PGIPs het gewys dat, afgesien van die feit dat PGIPs goeie antifungiese proteïene is wat lei tot beskerming van die plant teen Botrytis cinerea wanneer dit ooruitgedruk word, PGIPs ook moontlik addisionele funksies verrig wat verwant is aan selwandversterking. Hierdie werkshipotese vorm die motivering vir hierdie studie waarin ‘n sinnamiel alkohol dehidrogenase (SAD) (1.1.1.195) geen geteiken is vir funksionele analise in tabak (Nicotiana tabacum). Vorige navorsing en genetiese hulpbronne daardeur verkry is belangrik vir hierdie studie, spesifiek die gekarakteriseerde transgeniese tabaklyne wat die Vitis vinifera pgip1 (Vvpgip1) geen ooruitdruk. Hierdie lyne besit bevestigde PGIP-spesifieke weerstandsfenotipes teen B. cinerea, sowel as hoër vlakke van SAD transkripte in gesonde plante. Voorlopige analises het ook aangedui dat hierdie ooruitdrukkende lyne hoër lignien vlakke het, asook differensiële uitdrukking van verskeie ander selwandgene (in die afwesigheid van infeksie). In hierdie studie is ‘n transgeniese tabakpopulasie gegenereer wat die natuurlike tabak SAD14 geen ooruitdruk, deur middel van Agrobacterium transformasie. Die transgeen is ooruitgedruk deur die Blomkool Mosaïek Virus promoter (CaMV 35Sp). Die SAD transgeniese populasie is geanaliseer vir transgeen integrasie en uitdrukking en het aktiewe transkriptering getoon, selfs in blare waar daar normaalweg nie hoë vlakke van SAD uitgedruk word nie. Hierdie lyne, die ongetransformeerde wilde-tipe kontrole sowel as ’n verteenwoordigende transgeniese VvPGIP1 tabaklyn wat vooraf gekarakteriseerd was met hoë SAD uitdrukking, is gebruik vir alle verdere analises, spesifiek SAD aktiwiteitstoetse in stingels en blare, asook heelplantinfeksies met B. cinerea. Aktiwiteitsanalises van die SAD ensiem is gedoen op gesonde ongeinfekteerde plantlyne, sonder om natuurlike tabak SAD uitdrukking of weerstandsfenotipes te induseer (dus sonder Botrytis infeksie). SAD aktiwiteit is waargeneem in beide die blare en stingels, maar ‘n statisties betekenisvolle skeiding is slegs gevind tussen die SAD populasie en die ongetransformeerde kontrole in die stingels. Die SAD toetse het ook vorige resultate bevestig wat aangedui het dat SAD transkripsie opgereguleer word in die PGIP lyn in die afwesigheid van infeksie. Die stingels het oor die algemeen ‘n 10-voudige vermeerdering in SAD aktiwiteitsvlakke getoon in vergelyking met die blare, maar die transgeniese VvPGIP1 lyn het ‘n verdere 2-3-voudige verhoging in SAD aktiwiteit gehad in die stingels ,in vergelyking met die ongetransformeerde kontrole en die meerderheid van die SADooruitdrukkende lyne. Siekteweerstand ondersoeke deur middel van heelplantinfeksies met B. cinerea van die SAD transgeniese plante, het verminderde vatbaarheid aangedui ten opsigte van hierdie patogeen. ‘n Afname in siekte-vatbaarheid van 20 – 40% (gebaseer op wondgroottes) is waargeneem vir ‘n homoloë groep transgeniese lyne wat statisties betekenisvol geskei kon word van die ongetransformeerde kontrole plante na infeksie met Botrytis in ‘n infeksietoets wat 11 dae geduur het. Die VvPGIP1 transgeniese lyn het die mees weerstandbiedende fenotipe gehad, met ‘n afname in siekte-vatbaarheid van 47%. Die afname in plantweefselafbreking en wondgrootte was die duidelikste in die VvPGIP1 lyn in vergelyking met die SAD transgeniese plante, terwyl die SAD transgeniese plante ‘n groter afname aangedui het as die ongetransformeerde kontrole. In kombinasie het die data bevestig dat SAD opregulasie kan lei tot weerstandbiedende fenotipes. Hierdie data, in vergelyking met die vorige bevinding van opregulasie van SAD in die VvPGIP1-ooruitdrukkende lyne, bevestig dat hoër SAD aktiwiteit bydra tot die waargenome weerstandbiedende fenotipes, moontlik deur versterking van die plantselwand. Ter afsluiting, hierdie studie het ‘n gekarakteriseerde transgeniese populasie wat die SAD14 geen ooruitdruk gelewer; hierdie ooruitdrukking het bygedra tot hoër RNA transkripsie in vergelyking met die kontrole, verhoogde SAD aktiwiteit (veral in die stingels) en siekteweerstandbiedende fenotipes teenoor Botrytis. Hierdie bevindinge ondersteun die huidige werkshipotese in ons groep dat PGIPs moontlik ‘n rol speel in die voorbereiding van die plantsel teen infeksie deur spesifieke selwandveranderinge te veroorsaak. Die spesifieke meganismes is steeds onbekend en word verder ondersoek. Die transgeniese lyne wat tydens hierdie studie gegenereer is, sal baie belangrik wees in opvolgende analises waar hierdie verskillende fenotipes gebruik kan word om die profiel van selwandkomponente, maar ook die akkurate selwandsamestelling te bestudeer.

Sous l'effet de glycopeptides extraits de parois du mycelium de (phytophtora parantica nicotianae) et appeles eliciteurs, les cellules de tabac en culture synthetisent de grandes quantites d'ethylene. L'etude de l'hormone aux differentes etapes de sa voie de biosynthese montre une stimulation rapide et transitoire de l'activite acc-synthase, vraisemblablement par synthese de novo de l'enzyme, entrainant l'augmentation du taux intracellulaire d'acc sans pour autant modifier celui du n-malonyl-acc. (. . . )

Nous avons adopte une strategie, consistant en la modification qualitative et quantitative de la composition sterolique de plantes. Pour cefaire, nous avons traite des plantes a l'aide d'inhibiteurs de la biosynthese des sterols, possedant essentiellement deux cibles: la cycloeucalenol-obtusifoliol isomerase et la delta-huit-delta-sept-sterol isomerase. En agissant de la sorte, en bloquant diverses etapes de la biosynthese des phytosterols, nous accumulons de nouveaux sterols, en l'occurence des cyclopropylsterols. La molecule utilisee pour induire de telles modifications, est un fongicide systemique: le fenpropimorphe. Nous avons etudie le comportement de cette molecule en tant qu'inhibiteur chez des cellules animales (fibroblastes de souris) et chez des cellules vegetales (mais et ble)

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009.
Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide.

Agrobacterium tumefaciens transfers virulence effector proteins to infected host plants to facilitate the transfer and trafficking of a piece of its tumor inducing (Ti) plasmid, (T-[transfer] DNA), into and through plant cells. T-DNA integrates into the host genome where it uses the host’s gene expression machinery to express transgenes. Scientists have used this process to insert beneficial genes into plants by replacing native T-DNA in the bacteria with engineered T-DNA, making Agrobacterium-mediated transformation the preferred method for crop genetic engineering. In spite of its wide-spread use in research and agriculture, we still do not have a complete understanding of the transformation process. Consequently, many important crop species remain highly resistant to transformation. One of my lab’s major goals is to define the molecular interactions between Agrobacterium and its host plants which mediate transformation. I study the role of the Agrobacterium effector protein, VirE2, which is important for plant transformation. VirE2 likely coats the transferred DNA (T-DNA) after it enters the plant cell and protects it from degradation. VIP1 is a host transcription factor that interacts with VirE2 and is involved in activating plant defense responses. VIP1 localizes to both the cytoplasm and the nucleus. Under stress, VIP1 localizes to the nucleus where it activates expression of defense response genes. This observation led to the model that T-DNA-bound VirE2 binds VIP1 and uses VIP1 nuclear localization to deliver T-DNA into the nucleus (the “Trojan Horse” model). In contrast to this model, our lab has obtained data showing that VirE2 holds at least a portion of the VIP1 pool outside the nucleus. We also showed that VIP1 and its homologs are not necessary for transformation. VirE2 interacts with several host proteins in addition to VIP1, and these interactions could lead to changes in host gene expression and protein levels, possibly facilitating transformation. We investigated this model by placing VirE2 under the control of an inducible promoter in Arabidopsis and performing RNA-seq and proteomics under non-induced and induced conditions, and in the presence of Agrobacterium to determine its individual effect on plant RNA and protein levels during infection. Some genes differentially expressed after VirE2 induction are known to be important for transformation. Knockout mutant lines of some VirE2 differentially expressed genes showed altered transformation phenotypes. Protein levels of genes known to be important for transformation were also increased in response to VirE2 induction, and overexpression of some of these genes resulted in increased transformation susceptibility. We therefore conclude that VirE2 modulates both plant RNA and protein levels to facilitate transformation.

Fu Lai Hong.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 106-110).
Abstracts in English and Chinese.
Thesis/Assessment Committee --- p.ii
Statement --- p.iii
Acknowledgements --- p.iv
Abstract --- p.v
摘要 --- p.vi
Lists of Figures --- p.x
Lists of Tables --- p.xiii
List of Abbreviations --- p.xiv
Amino acid abbreviation --- p.xvi
Chapter Chapter 1 --- General Introduction --- p.1
Chapter 1.1 --- Human α-L-iduronidase (hIDUA) --- p.2
Chapter 1.1.1 --- Lysosomal storage disease --- p.2
Chapter 1.1.2 --- Treatments of MPS 1 --- p.4
Chapter 1.2 --- Plant cells as bioreactors --- p.5
Chapter 1.3 --- The Plant secretary pathway --- p.7
Chapter 1.3.1 --- Transport of soluble proteins --- p.9
Chapter 1.3.2 --- Transport of integral membrane proteins --- p.10
Chapter 1.4 --- Differences between plant and human proteins --- p.11
Chapter 1.5 --- Reducing the differences between plant and human proteins --- p.12
Chapter 1.6 --- Previous study: Expression of IDUA in transgenic tobacco plant --- p.13
Chapter 1.7 --- Project objectives --- p.14
Chapter 1.8 --- Long term significance --- p.14
Chapter Chapter 2 --- Materials and Methods --- p.15
Chapter 2.1 --- Introduction --- p.16
Chapter 2.2 --- Materials --- p.18
Chapter 2.2.1 --- Chemical --- p.18
Chapter 2.2.2 --- Plant materials --- p.18
Chapter 2.2.3 --- Plasmid vectors and bacterial strains --- p.18
Chapter 2.2.4 --- Human a-iduronidase (hIDUA) cDNA --- p.19
Chapter 2.2.5 --- Primers --- p.20
Chapter 2.3 --- Methods --- p.22
Chapter 2.3.1 --- Generation of IDUA antibodies --- p.22
Chapter 2.3.1.1 --- Synthetic peptide raised IDUA antibodies --- p.23
Chapter 2.3.1.1.1 --- Design of synthetic peptides --- p.23
Chapter 2.3.1.1.2 --- Immunization of rabbits --- p.25
Chapter 2.3.1.2 --- E. coli-derived rhIDUA protein --- p.25
Chapter 2.3.1.2.1 --- Cloning and expression of rhIDUA --- p.25
Chapter 2.3.1.2.2 --- Western analysis of E. coli-derived rhIDUA --- p.29
Chapter 2.3.1.2.3 --- MS/MS analysis of rhIDUA protein --- p.29
Chapter 2.3.1.2.4 --- Immunization of rabbits --- p.31
Chapter 2.3.2 --- Affinity-purified antibodies --- p.33
Chapter 2.3.3 --- Characterization of affinity-purified IDUA antibodies --- p.33
Chapter 2.3.4 --- Construction of chimeric gene constructs --- p.34
Chapter 2.3.5 --- Expression of IDUA in tobacco BY-2 cells --- p.39
Chapter 2.3.5.1 --- Electropoartion of Agrobacteria --- p.39
Chapter 2.3.5.2 --- Agrobacterium-mediated transformation --- p.39
Chapter 2.3.5.3 --- Screening of positive trans formants --- p.40
Chapter 2.3.6 --- Characterization of transgenic BY-2 cell expressing IDUA fusion --- p.40
Chapter 2.3.6.1 --- Genomic DNA polymerase chain reaction (Genomic DNA PCR) --- p.40
Chapter 2.3.6.1.1 --- Genomic DNA extraction from BY-2 callus --- p.40
Chapter 2.3.6.1.2 --- Genomic DNA PCR of tobacco BY-2 callus --- p.41
Chapter 2.3.6.2 --- Reverse transcription-PCR (RT-PCR) --- p.42
Chapter 2.3.6.2.1 --- Total RNA extraction from BY-2 cell --- p.42
Chapter 2.3.6.2.2 --- RT-PCR of BY-2 cell --- p.42
Chapter 2.3.6.3 --- Western blot analysis of BY-2 cell and medium --- p.43
Chapter 2.3.6.3.1 --- Protein extraction from tobacco BY-2 cells and culture medium --- p.43
Chapter 2.3.6.3.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44
Chapter 2.3.6.3.3 --- Immunodetection and Coomassie blue stain --- p.44
Chapter 2.3.7 --- Purification of IDUA from culture media --- p.46
Chapter Chapter 3 --- Results --- p.47
Chapter 3.1 --- Generation of IDUA antibodies --- p.48
Chapter 3.1.1 --- Cloning and expression of rhIDUA in E. coli --- p.48
Chapter 3.1.2 --- Characterization of IDUA antibodies --- p.51
Chapter 3.1.2.1 --- Specificity of IDUA antibodies towards hIDUA protein. --- p.51
Chapter 3.1.2.2 --- Cross-reactivity of IDUA antibodies with wild type tobacco BY-2 cell --- p.55
Chapter 3.2 --- Chimeric gene constructs construction and confirmation --- p.58
Chapter 3.3 --- Screening of transformed tobacco BY-2 callus with kanamycin-resistance --- p.66
Chapter 3.4 --- Genomic DNA PCR screening of transformed tobacco BY-2 callus . --- p.67
Chapter 3.5 --- RT-PCR screening of transformed BY-2 cells --- p.70
Chapter 3.6 --- Western blot analysis of transformed tobacco BY-2 cells and culture media --- p.72
Chapter 3.6.1 --- Tobacco BY-2 cells --- p.72
Chapter 3.6.2 --- Tobacco BY-2 cell culture media --- p.76
Chapter 3.7 --- Purification of IDUA protein in culture media --- p.81
Chapter Chapter 4 --- Discussion --- p.82
Chapter Chapter 5 --- Summary and Future Perspectives --- p.89
Chapter 5.1 --- Summary --- p.90
Chapter 5.2 --- Future perspectives --- p.92
Appendix Identification and Characterization of an Unknown Protein by 1B Antibody --- p.93
Chapter 6.1 --- Introduction --- p.94
Chapter 6.2 --- Objectives --- p.94
Chapter 6.3 --- Materials and Methods --- p.95
Chapter 6.3.1 --- Western blot analysis of different plant species --- p.95
Chapter 6.3.2 --- Subcellular localization of the unknown protein --- p.95
Chapter 6.3.3 --- Affinity-purification of the unknown protein --- p.95
Chapter 6.4 --- Results --- p.97
Chapter 6.4.1 --- Western blot analysis of different plant species --- p.97
Chapter 6.4.2 --- Subcellular localization of an unknown protein --- p.98
Chapter 6.4.3 --- Affinity-purification of 1B protein --- p.104
Chapter 6.5 --- Summary and Future Perspectives --- p.105
Chapter 6.5.1 --- Summary --- p.105
Chapter 6.5.2 --- Future Perspectives --- p.105
References --- p.106

Plant invertases are a class of proteins that have enzymatic function in cleaving sucrose to fructose and glucose. Cell wall invertase, located on the exterior of the cell wall of plant cells, plays a key role in the unloading of sucrose from the apoplast to the sink tissues. Cell wall invertase interacts with an inhibitor, cell wall invertase inhibitor, post-transcriptionally to regulate its activity. The inhibitor is constitutively expressed in pollen development, early developing seeds, and senescing leaves: indicative of sucrose allocation being a limiting factor at these stages of development. We introduced algal bicarbonate transporters LCIA/CCP1 to Camelina sativa for the purpose of increasing photosynthetic capacity. The bicarbonate transporters concentrate CO2 at RuBisCO by pumping CO2 in the form of bicarbonate through the membrane, then converting it back to CO2 at RuBisCO, increasing CO2 concentration. Results from these plants have shown an increase in seed number, but not seed mass, along with a faster rate of maturity and senescence. This is indicative of acclimation to high CO2 conditions, resulting from insertion of the bicarbonate transporters. RNA sequencing was performed and a putative invertase inhibitor was recognized as being expressed in the transgenic C. sativa but not in the wild type. Our strategy is to knock out two invertase inhibitors using induced RNA silencing, dramatically altering sucrose allocation into developing seeds and resulting in an increase in seed biomass. It is the aim of this research to increase the biomass of C. sativa seeds in order to increase its effectiveness as an agent to create sustainable biofuels.

Eucalyptus trees are a significant source of fuelwood, timber and raw material for the paper and pulp industry. In South Africa, Eucalyptus grandis and its hybrids are in high demand due to their fast growth and suitability of their timber for a wide range of products. Breeding and selection for superior quality eucalypts could sustain this high demand through selection and subsequent multiplication of superior genotypes and their use in controlled crosses. However, for this to be successful, a wide genepool must be available and maintained. Germplasm conservation of both vegetative and sexual material is therefore an integral part of such activities. However, in the case of trees, in vivo conservation practices are expensive and hazardous. The aim of this project was therefore to establish alternative conservation strategies for short-to-medium and long-term use of Eucalyptus spp. to facilitate on-going breeding and clonal programmes. For short-to-medium term storage, shoot cultures were subjected to various minimal growth conditions. Of the investigated treatments, reducing nutrients was the best storage method and shoots were maintained for 10 months with multiplication rates of 13.75 ± 7.05 shoots/explant. Encapsulated axillary buds were stored in jars at 10 °C or 28 °C. Of these two treatments, viability was sustained for longer (6 months) at 4 °C. Before establishing pollen storage regimes, viability assessment methods were evaluated and these consisted of in vitro germination on a BK (Brewbaker and Kwack, 1963) medium for 24 hours (26 ± 3.0%) and staining with two tetrazolium salts. Medium-term storage of pollen was best achieved by maintenance in the fridge (4 °C) without any desiccating substance (8 months at 6.73 ± 1.21%). Cryopreservation protocols were investigated for axillary buds and pollen. Buds that were 2 mm long were pretreated with chemical cryoprotectants, and a mixture of DMSO (dimethylsulphoxide) and glycerol was found to induce high survival rates (63%) after washing with MS (Murashige and Skoog, 1962) and 4 g.l-1 sucrose solution. Explants precultured in 1M sucrose showed increased tolerance (explant retained high survival rates of 80%) when desiccated to 20% moisture content fresh weight basis (fwb). Although pretreatments were successfully established, explants did not survive storage in liquid nitrogen indicating the need for further optimization of protocols. Pollen was successfully cryopreserved for 12 months with 23% survival rates. Applications and future research strategies of the developed protocols to Eucalyptus breeding programmes are discussed.
Thesis (M.Sc.)-University of Natal, Durban, 1998.

Orientação: Ana Teresa de Carvalho Negrão Serra ; co-orientação: Maria Margarida de Carvalho Negrão Serra
Actualmente, os compostos bioactivos de alimentos têm vindo a ser comercializados na forma de produtos farmacêuticos, cosméticos e aditivos alimentares, reclamando efeito benéfico na saúde. Para o desenvolvimento e comercialização destes produtos é necessário a avaliação das propriedades biológicas. Este trabalho teve como objectivo a avaliação da capacidade antioxidante e neuroprotectora de extractos naturais de maçã e azeitona utilizando ensaios pré-clinicos, nomeadamente métodos químicos e modelos celulares em 2D e 3D. Foram analisados 11 extractos de maçã (variedade Bravo de Esmolfe) obtidos por diferentes tecnologias. A extracção com fluídos pressurizados (EFP) demonstrou ser mais eficiente do que a extracção convencional em termos de rendimento e de compostos extraídos. As condições de EFP 60:40:0 e 90:5:5 (v/v CO2:EtOH:H2O) permitiram obter fracções com maior concentração em compostos antioxidantes e maior capacidade de resgate de radicais livres (EROs) no modelo celular Caco-2, respectivamente. Os principais compostos responsáveis pela actividade antioxidante foram: epicatequina, glucósidos de quercetina e procianidina B2. Para o extracto de bagaço de azeitona, rico em hidroxitirosol, foi demonstrada a capacidade de resgate de EROs e de prevenção da morte celular em agregados de células NT2 diferenciadas em neurónios, sendo que este efeito neuroprotector foi superior ao obtido para o composto puro.
Nowadays, bioactive compounds from foods have been commercialized as pharmaceuticals, cosmetics and food additives products, claiming health promoting effects. For the development of these products the evaluation of their biological properties is required. The main aim of this work was the evaluation of antioxidant and neuroprotective effect of apple and olive natural extracts using preclinical assays, namely chemical methods and cellular models in 2D and 3D. Eleven extracts obtained by different technologies from ‘Bravo de Esmolfe’ apple variety were analyzed. High pressure extraction (HPE) proved to be more efficient in yield and extracted compounds than conventional method. The solvent ratio of 60:40:0 and 90:5:5 (v/v CO2:EtOH:H2O) allowed to obtain fractions with the highest concentration of antioxidants and increased ability in scavenging free radicals (ROS) in Caco-2 cell model, respectively. Epicatechin, quercetin glycosides and procyanidin B2 were identified as the main compounds responsible for the antioxidant of samples. The olive mill residues extract rich in hydroxytyrosol, demonstrated ability to scavenge ROS and prevent death of neurons derived from NT2 cells cultured as aggregates. This neuroprotective effect was superior to that obtained for the pure compound.