Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Machine learning techniques have been widely used for classification problems in computational biology. They require that the input must be a collection of fixedlength feature vectors. Since proteins are of varying lengths, there is a need for a means of representing protein sequences by a fixed-number of features. This thesis introduces three novel methods for this purpose: n-peptide compositions with reduced alphabets, pairwise similarity scores by maximal unique matches, and pairwise similarity scores by probabilistic suffix trees. New sequence representations described in the thesis are applied on three challenging problems of computational biology: remote homology detection, subcellular localization prediction, and solvent accessibility prediction, with some problem-specific modifications. Rigorous experiments are conducted on common benchmarking datasets, and a comparative analysis is performed between the new methods and the existing ones for each problem. On remote homology detection tests, all three methods achieve competitive accuracies with the state-of-the-art methods, while being much more efficient. A combination of new representations are used to devise a hybrid system, called PredLOC, for predicting subcellular localization of proteins and it is tested on two distinct eukaryotic datasets. To the best of author&rsquo / s knowledge, the accuracy achieved by PredLOC is the highest one ever reported on those datasets. The maximal unique match method is resulted with only a slight improvement in solvent accessibility predictions.

The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375755 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Catharanthus roseus est une plante médicinale produisant divers types d’alcaloïdes indoliques monoterpéniques (AIM) d’intérêt en santé humaine. Ainsi, les AIM dimères comme la vinblastine et la vincristine sont utilisés en chimiothérapie anticancéreuse et les alcaloïdes monomères de type hétéroyohimbine présentent diverses activités pharmacologiques. La fabrication de ces molécules dans la plante est fort complexe. Elle requiert un haut niveau de compartimentation tissulaire et subcellulaire et met en jeu plus d’une trentaine d’étapes enzymatiques, dont certaines sont encore très mal connues. Dans ce contexte, l’objectif de la thèse a consisté à élucider plusieurs étapes enzymatiques de la voie de biosynthèse des AIM. Nos travaux ont permis de caractériser de nouvelles isoformes enzymatiques de la famille des cytochromes P450 ainsi que les réductases qui leur sont associées. Ils ont abouti à l’identification de nouvelles déshydrogénases et mis en évidence, in planta, leurs interactions avec la strictosidine synthase suggérant une biosynthèse orientée vers les divers alcaloïdes de type hétéroyohimbine. Enfin, en ayant recours à l’ingénierie métabolique, un segment de la voie de biosynthèse a été transféré dans la levure Saccharomyces cerevisiae, lui conférant la capacité de bio-transformer la tabersonine en vindoline, l’un des deux précurseurs finaux des alcaloïdes dimères. / Catharanthus roseus is a medicinal plant producing various types of monoterpene indole alkaloids (MIA) with a great interest in human health. Dimeric alkaloids such as vinblastine and vincristine are used in cancer chemotherapy and monomeric heteroyohimbine alkaloids exhibit various pharmacological activities. The production of these molecules in the plant is very complex. It requires a high level of tissular and subcellular compartmentalization and involves more than thirty enzymatic steps, some of which are largely unknown. In this context, the aim of this thesis was to elucidate several enzymatic steps of the MIA biosynthetic pathway. Our work allowed us to characterize new enzyme isoforms of cytochrome P450 and their associated reductases. They also resulted in the identification of new dehydrogenases and highlighted their interactions with the strictosidine synthase suggesting a directed biosynthesis towards various heteroyohimbine type of alkaloids. Finally, engineered yeast containing a segment of the MIA biosynthetic pathway was able to convert tabersonine into vindoline, one of the two final precursors of the dimeric alkaloids.

This thesis explores localisations and roles of three auxiliary protein factors involved in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 and contributes to subcellular localisation of the initial steps of PSII biogenesis and repair-related D1 synthesis. The main results consist in i) identification of a functional interaction of the protein factor Psb27 with a lumenal domain of the Photosystem II subunit CP43, ii) discovery of a novel pigment binding complex formed by the Ycf39 protein and high-light-inducible proteins implicated in photoprotection and delivery of recycled chlorophyll to newly synthesized D1 protein during the PSII reaction centre formation, iii) providing evidence that the early steps of PSII assembly and the repair-related D1 synthesis occur in the thylakoid membrane of Synechocystis, and iv) revealing that the cyanobacterial PsbP orthologue, CyanoP, assists in the early phase of PSII biogenesis as an assembly factor facilitating the association of D2 and D1 assembly modules.

En tant qu’acteurs moléculaires impliqués dans le transport d’eau au travers des membranes biologiques, les aquaporines (AQP) jouent un rôle et sont régulées en réponse à des stress. Ils sont soumis à d’intenses recherches, en particulier chez le riz. En utilisant une approche de génomique fonctionnelle, nous avons généré 5 lignées transgéniques de riz dans le fond génétique Nipponbare, en sur-exprimant des AQP uniques fusionnées à un marqueur fluorescent ; parmi celles-là, figurent trois AQP de la membrane plasmique (OsPIP1;1, OsPIP2;4, OsPIP2;5) fusionnées à la GFP et deux AQP de la membrane tonoplastique (OsTIP1;1 and OsTIP2;2) fusionnées à la mCherry. Leurs localisations subcellulaires en condition contrôle ont été observées. Chez le riz, les isoformes OsPIP présentaient un marquage homogène typique de la membrane plasmique, tandis que les isoformes OsTIP ont été trouvées avec un marquage des invaginations intracellulaires qui entourent le noyau, typique du tonoplaste. Le comportement des AQP de la membrane plasmique a été testé en conditions de stress salin et osmotique. Les stress abiotiques ont provoqué une relocalisation des AQP et le stress salin a augmenté l’endocytose de l’isoforme OsPIP2;5 dans les cellules de la racine. Par ailleurs, la sur-expression de tels transgènes ne semblait pas affecter la morphologie des plantes et ne conférait pas un effet bénéfique sur la production de graines, aussi bien en condition contrôle que stressée. Enfin, nous nous sommes focalisés sur la contribution des AQP dans la racine de riz en relation avec la morphologie racinaire. Nous avons trouvé que les AQP contribuaient à un pourcentage relativement important dans le transport de l’eau dans la racine entière (44-58%) et que cette contribution semblait plus importante dans les racines primaires que latérales. / As molecular players involved into the water transport through biological membranes, aquaporins (AQPs) have a role and are regulated in stress response. They were deeply investigated in plants and particularly in rice. Using functional genetic approach, we generated 5 transgenic rice lines based on Nipponbare cultivar, by overexpressing a single AQP in fusion with a fluorescent marker; among them, 3 plasma membrane AQPs (OsPIP1;1, OsPIP2;4, OsPIP2;5) fused with GFP and 2 tonoplast AQPs (OsTIP1;1 and OsTIP2;2) fused with mCherry. Their subcellular localizations in resting condition were investigated. In rice, OsPIP isoforms showed typical homogeneous labelling of the plasma membrane, whereas OsTIP isoforms were observed localized in the tonoplast with a typical labelling of intracellular invaginations that skirted the nucleus. The behaviors of plasma membrane AQPs were tested in salt and drought stress-mimicked-conditions. Abiotic stresses triggered a re-localization of plasma membrane AQPs and salt stress enhanced endocytosis process of OsPIP2;5 in rice root cells. Overexpressing such transgenes did not seem to affect the plant morphology and showed no beneficial effects for grain yield in both non-stress and stress conditions. We took more focus on the contribution of AQPs in rice root water transport in link with root morphology. AQPs contributed a relative high percentage of water transport in whole root system (44-58%) and seemed to contribute more in primary roots rather than in lateral roots.

L’acide phosphatidique (PA) est un lipide simple qui peut exister sous différentes formes. A partir des sondes que j’ai préparé en se basant sur des domaines de liaison au PA : PDE4A1, Spo20p, et OpiQ2, j’ai pu étudier la localisation subcellulaire du PA dans les cellules PC12 et les macrophages RAW264.7. Ces sondes lient différents formes de PA dans les membranes de différents compartiments subcellulaires. De plus, j’ai pu montrer qu’il y a une néosynthèse de PA et de certaines espèces de PA mono- ou bi-insaturé à la membrane plasmique lors de la stimulation de l’exocytose. Nous avons ainsi observé que la PLD1 semble être la source principale de PA dans les glandes surrénales. D’autre part, mes travaux indiquent une augmentation du niveau global de PA à la membrane plasmique et une diminution importante du PA au niveau du RE dans les macrophages après stimulation de la phagocytose frustrée. Ce qui pourrait ainsi valider le concept d’une fusion d’une partie de la membrane du RE avec la membrane plasmique lors de la phagocytose. / The phosphatidic acid (PA) is a simple lipid which may exist in various forms. I have generated probes based on PA binding domains: PDE4A1, Spo20p and OpiQ2 to study the subcellular localization of PA in PC12 cells and RAW264.7 macrophages. These probes bind different form of PA in different subcellular compartments. In addition, I show that PA and certain species mono- or bi-unsaturated of PA are synthesized at the plasma membrane upon stimulation of exocytosis. We observed that the PLD1 seems to be the main source of PA in the adrenal glands. On the other hand, my research indicates an increase in the level of PA at the plasma membrane and a significant decrease in the ER in macrophages after stimulation of phagocytosis frustrated. Thus these results could validate the concept of a fusion of a portion of the ER membrane with the plasma membrane during phagocytosis.

Solute carriers (SLCs) constitute the largest family of membrane-bound transporter proteins in humans, and they convey transport of nutrients, ions, drugs and waste over cellular membranes via facilitative diffusion, co-transport or exchange. Several SLCs are associated with diseases and their location in membranes and specific substrate transport makes them excellent as drug targets. However, as 30 % of the 430 identified SLCs are still orphans, there are yet numerous opportunities to explain diseases and discover potential drug targets. Among the novel proteins are 29 atypical SLCs of major facilitator superfamily (MFS) type. These share evolutionary history with the remaining SLCs, but are orphans regarding expression, structure and/or function. They are not classified into any of the existing 52 SLC families. The overall aim in this thesis was to study the atypical SLCs with a focus on their phylogenetic clustering, evolutionary conservation, structure, protein expression in mouse brains and if and how their gene expressions were affected upon changed food intake. In Papers I-III, the focus was on specific proteins, MFSD5 and MFSD11 (Paper I), MFSD1 and MFSD3 (Paper II), and MFSD4A and MFSD9 (Paper III). They all shared neuronal expression, and their transcription levels were altered in several brain areas after subjecting mice to food deprivation or a high-fat diet. In Paper IV, the 29 atypical SLCs of MFS type were examined. They were divided into 15 families, based on phylogenetic analyses and sequence identities, to facilitate functional studies. Their sequence relationships with other SLCs were also established. Some of the proteins were found to be well conserved with orthologues down to nematodes and insects, whereas others emerged at first in vertebrates. The atypical SLCs of MFS type were predicted to have the common MFS structure, composed of 12 transmembrane segments. With single-cell RNA sequencing and in situ proximity ligation assay, co-expression of atypical SLCs was analysed to get a comprehensive understanding of how membrane-bound transporters interact.   In conclusion, the atypical SLCs of MFS type are suggested to be novel SLC transporters, involved in maintaining nutrient homeostasis through substrate transport.

Les récepteurs ionotropiques du glutamate peuvent être compartimentés de manière très spécifique au niveau des différentes afférences synaptiques d’un neurone. Dans les neurones pyramidaux de CA3, les récepteurs de type kaïnate (rKA) post-synaptiques sont localisés à la synapse formée entre les fibres moussues et les cellules pyramidales de CA3 (synapse FM-CA3) mais ils sont totalement absents des autres afférences glutamatergiques sur ce même neurone. Nous avons cherché à comprendre les mécanismes moléculaires de cette compartimentation subcellulaire. En réalisant une cartographie fonctionnelle des récepteurs du glutamate par décageage focalisé de glutamate dans les cellules pyramidales de CA3, nous avons montré que les rKA présentent une localisation subcellulaire strictement confinée dans les excroissances épineuses, éléments post-synaptiques des synapses FM-CA3, et sont exclus des compartiments somato-dendritiques, contrairement aux récepteurs AMPA. Nous avons identifié une séquence du domaine C-terminal de GluK2a nécessaire pour la stabilisation des rKA. Cette séquence est responsable d’une interaction avec la protéine d’adhérence N-cadhérine. L’altération de la fonction de la N-cadhérine dans les cellules pyramidales de CA3 entraine une déstabilisation des rKA à la synapse FM-CA3. Ces travaux suggèrent que plusieurs mécanismes participent à la compartimentation des rKA à la synapse FMCA3 impliquant le recrutement et la stabilisation des rKA par les N-cadhérines. / Distinct subtypes of ionotropic glutamate receptors can be segregate to specific synaptic inputs in a given neuron. In CA3 pyramidal cells (PCs), kainate receptors (KARs) are present at mossy fiber (mf) synapses and absent from other glutamatergic inputs. The mechanisms for such a constrained subcellular segregation is not known. We have investigated the molecular determinants responsible for the subcellular segregation of KARs at mf-CA3 synapses. Using functional mapping of glutamate receptors by focal glutamate uncaging we show that KARs display a strictly confined expression on thorny excrescences, the postsynaptic elements of mf-CA3 synapses, being excluded from extrasynaptic somatodendritic compartments, at variance with AMPA receptors. We have identified a sequence in the GluK2a C-terminal domain necessary for restricted expression of KARs which is responsible for GluK2a interaction with N-Cadherin. Targeted deletion of N-Cadherin or overexpression of a dominant negative N-Cadherin in CA3 PCs greatly induce a destabilization of KARs at the mf-CA3 synapses. Our findings suggest that multiple mechanisms combine to control the compartmentalization of KARs at mf-CA3 synapses, including a stringent control of the amount of GluK2 subunit in CA3 PCs, a limited number of slots for KARs, and the recruitment/stabilization of KARs by N-Cadherins.