Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11. / Cell to cell communication is paramount during plant developmental processes, from cellular identity in early organogenesis to pollen tube guidance. In response to this requirement, molecular cell signalling is used to perceive an external signal and transduce the response by an intracellular signalling cascade leading to specific gene activation. The sensing protein is typically a receptor kinase, which will transduce the stimulus by phosphorylation of a cytoplasmic interaction partner. Although plant receptor kinases represent the largest protein kinase family, only handfuls are well characterized. By sequence identity (orthology), a family of leucine-rich repeat receptor kinases from Arabidopsis thaliana was identified as AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11). Based upon previous results from its ortholog in Solanum chacoense, the ovary- specific ScORK11 receptor kinase, we hypothesized that members of the AtORK11 receptors would be involved in gametophyte development and reproduction. In order to characterize the role of the four family members (AtORK11a, AtORK11b, AtORK11c and AtORK11d), a T-DNA insertional mutant strategy was undertaken, as well as bimolecular fluorescence complementation assays (BiFC). No precise function could be assigned to the double mutants although a dominant negative strategy revealed an involvement in gametophytic development. It was also shown that all of the receptors could form homodimers as well as heterodimers in a heterologous system, suggesting high functional redundancy for the AtORK11 family.

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11. / Cell to cell communication is paramount during plant developmental processes, from cellular identity in early organogenesis to pollen tube guidance. In response to this requirement, molecular cell signalling is used to perceive an external signal and transduce the response by an intracellular signalling cascade leading to specific gene activation. The sensing protein is typically a receptor kinase, which will transduce the stimulus by phosphorylation of a cytoplasmic interaction partner. Although plant receptor kinases represent the largest protein kinase family, only handfuls are well characterized. By sequence identity (orthology), a family of leucine-rich repeat receptor kinases from Arabidopsis thaliana was identified as AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11). Based upon previous results from its ortholog in Solanum chacoense, the ovary- specific ScORK11 receptor kinase, we hypothesized that members of the AtORK11 receptors would be involved in gametophyte development and reproduction. In order to characterize the role of the four family members (AtORK11a, AtORK11b, AtORK11c and AtORK11d), a T-DNA insertional mutant strategy was undertaken, as well as bimolecular fluorescence complementation assays (BiFC). No precise function could be assigned to the double mutants although a dominant negative strategy revealed an involvement in gametophytic development. It was also shown that all of the receptors could form homodimers as well as heterodimers in a heterologous system, suggesting high functional redundancy for the AtORK11 family.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

Nous avons découvert que TRF2 est aussi sur-exprimée au niveau des cellules endothéliales de nombreux types de cancers humains alors qu’elle n’est pas détectable dans les vaisseaux des tissus sains adjacents. Des cellules endothéliales extraites de tumeurs ex-vivo manifestent une expression supérieure de TRF2, une migration et une prolifération accrues et une aptitude à former des tubules élevée, comparées aux endotheliums isolées de tissus sains. La sur-expression de cette protéine in vitro dans des cellules endothéliales primaires et ex-vivo entraine l’augmentation de la prolifération, de la migration et de la capacité de ces dernières à former des tubules. La diminution de l’expression de TRF2 conduit à l’effet inverse. Par ailleurs, la modulation de l’expression de TRF2 n’affecte pas la proportion de cellules apoptotiques. De même, les variations des niveaux d’expression de TRF2 n’induisent aucune réponse aux dommages à l’ADN et les modifications des facultés angiogéniques sont indépendantes d’ATM. Les effets angiogéniques de TRF2 semblent donc distincts des fonctions télomériques. Etant donné que le facteur de transcription WT1 (Wilms’ tumour suppressor 1) est fortement exprimé dans les vaisseaux de tumeurs humaines et régule les propriétés angiogéniques des cellules endothéliales, nous nous sommes penché sur la régulation potentielle de TRF2 par WT1. WT1 se lie en effet sur le promoteur de TRF2 pour activer sa transcription. Enfin, nous avons démontré que l’activité angiogénique de TRF2 réside en partie dans sa capacité à se fixer sur le promoteur du gène codant pour le récepteur angiogénique à activité tyrosine kinase PDGFRβ et à activer sa transcription. / We discovered that TRF2 is expressed in endothelial cells of many human cancer types but not in the vessels of healthy adjacent tissues. Endothelial cells derived from tumours ex vivo exhibited a significantly increased TRF2 expression, and a higher migration, proliferation and tube formation potential as endothelium obtained from healthy tissues. In vitro TRF2 over-expression in primary or ex vivo endothelial cells resulted in an increased proliferation, migration and tube formation, while silencing of TRF2 led to the opposite results. No changes in apoptosis could be observed. Interestingly, modulation of TRF2 in endothelium does not induce DNA damage responses and the observed changes in the angiogenic behaviour are ATM –independent. The angiogenic effects of TRF2 seem therefore to be uncoupled from its telomeric function. Since the transcription factor WT1 (Wilms’ tumour suppressor 1) is highly expressed in human tumour vessels and mediates angiogenic properties of endothelial cells, we investigated whether TRF2 expression could be regulated by WT1. Indeed, WT1 binds the TRF2 promoter and activates its transcription. Finally, we demonstrated that TRF2 promotes angiogenesis by binding to the promoter of the gene encoding for the angiogenic tyrosine kinase receptor PDGFRβ and activating its transcription.

This study is set out to assess the effectiveness of customer relationship management (CRM) in the hotel industry. Relying on the integration of several CRM scholars, the study seeks to answer questions about the relationship between CRM and performance. A mixed research method approach was adopted. The research targeted 106 hotels in Pretoria. 47 responses were received from the hotel personnel who answered the questionnaire. The findings of the survey clearly indicate that hotel performance is dependent on CRM, as shown in the variance of performance levels in line with the level of CRM practised by a specific, named hotel. The study further suggests that hotels’ management should continuously embark on various CRM practices in order to improve their performance. Responses will be captured and analysed using the SPSS computer software. The results will be subjected to quantitative data analysis. It is expected that the findings will highlight important implications for hotels and other hospitality and tourism stakeholders in Pretoria and beyond. Stakeholders wishing to improve their understanding of the effectiveness of CRM in hotels will find this study helpful. / Business Management / M. Tech. (Business Administration)

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.

This study is set out to assess the effectiveness of customer relationship management (CRM) in the hotel industry. Relying on the integration of several CRM scholars, the study seeks to answer questions about the relationship between CRM and performance. A mixed research method approach was adopted. The research targeted 106 hotels in Pretoria. 47 responses were received from the hotel personnel who answered the questionnaire. The findings of the survey clearly indicate that hotel performance is dependent on CRM, as shown in the variance of performance levels in line with the level of CRM practised by a specific, named hotel. The study further suggests that hotels’ management should continuously embark on various CRM practices in order to improve their performance. Responses will be captured and analysed using the SPSS computer software. The results will be subjected to quantitative data analysis. It is expected that the findings will highlight important implications for hotels and other hospitality and tourism stakeholders in Pretoria and beyond. Stakeholders wishing to improve their understanding of the effectiveness of CRM in hotels will find this study helpful. / Business Management / M. Tech. (Business Administration)

Diante da explicação freudiana para o fenômeno da compulsão à repetição através da tese da pulsão de morte concebida enquanto retorno ao estado inorgânico, formula-se a seguinte questão: seria possível, por um lado, recusar a explicação freudiana pautada em seu biologismo mítico e, por outro lado, aceitar a tese de que a pulsão de morte seja uma força de desligamento? Para responder a essa pergunta encontramos um vasto material na obra publicada por André Green ao longo de sua carreira de mais de quarenta anos sob a rubrica trabalho da pulsão de morte, mais tarde, substituída por trabalho do negativo. Este texto tem como objetivo sistematizar a leitura e os aportes de Green à teoria das pulsões freudiana, sobretudo em relação ao segundo dualismo pulsional. Ele se divide em duas partes. Na primeira, destaca-se a análise de Green sobre as relações entre a pulsão de morte e a teoria do narcisismo na obra freudiana; na segunda, sua crítica em relação ao solipsismo freudiano e a necessidade de sua superação através das teorizações contemporâneas em torno às noções de objeto e de espaço potencial. Estas são complementadas por uma teoria da temporalidade do psiquismo. O trabalho defende a tese de que Green aceita o conceito freudiano de pulsão de morte enquanto força de desligamento, mas recusa o biologismo mítico subjacente à ideia do retorno ao estado inorgânico. Além disso, se Green concorda com a explicação freudiana para a pulsão de morte enquanto força de desligamento expressada no narcisismo negativo , ele se recusa a conceber que o processo de desligamento possa se instaurar de maneira espontânea ou automática. Para ele, este processo deve ser pensado mediante a articulação do funcionamento pulsional e da reposta do objeto que, neste caso, falha no estabelecimento do princípio do prazer; em outras palavras, o fracasso na instalação da espera institui a compulsão à des-fazer e des-ligar. De tal modo, a compulsão à repetição mortífera, ao contrário de repetir o desejo inconsciente e, portanto, estar referida à intemporalidade do inconsciente e à lógica da esperança é, na verdade, um anti-tempo. Nesse sentido, presente, passado e futuro ficam reduzidos ao instante da descarga completa de toda tensão, impossibilitando qualquer projeto. Dado o anterior, resulta que as teorizações de Green em relação ao trabalho do negativo, ainda que avessas à tese freudiana do retorno ao estado inorgânico, aceitam, não obstante, a tese da pulsão de morte enquanto processo de desligamento desde que esta seja pensada através da articulação das dimensões intrapsíquica e intersubjetiva. Isso implica pensar na resposta do objeto e fazê-lo responsável pelo malogro na instalação da heterocronia no psiquismo. 8 Conclui-se que a obra de Green oferece uma alternativa original ao biologismo mítico para a explicação da pulsão de morte / Given the Freudian explanation for the phenomenon of compulsion to repeat based on the death drives thesis, conceived as a return to the inorganic state, one formulates the following question: would it be possible, on the one hand, to refuse the Freudian explanation guided by its mythical biologism, and on the other hand accept the thesis that the death drive is a disengagement force? To answer this question we find a vast amount of material on the work published by André Green throughout his career of more than forty years under the title \"work of the death drive\", later renamed \"work of the negative\". This thesis aims to systematize Greens reading and contributions to the Freudian drive theory, especially regarding the second drive dualism. It is divided into two parts. The first one is Green\'s analysis of the relationship between death drive and theory of narcissism on Freud\'s work; the second one is about his criticism of Freud\'s solipsism and the need to its overcome through contemporary theories around the notions of object and potential space. These will be complemented by a theory of the temporality of the psyche. The present work supports the thesis that Green accepts the Freudian concept of death drive as a disengagement force, but refuses the mythical biologism subjacent to the return to the inorganic states idea. Furthermore, if Green agrees with the Freudian explanation of the death drive as a disengagement force expressed in the negative narcissism he will refuse to conceive that the disengagement process will be established spontaneously or automatically. To him, this process should be thought through the articulation of instintual functioning and the objects response that in this case fails in establishing the principle of pleasure; in other words, the failure of the waiting installation establishes the compulsion to disengage and to disconnect. Insomuch, the deadly compulsion to repeat, instead of repeating the unconscious desire and therefore be referred to the intemporality of the unconscious and to the logic of hope is actually an anti-time. In this sense, present, past and future are reduced to the instant of total discharge of all tension, precluding any project. Given the above it follows that Greens theorization regarding the work of the negative, though averse to the Freudian thesis of the return to the inorganic state, accept however the thesis of death drive as a shutdown process provided that this is thought through the articulation of the intrapsychic and intersubjective dimensions. This implies thinking of the objects response and make it responsible for the failure in the installation of heterochrony in the psyche. It concludes that the work of Green offers an original alternative to the mythical biologism regarding the explanation of the death drive

The primary goal of this study was to evaluate the effectiveness of a program being run by the Bedford Police Department’s Repeat Victimization Unit on domestic violence and mental health and mental retardation. The study sought to determine whether the program was effective in reducing instances of repeat victimization in domestic violence and MHMR victims. Additionally the program investigated whether or not the program was effective at reducing victimization severity, and which demographic could be identified as the most victimized. Participants consisted of 157 domestic violence and MHMR victims in the city of Bedford, Tx between November 11, 2012 to July 30, 2013. Findings indicate that levels of repeat victimization for domestic violence and MHMR are relatively low regardless of whether the victim received services through the repeat victimization program or not. Additionally the severity of these repeat victimizations remains relatively constant regardless of whether services were received through the program or not. Implications and findings are discussed.

<p>In many forensic cases, such as mass disasters reconstruction cases, the recovered DNA is highly degraded. In such incidences, typing of STR loci has become one of the most powerful tools for retrieving information from the degraded DNA. However, as DNA degradation proceeds, three phenomena occur consecutively: loci imbalance, allele dropout and no amplification. To solve the problem of degraded DNA, redesigned primer sets have been developed in which the primers were positioned as close as possible to the STR repeat region. These reduced primer sets were called Miniplexes. Unfortunately, a few of the CODIS STR loci cannot be made into smaller amplicons. For this reason non-CODIS miniSTRs have been developed. The present study was undertaken for the population genetic analysis of microsatellite variation in four South African populations / Afrikaner, Xhosa, Mixed Ancestry and Asian Indian using eight non-CODIS miniSTR loci. These miniSTRs loci were characterized within the populations by estimating the levels of diversity of the markers, estimating the population genetic parameters, and studying the inter-population relationships. All of the miniSTRs were amplified successfully and the genetic variability parameters across all loci in Afrikaner, Mixed Ancestry, Asian Indian and Xhosa were estimated to be in the range of 3 (D4S2364) to 12 (D9S2157) alleles, the total number of alleles over all loci ranged from 100 to 204, the allelic richness ranged from 3.612 to 10.307 and the heterozygosity ranged from 0.4360 to 0.8073. Genetic distance was least between Afrikaner and Asian Indian and highest between Xhosa and Mixed Ancestry. Deviations from Hardy-Weinberg equilibrium were not observed for most of the loci. The low mean FIS (-0.027) and FIT (-0.010) and FST (0.017) values across the populations indicated low level of inbreeding within (FIS) and among (FST) the populations. The Asian Indian population showed higher levels of the inbreeding coefficient, indicating less gene exchange between it and other populations. These 8 markers can be used for genetic investigations and assessing population structure. The study contributed to the knowledge and genetic characterization of four South African populations. In addition, these MiniSTRs prove to be useful in cases where more genetic information is needed.</p>