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S179D prolactina (hPRL) é uma mímica molecular da prolactina humana fosforilada. Demonstrou-se que a S179D-hPRL era anti angiogênica nos ensaios de angiogênese baseados na membrana corialantóica de galinha e na córnea de camundongos. Investigações posteriores realizadas empregando modelos in vitro demonstraram que o tratamento com S179D-hPRL diminuiu o número de células viáveis, reduziu a formação de túbulos em Matrigel e interferiu com a migração e invasão da matriz extracelular. A análise dos fatores de crescimento de células endoteliais humanas tratadas com S179D-hPRL revelou: uma diminuição na expressão ou liberação da PRL endógena, da heme-oxigenase-1, do fator de crescimento de fibroblasto básico (bFGF) e um aumento na expressão de dois inibidores teciduais de metaloproteases. A S179D-hPRL também bloqueou a sinalização provocada por bFGF nessas células. Nós concluímos que essa mímica molecular do hormônio pituitário fosforilado é uma potente proteína anti-angiogênica, em parte devido á sua habilidade de reduzir o estímulo autócrino de fatores de crescimento de células endoteliais de cordão umbilical humano (HUVEC), por sua capacidade de bloquear a sinalização promovida pelo bFGF e por sua habilidade de interferir na migração endotelial. Também foi estudada a influência da S179D-hPRL na apoptose em células endoteliais humanas, empregando caspase-8 como um marcador da via extrínseca, e a liberação de citocromo C como um marcador da via intrínseca. As duas cascatas convergem na ativação da caspase-3, que cliva a fator de fragmentação de DNA (DFF45). Uma incubação de três dias com 50 ng/mL de S179D-hPRL quadruplicou o número de células apoptóticas; esse efeito duplicou-se com uma concentração de 100 ng/mL e atingiu um ápice com 500 ng/mL. A clivagem de DFF45 e da pro-caspase-8 foi detectado com 100 ng/mL. Citocromo C, porém, só foi observado com concentrações de 500 ng/mL. O regulador de ciclo celular p21 (um marcador pró-apoptótico) elevou-se com 100 ng/mL, enquanto que um incremento do supressor tumoral p53 necessitou três vezes o tempo de incubação e 500 ng/mL. A atividade do promotor de p21 foi máxima com 50 ng/mL do análogo de hPRL, enquanto que 500 ng/mL foram necessários para se visualizar uma alteração significativa na atividade do promotor de Bax (um indicador da atividade de p53). Como previamente demonstrado na literatura, S179D-hPRL bloqueou a fosforilação da quinase regulada extracelularmente (ERK) em resposta ao bFGF, mas também causou uma ativação tardia e prolongada da ERK. PD 98059 [inibidor específico da proteína quinase ativada por mitógeno (MAPkinase)] inibiu essa ativação tardia e sustentada assim como outros efeitos da S179D-hPRL, exceto aquele sobre a indução de p53 e ativação do promotor de Bax. Podemos concluir que baixas doses de S179D-hPRL bloqueiam a sinalização de ERK induzida por bFGF e concomitantemente ativam a ERK em um tempo diferente, resultando na elevação de p21 e ativando a via extrínseca de apoptose. Maiores tempos de incubação e concentração, entretanto, ativam a via intrínseca empregando uma cascata intracelular diferente. Esses achados sugerem que níveis circulantes de PRL fosforilada podem inibir a progressão do câncer e, portanto, S179D-hPRL poderia ser um agente anti-angiogênico útil na terapêutica. / S179D-prolactin (hPRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D-hPRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D-hPRL. Analysis of growth factors in human endothelial cells in response to S179D-hPRL showed a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor and an increased expression of inhibitors of matrix metalloproteases. S179D-hPRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. We also examined the influence of S179D-hPRL on apoptosis in human endothelial cells, using procaspase-8 as a marker of the extrinsic pathway, and cytochrome C release as a marker of the intrinsic pathway. Both pathways converge at caspase-3, which cleaves DNA fragmentation factor (DFF45). A 3-day incubation with 50 ng/ml S179D-hPRL quadrupled the early apoptotic cells; this effect was doubled at 100 ng/ml and maximal at 500 ng/ml. DFF45 and pro-caspase 8 cleavage were detectable at 100 ng/ml. Cytochrome C, however, was unaffected until 500 ng/ml. p21 increased at 100 ng/ml, whereas a change in p53 activity required both triple the time and 500 ng/ml. p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). As previously shown, S179D-hPRL blocked extracelular regulated kinase (ERK) phosphorylation in response to bFGF, but, in addition, continued co-incubation showed a delayed and prolonged activation of ERK. PD98059 [a specific mitogen-activated protein kinase (MAPkinase) inhibitor] inhibited this delayed activation of ERK and the effects of S179D-hPRL on all parameters except p53, or activity of the Bax promoter. We conclude that low doses of S179D-hPRL block bFGF-induced ERK signaling and yet activates ERK in a different time frame to elevate p21, and activate the extrinsic pathway. Longer incubations and higher concentrations, however, additionally activate the intrinsic pathway using an alternate intracellular signal. These findings suggest that circulating levels of phosphorylated hPRL may reduce the progression of cancer and, furthermore, that S179D-hPRL may be a useful anti-angiogenic therapeutic.

S179D prolactina (hPRL) é uma mímica molecular da prolactina humana fosforilada. Demonstrou-se que a S179D-hPRL era anti angiogênica nos ensaios de angiogênese baseados na membrana corialantóica de galinha e na córnea de camundongos. Investigações posteriores realizadas empregando modelos in vitro demonstraram que o tratamento com S179D-hPRL diminuiu o número de células viáveis, reduziu a formação de túbulos em Matrigel e interferiu com a migração e invasão da matriz extracelular. A análise dos fatores de crescimento de células endoteliais humanas tratadas com S179D-hPRL revelou: uma diminuição na expressão ou liberação da PRL endógena, da heme-oxigenase-1, do fator de crescimento de fibroblasto básico (bFGF) e um aumento na expressão de dois inibidores teciduais de metaloproteases. A S179D-hPRL também bloqueou a sinalização provocada por bFGF nessas células. Nós concluímos que essa mímica molecular do hormônio pituitário fosforilado é uma potente proteína anti-angiogênica, em parte devido á sua habilidade de reduzir o estímulo autócrino de fatores de crescimento de células endoteliais de cordão umbilical humano (HUVEC), por sua capacidade de bloquear a sinalização promovida pelo bFGF e por sua habilidade de interferir na migração endotelial. Também foi estudada a influência da S179D-hPRL na apoptose em células endoteliais humanas, empregando caspase-8 como um marcador da via extrínseca, e a liberação de citocromo C como um marcador da via intrínseca. As duas cascatas convergem na ativação da caspase-3, que cliva a fator de fragmentação de DNA (DFF45). Uma incubação de três dias com 50 ng/mL de S179D-hPRL quadruplicou o número de células apoptóticas; esse efeito duplicou-se com uma concentração de 100 ng/mL e atingiu um ápice com 500 ng/mL. A clivagem de DFF45 e da pro-caspase-8 foi detectado com 100 ng/mL. Citocromo C, porém, só foi observado com concentrações de 500 ng/mL. O regulador de ciclo celular p21 (um marcador pró-apoptótico) elevou-se com 100 ng/mL, enquanto que um incremento do supressor tumoral p53 necessitou três vezes o tempo de incubação e 500 ng/mL. A atividade do promotor de p21 foi máxima com 50 ng/mL do análogo de hPRL, enquanto que 500 ng/mL foram necessários para se visualizar uma alteração significativa na atividade do promotor de Bax (um indicador da atividade de p53). Como previamente demonstrado na literatura, S179D-hPRL bloqueou a fosforilação da quinase regulada extracelularmente (ERK) em resposta ao bFGF, mas também causou uma ativação tardia e prolongada da ERK. PD 98059 [inibidor específico da proteína quinase ativada por mitógeno (MAPkinase)] inibiu essa ativação tardia e sustentada assim como outros efeitos da S179D-hPRL, exceto aquele sobre a indução de p53 e ativação do promotor de Bax. Podemos concluir que baixas doses de S179D-hPRL bloqueiam a sinalização de ERK induzida por bFGF e concomitantemente ativam a ERK em um tempo diferente, resultando na elevação de p21 e ativando a via extrínseca de apoptose. Maiores tempos de incubação e concentração, entretanto, ativam a via intrínseca empregando uma cascata intracelular diferente. Esses achados sugerem que níveis circulantes de PRL fosforilada podem inibir a progressão do câncer e, portanto, S179D-hPRL poderia ser um agente anti-angiogênico útil na terapêutica. / S179D-prolactin (hPRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D-hPRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D-hPRL. Analysis of growth factors in human endothelial cells in response to S179D-hPRL showed a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor and an increased expression of inhibitors of matrix metalloproteases. S179D-hPRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. We also examined the influence of S179D-hPRL on apoptosis in human endothelial cells, using procaspase-8 as a marker of the extrinsic pathway, and cytochrome C release as a marker of the intrinsic pathway. Both pathways converge at caspase-3, which cleaves DNA fragmentation factor (DFF45). A 3-day incubation with 50 ng/ml S179D-hPRL quadrupled the early apoptotic cells; this effect was doubled at 100 ng/ml and maximal at 500 ng/ml. DFF45 and pro-caspase 8 cleavage were detectable at 100 ng/ml. Cytochrome C, however, was unaffected until 500 ng/ml. p21 increased at 100 ng/ml, whereas a change in p53 activity required both triple the time and 500 ng/ml. p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). As previously shown, S179D-hPRL blocked extracelular regulated kinase (ERK) phosphorylation in response to bFGF, but, in addition, continued co-incubation showed a delayed and prolonged activation of ERK. PD98059 [a specific mitogen-activated protein kinase (MAPkinase) inhibitor] inhibited this delayed activation of ERK and the effects of S179D-hPRL on all parameters except p53, or activity of the Bax promoter. We conclude that low doses of S179D-hPRL block bFGF-induced ERK signaling and yet activates ERK in a different time frame to elevate p21, and activate the extrinsic pathway. Longer incubations and higher concentrations, however, additionally activate the intrinsic pathway using an alternate intracellular signal. These findings suggest that circulating levels of phosphorylated hPRL may reduce the progression of cancer and, furthermore, that S179D-hPRL may be a useful anti-angiogenic therapeutic.

Cancer is a group of diseases that are the second leading cause of human mortality in the United States. Discovering new therapies is vital to conquer cancer. Thymoquinone (TQ) is found in the plant Nigella sativa. TQ was found to be cytotoxic to the human ovarian cancer cell lines PA-1, CAOV-3 and SKOV-3, which have varying p53 status. PA-1 cells were the most sensitive, indicating that TQ was effective against cells having wild-type (WT) p53. Western blots indicated an increase in p53 in cell lines having WT p53. TQ when given concurrently with cisplatin resulted in antagonism for PA-1, A172 and H460 cell lines. Sequential exposure to TQ followed by cisplatin resulted in synergy or additive effects in these cell lines. Sequential exposure to cisplatin followed by TQ resulted in additive or moderate antagonism in these cell lines. Concurrent exposure to TQ and paclitaxel showed synergy in PA-1 and H460 cells. Sequential exposure to TQ followed by paclitaxel resulted in synergism or antagonism in A172, PA-1, and H460 cells. Paclitaxel followed by TQ resulted in antagonism or synergism in these cells. These results demonstrate that TQ has a potential as an antineoplastic agent and may affect p53 levels.

Narcissism is currently being redefined as a multidimensional construct. While some researchers hold that narcissism remains a unidimensional phenomenon, others have suggested that bi- and tridimensional conceptualizations may better represent the construct as well as align more succinctly with the experiences of individuals with varying levels of narcissistic traits. Also, since the latter conceptualizations offer a broader assessment of narcissistic tendencies, they may provide greater accuracy in identifying differing narcissistic phenotypes (e.g., malignant vs vulnerable). Given the variety in conceptualizations of narcissism, it remains an open area as to how their respective domains are associated with various risk factors and behavioral outcomes, particularly within antagonistic forms of narcissism. This thesis explored the multidimensional approaches to defining narcissism and examined the associations between narcissistic traits and attachment style, self-concept, self-esteem, emotion regulation, and aggression. Also, the current study explored how differences in college degree (business vs. psychology) and gender may influence the expression of narcissism and associations with attachment style. Overall, the study provides results relevant for a tridimensional view of narcissism and adds to the literature on narcissism's link with factors involved in personality pathology, gender, and choice of college degree.

Natural selection acts on multiple traits simultaneously. How mechanisms underlying such traits enable or constrain their response to simultaneous selection is poorly understood. We show how antagonism and synergism among three traits at the developmental level enable or constrain evolutionary change in response to simultaneous selection on two focal traits at the phenotypic level. After 10 generations of 25% simultaneous directional selection on all four combinations of body size and development time in Manduca sexta (Sphingidae), the changes in the three developmental traits predict 93% of the response of development time and 100% of the response of body size. When the two focal traits were under synergistic selection, the response to simultaneous selection was enabled by juvenile hormone and ecdysteroids and constrained by growth rate. When the two focal traits were under antagonistic selection, the response to selection was due primarily to change in growth rate and constrained by the two hormonal traits. The approach used here reduces the complexity of the developmental and endocrine mechanisms to three proxy traits. This generates explicit predictions for the evolutionary response to selection that are based on biologically informed mechanisms. This approach has broad applicability to a diverse range of taxa, including algae, plants, amphibians, mammals, and insects.

Un des freins majeurs au développement de la manipulation d'objet avec une main robotisée est sans aucun doute leur fragilité. C'est l'une des raisons pour laquelle un système bras-main anthropomorphe, extrêmement robuste, est développé au centre de robotique et de mécatronique de DLR. Le système est unique à la fois par sa complexité, utilisant 52 moteurs et plus de200 capteurs, ainsi que par ses capacités dynamiques. En effet, ce nouveau système a la particularité d'être mécaniquement flexible ce qui offre la possibilité de stocker de l'énergie à court terme et remplit ainsi deux fonctions essentielles pour un robot humanoïde: les impacts sont filtrés et les performances dynamiques sont augmentées.Dans cette thèse, on se concentre plus particulièrement sur la main. Elle dispose de 19 degrés de liberté dont chacun est actionné par deux tendons flexibles antagonistes. La rigidité des tendons étant non linéaire il est possible, tout comme peut le faire l'être humain, de co-contracter les <<muscles>> et donc d'ajuster la rigidité des doigts afin de s'adapter au mieux aux tâches à effectuer. Cependant, cette flexibilité entraine de nouveau défis de modélisation et de contrôle. L'état de l'art se concentre majoritairement sur le problème de la répartition des forces internes ou du contrôle d'articulation flexible mais peu de travaux considèrent les deux problèmes simultanément.Le travail présenté dans la première partie de la thèse se concentre sur la modélisation de la main et du poignet. Les problématiques spécifiques aux systèmes actionnés par des tendons, tels que les matrices de couplage et l'estimation du déplacement des articulations à partir du déplacement des tendons, sont étudiées.La seconde partie se concentre sur le contrôle d'articulations actionnées par des tendons flexibles antagonistes. Les problèmes de distribution des forces internes et de correction de la rigidité perçue par l'utilisateur sont présentés.Des approches de contrôle linéaire et non linéaire sont utilisées et des expériences sont réalisées pour comparer ces approches. En particulier, il est montré que le <<backstepping>>, une méthode de contrôle non linéaire peut être utilisée et permet d'obtenir le comportement d'impédance souhaité tout en garantissant la stabilité en boucle fermée. / One of the major limitations of object manipulation with a robotic hand is the fragility of the hardware.This is one of the motivations for developing the new anthropomorphic and extremely robust Hand Arm System at the robotics and mecatronics center of DLR.The system is unique in terms of complexity, with 52 motors and more than 200 sensors, and also in terms of dynamics.Indeed, the system is mechanically compliant, thus offers the possibility to store and release energy, thereby providing two essential functions: The impacts are filtered and the dynamics are enhanced.This thesis focuses on the hand. It has 19 degrees of freedom, each being actuated by two flexible antagonistic tendons. Because the stiffnes of the tendons is not linear, it is possible to adjust the mechanical stiffness of the joints, similar to the co-contraction of human muscles, in order to adapt to a task. However, the stiffness adjustability rises new challenges in modeling and control. The state of the art usually focuses on the problems of tendon-driven systems or flexible joint robots but seldomly both simultaneously.In the first part, the modeling of the hand and the wrist is conducted. Several problems specific to tendon-driven systems are presented, such as the coupling matrices and the joint position estimation based on the tendon displacement. The second part focuses on the control of a single joint actuated by two flexible tendons. The distribution of the tendon forces and the correction of the effective stiffness are reported. Linear and nonlinear approaches are used and multiple experiments are realised to compare them. The major result is that the backstepping, a nonlinear control method, can be used and provides the desired impedance behavior while guaranting closed-loop stability.

The antagonistic relationship of proteins describes the opponent interactions that result in one protein suppressing the function of another. Developmental genetic studies of Antirrhinum majus demonstrated that two transcription factors from the MYB gene family, RAD and DIV, interact through antagonism to regulate floral dorsoventral asymmetry. Interestingly, similar antagonistic interactions were found among proteins of FSM1 (RAD-like), MYBI (DIV-like), and DRIF in Solanum lycopersicum, which is involved in fruit development. Here, we report on the homology of these antagonistic MYB proteins based on reconstruction of the phylogeny of I-box-like and R-R-type clades, where RAD- and DIV-like belong, respectively. Three paralogs of RAD-/I-box-like genes, RAD1, RAD2, and RAD3 are reprensent in the phylogeny, which originated in the common ancestor of the core eudicots. In contrast, R-R-type sequences fall into two major clades, RR1 and RR2, which are the result of gene duplication in the common ancestor of monocots and dicots. RR1 was divided into clades, RR1A, RR1B, and RR1C, while RR2 divided into clades, RR2A/DIV1, RR2B/DIV2, and RR2C/DIV3. We demonstrate that among similar antagonistic interactions in A. majus and So. lycopersicum, RAD-like genes originate from the RAD2 clade, while DIV-like genes originate from distantly related paralogs of the R-R-type lineage.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Designing a mechanistic model that can give rise to realistic architecture of ecological networks is central to the understanding of how species assemble and function in ecosystems. As species are constantly adjusting their diets in an antagonistic network, we here incorporate this adaptive behaviour of diet choice into a bipartite network model, with the effect of antagonistic interactions between species depicted by Holling’s type II functional response. Predictions of this model fit extremely well with the observed levels of nestedness, modularity and node-degree distributions for 61 real host-parasitoid and plant-herbivore networks. We further examined two specific scenarios of our model (species with identical [neutral] demographic parameters and interactions with identical [neutral] benefit in the network) and found that the demography-neutral scenario overestimated observed modularity, whilst the benefit-neutral scenario over-estimate observed nestedness. Relationships between nestedness, modularity and connectance were found strong. Moreover, in contrast to the common belief of the high modularity in antagonistic networks, most real networks (> 80%) are significantly nested, whilst nearly 40% of the real networks are surprisingly less compartmentalized than random networks generated from null models. Regardless of the controversy on whether antagonistic networks are nested or compartmentalized, the proposed model captured the essence of the dynamic nature of structural emergence in antagonistic networks. Due to its predictive power, this model was further used to investigate robustness in antagonistic networks. Predictions showed that the robustness of a network is determined by many factors, such as connectance, resource degree distribution, resource-consumer ratio, diversity, nestedness and compartmentalisation. Surprisingly, the manner of network response to species loss was independent of the sequence followed while removing species from a network. Variations were only noticed in the intensity of the effect resulting from the removals. In addition, we also showed that species extinction procedures which ignore the interaction switch underestimate the effect of any loss of species in these networks. We must therefore value our knowledge of possible adaptive processes in the ecosystem as they may be important for resolving the diversity-stability debate. / AFRIKAANSE OPSOMMING: Die ontwerp van ’n meganistiese model wat aanleiding kan gee tot realistiese argitektuur van ekologiese netwerke is sentraal tot die begrip van hoe spesies bymekaar kom en funksioneer in ekosisteme. Soos spesies voortdurend hul dieet aanpas in ’n antagonistiese netwerk, het ons hierdie aanpasbare gedrag van dieet keuse in ’n bipartiet netwerk model ingewerk, met die effek van antagonistiese interaksies tussen spesies wat uitgebeeld word deur Holling se tipe II funksionele reaksie. Voorspellings van hierdie model pas baie goed met die waargenome vlakke van nestedness, modulariteit en node-graad uitkerings vir 61 ware gasheer-parasiet en plant-herbivoor netwerke. Verder het ons twee spesifieke gevalle van ons model (spesies met identiese [neutrale] demografiese parameters en interaksies met identiese [neutrale] voordeel in die netwerk) ondersoek en gevind dat die demografie-neutrale geval waargenome modulariteit oorskat, terwyl die voordeelneutraal geval waargenome nestedness oorskat. Verhoudings tussen nestedness, modulariteit en konnektiwiteit is sterk bevind. Verder, in teenstelling met die algemene verwagting van hoe modulariteit in antagonistiese netwerke, is oorhoofse werklike netwerke (> 80%) aansienlik geneste, terwyl byna 40% van die werklike netwerke is verbasend minder gekompartimenteerd as ewekansige netwerke gegenereer uit null modelle. Ongeag van die omstredenheid oor of antagonistiese netwerke geneste of gekompartimenteerd is, die voorgestelde model vang die essensie van die dinamiese aard van die strukturele opkoms in antagonistiese netwerke. As gevolg van sy voorspellende krag, is hierdie model verder gebruik om robuustheid te ondersoek in antagonistiese netwerke. Voorspellings het getoon dat die robuustheid van ’n netwerk word bepaal deur verskeie faktore, soos konnektiwiteit, hulpbron-graad verspreiding, hulpbron-verbruiker verhouding, diversiteit, nestedness en kompartementasie. Verrassend, die wyse van die netwerk reaksie op die verlies van spesies was onafhanklik van die reeks wat gevolg het toe die spesies verwyder is uit ’n netwerk. Variasies is slegs opgemerk in die intensiteit van die effek van die verskuiwings. Benewens, ons het ook aangetoon dat die prosedures van spesies se uitsterwing wat die interaksie skakelaar geignoreer het, onderskat die effek van ’n verlies van spesies in hierdie netwerke. Ons moet dus die waarde van ons kennis van die moontlike aanpassing prosesse in die ekosisteem in agneem, aangesien dit belangrik kan wees vir die oplossing van die diversiteit-stabiliteit debat.

Thesis (MScAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Several attempts have been made to reduce Botrytis cinerea grey mould in vineyards and in storage by means of biological control. However, the so called "silver bullet" approach in utilising a single antagonist, has its limitations when compared with synthetic fungicides. Often the antagonist has a limited spectrum of activity and the duration of its effectiveness is less than that provided by synthetic fungicides. Furthermore, antagonists are more likely to be effective in preventing initial infection rather than resumption of latent infection. Therefore, due to the various infection sites in grape bunches utilised by B. cinerea and the fact that the pathogen can remain latent in the grapevine tissue, it may be possible to obtain effective control of the pathogen by integrating fungicides and different biological control agents each aimed at a different site in grape bunches, protecting the bunch at the various phenological stages of growth and under different micro climatic conditions. In this study the potential of three fungal antagonists (Glioc/adium roseum, Uloc/adium atrum and Trichoderma harzianum) and one yeast (Trichosporon pullulans) to colonise different sites in grape bunches, and to reduce B. cinerea infection, was investigated in commercial vineyards. As the biological control agents were used in an integrated system, the effect of various fungicides frequently applied to local vineyards on the organisms was also investigated. Fungicide trials were conducted taking into account two possible scenarios. Firstly, the possible effect of fungicides applied to the vineyard after an application of the biological control agent or shortly before the application of the biocontrol agent. This entailed exposing the biocontrol agents to relatively low concentrations of the active ingredient of the fungicides, similar to the residue levels to which these organisms would be exposed under field conditions. Secondly, the possibility of applying the organisms and the fungicides at the same time by making use of spray tank mixtures. This meant exposing the biocontrol agents to relatively high doses of the active ingredient of the various fungicides. Mycelial growth and germination tests were performed on agar in Petri dishes to determine the effect of fungicides. It was assumed that if the fungicide effectively inhibits the antagonist at 2.5 !-lg a.Uml, the fungicide and antagonist can not be used in an integrated programme. Based on this criterium, T harzianum can not be applied to vineyards with penconazole, mancozeb/metalaxyl, pyrifenox or mancozeb. In addition T harzianum can not be applied as tank mixtures with iprodione. However, T harzianum can be used in conjunction with pyrimethanil, folpan, iprodione, fosetyl-Al and copperhydroxide, provided the chemicals and the antagonist are applied alternately. Gliocladium roseum can not be applied in a tank mixture with pyrimethanil and penconazole, but can be used on grapevine in conjunction with penconazole, pyrifenox, pyrimethanil, iprodione and fosetyl-Al. Ulocladium atrum can not be applied with pyrimethanil and iprodione. Ulocladium atrum can be applied in conjunction with penconazole, pyrifenox, pyrimethanil, iprodione, fosetyl-Al and mancozeb. The fungus can be applied in a tank mixture with penconazole and pyrifenox. The antagonists were applied as conidial suspensions to bunches at various phenological stages in commercial vineyards planted with the wine grape cultivar Chardonnay in the Stellenbosch region, or the table grape cultivar Dauphine planted in Paarl region. Bunches were collected 2 wk after application, surface-sterilised and used for determining antagonist colonisation and B. cinerea infection at specific sites in the bunches. In Chardonnay, the antagonists colonised the different sites, but colonisation during the three seasons was inconsistent and sporadic. Ulocladium atrum and G. roseum colonised floral debris to a degree in the 1996 season. However, in the 1997 season these two antagonists did not develop from floral debris. Trichoderma harzianum colonised floral debris extensively in the 1996 season. In the 1997 season colonisation by T harzianum dropped, but unlike G. roseum and U atrum, T harzianum occurred at a low level in flowers. Ulocladium atrum only colonised bunches during bloom, and was not found in bunches monitored from pea-size stage to véraison. This finding suggests that the saprophyte colonised moribund and dead flower parts occurring in bunches during full bloom to the pre-pea size stage, and is not likely to be found in living tissue. Gliocladium roseum colonised grape berries and pedicels to some degree and T harzianum colonised these grape parts extensively. Botrytis cinerea occurred inconsistently and at low frequencies in the different sites in bunches. It was therefore not possible to comment on the effectivity of the various antagonists in the three seasons during which the trials were performed. However, it was noted that, during the peasize stage in 1996, when high levels of B. cinerea were recorded, T harzianum controlled these infections in the pedicels more effectively than any other treatment. / AFRIKAANSE OPSOMMING: ONDERDRUKKING VAN BOTRYTIS CINEREA DEUR ANTAGONISTE IN LEWENDE, AFSTERWENDE EN DOOIE WINGERDWEEFSEL Die benadering om Botrytis cinerea verrotting van wingerd met behulp van 'n enkele biologiese beheeragent in plaas van met sintetiese fungisiede te beheer, het sekere beperkinge. Antagoniste het dikwels 'n beperkte spektrum van aktiwiteit, en die duur van hul effektiwiteit is minder as dié van fungisiede. Antagoniste is gewoonlik ook minder effektief in die beheer van latente infeksie. Die patogeen het verder die opsie om druiwetrosse deur verskillende infeksieweë te koloniseer. Fungisiede kan druiwetrosse beter teen infeksie deur veelvuldige infeksieweë beskerm as 'n enkele antagonis. In die lig hiervan is die beheer van die patogeen deur 'n kombinasie van fungisiede en verskillende biologiese beheeragente, wat elk gemik is om 'n ander infeksiepunt in die druiwe te beskerm, ondersoek. Drie swamagtige antagoniste (Glioc/adium roseum, Uloc/adium atrum en Trichoderma harzianum) en een gis (Trichosporon pullulans) is in die ondersoek gebruik. Voorloper ondersoeke, waar twee moontlike scenarios in ag geneem is, is met fungisiede uitgevoer. In die eerste scenario is die effek van fungisiede, aangewend op wingerd kort vóór aanwending van die biologiese beheeragent, of kort ná aanwending, ondersoek. Hierdie proef het die blootstelling van die biologiese beheeragent aan relatief lae konsentrasies van die aktiewe bestanddeel van die fungisied, vergelykbaar met residuvlakke waaraan die organismes onder veldtoestande blootgestel sou word, behels. Tweedens is die moontlikheid om antagoniste en fungisiede gelyktydig as spuitpompmengsels toe te dien, ondersoek. In hierdie proef is die biologiese beheeragente aan relatief hoë dosisse van die aktiewe bestanddeel van verskillende fungisiede blootgestel. Miseliumgroei en ontkiemingstoetse is op agar in Petribakkies uitgevoer om die effek van die fungisiede te bepaal. As kriterium is aanvaar dat indien 'n fungisied die antagonis effektief by 2.5J..lglml aktiewe bestanddeel inhibeer, die fungisied en antagonis nie in 'n geïntegreerde program gebruik kan word nie. Gebaseer op hierdie kriterium kan T harnzianum nie aangewend word in 'n wingerd wat met penconazole, mancozeb/metalaxyl, pyrifenox of mancozeb behandel is nie. Ook kan T harzianum nie in 'n spuitpompmengsel met iprodione aangewend word nie. Trichoderma harzianum kan egter saam met pyrimethanil, folpan, iprodione en fosetyl-Al gebruik word, mits dié chemikalieë en die antagonis afwisselend aangewend word. Glioc/adium roseum kan nie in 'n spuitpompmengsel met pyrimethanil en penconazole aangewend word nie, maar kan saam met penconazole, pyrifenox, pyrimethanil, iprodione en fosetyl-Al gebruik word. Uloc/adium atrum kan nie saam met pyrimethanil, iprodione en fosetyl-Al gebruik word nie. Die swam kan wel in 'n spuitpompmengselmet penconazole en pyrifenox aangewend word. In verdere proewe is die antagoniste as spoorsuspensies op trosse op verskillende groeistadia in kommersiële wingerde, wat met die wyndruitkultivar Chardonnay of die tafeldruifkultivar Dauphine aangeplant is, ondersoek. Trossies is twee weke na toediening versamel, oppervlakkig gesteriliseer en gebruik om vlakke van antagoniskolonisasie en B. cinerea infeksie op spesifieke nisse in die trosse te bepaal. In die geval van Chardonnay het die antagoniste die verskillende nisse gekoloniseer, maar die kolonisasie was sporadies en nie konstant gedurende die drie seisoene van ondersoek nie. Uloc/adium atrum en G. roseum het blomdeeltjies tot 'n beperkte mate in die 1996 seisoen gekoloniseer, maar nie in die daaropvolgende seisoen nie. Daarteenoor het T. harzianum blomdeeltjies ekstensief in die 1996 seisoen gekoloniseer, en in 'n beperkte mate in die daaropvolgende seisoen. Uloc/adium atrum kon nie trosse van ertjiekorrelgrootte tot deurslaan vestig nie. Hierdie bevinding dui daarop dat die saprofiet afsterwende en dooie blomdeeltjies, wat van volblom tot ertjiekorrelstadium in die trosse voorkom, koloniseer, maar dat dit nie in lewende weefsel voorkom nie. Daarteenoor het T. harzianum die verskillende trosdele ekstensief gekoloniseer. Botrytis cinerea het gedurende die drie seisoene wisselvallig en teen lae frekwensies in die verskillende nisse in die trosse voorgekom. Dit was gevolglik nie moontlik om 'n konkrete afleiding oor die effektiwiteit van die verskillende antagoniste as biobeheeragente van B. cinerea te maak nie. In die geval van Dauphine was die onderskeie organismes swak koloniseerders van blomdeeltjies. Trichoderma harizanum kon egter die lewende trosdele koloniseer. Kolonisasievlakke was laag en was nooit meer as 50% nie. In beide seisoene het die kolonisasievermoë van T. harzianum drasties ná trostoemaak gedaal. Daarteenoor het beide G. roseum en U atrum tydens al die ontwikkelingstadia die lewende trosdele swak gekoloniseer. Botrytis cinerea het ook uiters sporadies en teen baie lae vlakke voorgekom. Die bevindinge het getoon dat klimaatsomstandighede wat in tafeldruifwingerde in die Wes-Kaap heers, nie geskik is vir die vestiging van die biologiese beheeragente wat in die studie ondersoek is nie.