Includes list of publications by the author. / Includes bibliographical references (leaves 103-123) / x, 123 leaves : ill.;c 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Evaluates the nature of the interactions occurring between semen and cells of the uterus that occur following mating in pigs. Describes a novel ability of porcine seminal plasma to induce dose dependent cell-cell adhesion and mitogenesis amongst peripheral blood lymphocytes in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1993

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Bibliography: leaves 214-236. / xviii, 238 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1999

Bacteria swim towards improving conditions by controlling flagellar activity via signals (CheY) sent from chemosensory protein clusters, which respond to changing stimuli. The best studied chemotactic bacterium, E. coli, has one transmembrane chemosensory protein cluster controlling flagellar behaviour. R. sphaeroides has two clusters, one transmembrane and one cytoplasmic. The roles of the two clusters in regulating swimming and chemosensory behaviour are explored here. Newly-developed software was used to measure the effect of deleting or mutating each chemotaxis protein on unstimulated swimming and on the chemosensory response to dynamic change. New behaviours were identified by using much larger sample sizes than previous studies. R. sphaeroides chemotaxis mutants were classified as (i) stoppy unresponsive; (ii) smooth unresponsive or (iii) stoppy inhibited compared to wildtype swimming and chemosensory behaviour. The data showed that the ability to stop during free-swimming is not necessarily connected to the ability to respond to a chemotaxis challenge. The data suggested a new model of connectivity between the two chemosensory pathways. CheY₃ and CheY₄ are phosphorylated by the transmembrane polar cluster in response to external chemoeffector concentrations. CheY₆-P produced by the cytoplasmic cluster is a requirement for chemotaxis, whether or not the polar cluster is able to produce CheY₆-P. CheY₆-P stops the motor, whereas CheY_3,4-P allow smooth swimming. When chemoeffector levels fall, the signals through CheY_3,4 fall, allowing CheY₆-P to bind and stop the motor. As the polar cluster adapts to the fall by the action of the adaptation proteins CheB₁ and CheR₂, the concentration of CheY_3,4-P increases again, to compete with CheY₆-P and allow periods of smooth swimming. Under aerobic conditions, the cytoplasmic cluster controls the basal stopping frequency and does not appear to respond to external chemoeffector changes. The role of the adaptation proteins in resetting the signalling state in R. sphaeroides is unclear, particularly the roles of the proteins associated with the cytoplasmic cluster, CheB₂ and CheR₃. Tandem mass spectrometry was used to identify glutamate and glutamine (EQ) sites on the cytoplasmic R. sphaeroides chemoreceptor TlpT that are deamidated and methylated by the R. sphaeroides adaptation homologues. In E. coli, adaptation sites are usually EQ/EQ pairs. However the sites reported in TlpT vary at the first residue in the pair. Mutation of the putative EQ adaptation sites caused changes in adaptation, suggesting that CheY₆-P levels are controlled and reset by CheB₂ and CheR₃ controlling the adaptation state of TlpT.

The dispersion and reaggreation [sic] stages have been described as a unique feature of embryonic development in annual killifish such as Austrofundulus limnaeus, a killifish that inhabits ephemeral ponds in the Maracaibo basin of Venezuela. These stages have previously been described as an atypical developmental progression in which blastomeres completely disperse on the surface of the yolk and then reaggregate into a mass of cells to form the embryo. Temperature affects the onset as well as the duration of this stage in related annual fishes. We have undertaken this study to show in detail the behavior of blastomere cells and their distributions as a function of developmental temperature. Embryos incubated at either 25 or 30°C were fixed and stained with Hoescht dye to allow visualization and quantification of cell number during the dispersion and reaggregation phases of development. The location of every cell nucleus on the embryo was assessed through photomicroscopy using inverted epifluorescent microsopy [sic]. This analysis revealed that the rate of cell division during the process of dispersion/reaggergation [sic] has a typical sensitivity to temperature with Q10 values of about 2-3. There is no indication that the pattern of blastomere movement and distribution is different in embryos incubated at 25°C versus 30°C. The primary developmental difference was observed as a temporary plateau in blastomere counts at 25°C followed by great variation of blastomere numbers in subsequent timepoints compared to the degree of variation observed in embryos incubated at 30°C. This trend fits the model that embryos developing at 25°C enter into a brief diapause-like event at the dispersion stage from which they emerge at a variable rate. Of great interest, at both temperatures examined, the majority of the dispersed blastomeres do not reaggregate and contribute to the formation of the primary embryonic axis. Prior studies have overemphasized blastomere reaggregation in A. limnaeus due to the limitations of the sampling methods used as well as overdependence upon a statistical test, the coefficient of dispersion. Thus, the present study sheds light on these early mischaracterizations of A. limnaeus development.

Il a été mis en évidence que le canal SK3 est un médiateur de la migration de cellules cancéreuses mammaires, une propriété essentielle à la formation de métastases. Par ailleurs, ce canal est inhibé par l’édelfosine, un éther-lipide ayant des propriétés anti-tumorales in vitro mais son usage en clinique a été abandonné en raison d’effets secondaires. Une première partie de ce travail a permis de déterminer les parties de l’édelfosine nécessaires à l’inhibition du canal SK3 et de la migration cellulaire. Ceci nous a permis de sélectionner l’Ohmline (1-O-Hexadécyl-2-O-Méthyl-sn-glycéro-lactose), un analogue non toxique de l’édelfosine qui conserve son activité inhibitrice de SK3 et de la migration. Dans un deuxième temps, nous avons testé ce lipide dans un modèle murin de développement tumoral et nous avons montré qu’il réduisait le développement des métastases sans modifier la tumeur primaire. En conclusion, nous avons décrit l’Ohmline qui est le premier inhibiteur lipidique de SK3 et qui pourrait devenir le premier membre d’une famille de composés lipidiques inhibiteurs de la formation de métastases. / It has been shown that SK3 channel was a mediator of breast cancer cells migration, a fundamental property for metastasis formation. In addition, edelfosine inhibits SK3 channel. This ether-lipid owns a high anti cancerous potential in vitro but its clinical use was hampered by some side effects, Firstly, we showed the structural parts of edelfosine required for SK3 channel inhibition and cell motility inhibition. Moreover, we selected Ohmline (1-O-Hexadécyl-2-O-Méthyl-sn-glycéro-lactose), an edlefosine’s analogue that preserves SK3 channel and motility inhibitory properties. Secondly, we evaluated this lipid on tumor development in nude mice model. We showed that this lipid reduces metastasis formation without effect on primary tumor. To conclude, we described Ohmline, the first lipid inhibitor of SK3. This compound should become the first member of a new family of metastasis lipid inhibitors.

[Truncated abstract] For this thesis, the molecular biology of cancer was approached from two directions. Firstly, an investigation was conducted on the role of growth factor receptor-bound protein 7 (Grb7) in breast cancer. Grb7 is an adapter molecule that binds to a variety of proteins, including the growth factor receptor and proto-oncogene, ErbB2, and mediates signalling to downstream pathways. It has been linked to cell migration and an invasive phenotype, and is of interest as a therapeutic target. To investigate the role of Grb7 in breast cancer, preliminary experiments were performed that, firstly, determined the expression of wild-type Grb7 and a splice variant, Grb7V, in a range of cell lines, and secondly, aided the development of a protocol for treating cells with short interfering RNA (siRNA) against Grb7 and the ErbB ligand, heregulin (HRG), in a cell system appropriate for measuring the functional outcomes. Using this protocol in conjunction with CellTitre (CT) proliferation assays, it was demonstrated that Grb7 does not play a role in the proliferation of either unstimulated or HRG-stimulated SK-BR-3 breast cancer cells. Furthermore, using the protocol in conjunction with Boyden chamber migration assays, it was shown that inhibition of Grb7 expression has a slight stimulatory effect on HRG-stimulated SK-BR-3 cell migration. Thus, Grb7 was found to play only a minor role in the migration of SK-BR-3 cells, suggesting that it is not an ideal anti-cancer target for breast cancers modelled by this cell system. Concurrently, a second investigation was conducted, which similarly sought insight into the molecular biology of cancer, but adopted a more strategic approach. ... These results provide evidence for a biologically significant role for the miR-7-mediated regulation of EGFR expression. A microarray experiment was also performed to identify genes that were down-regulated following treatment with miR-7 compared to NS precursor. Of 248 down-regulated genes, including EGFR, 37 promising new miR-7 target candidates were identified. Functional clustering of down-regulated genes and promising target candidates suggested that miR-7 may have functionally-related targets involved in processes including cell motility and brain-associated functions. This investigation thus yielded a program capable of accurately predicting a miRNA target not predicted by any other target prediction program, verified a previously unknown miRNA:target interaction with functional consequences in cancer cells and provided the first steps towards investigating miR-7-mediated regulation in greater depth. Furthermore, EGFR was, to our knowledge, the first example of a verified miRNA target with target sites that are not conserved across mammals, an observation with important implications for computational target prediction and the evolution of miRNA regulatory systems. In addition, the demonstrated growth inhibitory and cytotoxic effects of miR-7 on lung cancer cells raise the possibility of a miR-7-based therapeutic for the treatment of EGFR-overexpressing tumours.

Pour lutter efficacement contre une attaque, l’organisme est doté d’un système immunitaire lui permettant de reconnaître le danger et de se défendre contre celui-ci. Les lymphocytes T jouent le rôle de chef d'orchestre de la réponse immunitaire, organisant l'activité des autres cellules nécessaires à la défense contre les infections. Pour accomplir ce rôle, les cellules T sont dotées d’une forte mobilité, leur permettant ainsi de circuler constamment dans diverses régions de l’organisme et d’y établir de nombreuses interactions.<p><p>Durant ce travail, nous nous sommes intéressé aux mécanismes moléculaires responsables de la motilité des cellules T. En particulier, nous avons investigué le rôle de la protéine S100A4, dont l’expression a été démontrée au sein de cellules T, dans la motilité de ces cellules ainsi que son implication dans l’inflammation. La protéine S100A4 est connue pour son implication dans la motilité de divers types cellulaires tels que les fibroblastes, les macrophages ou encore les cellules cancéreuses. En outre, S100A4 est capable d’interagir avec de nombreuses protéines cruciales pour la migration cellulaire telles que la myosine-IIA, l’actine, la tropomyosine, la rhotékine, les septines 2,6 et 7, CCN3 ou encore la transglutaminase 2.<p><p>Nous avons montré que des souris déficientes pour S100A4 ne présentent aucune modification majeure au niveau des cellules T situées dans le thymus ou en périphérie. Nous avons observé que la protéine S100A4 est principalement exprimée par les cellules T mémoires effectrices des populations de LT CD4+ ou CD8+. Cependant, la présence de la protéine ne semble pas requise pour la migration in vitro des LT mémoires. De plus, des expériences d’infections bactériennes par Listeria monocytogenes nous ont permis de démontrer que la réponse immunitaire mémoire des cellules T n’est pas affectée par l’absence de S100A4. En outre, la différenciation in vitro de cellules T CD4+ naïves en diverses sous-populations effectrices n’est pas modifiée suite à l’absence de la protéine dans les cellules. Finalement, nous avons étudié l’implication de la protéine S100A4 dans le développement de maladies immunitaires impliquant la migration de cellules T. Nos modèles d’intérêts ont été la colite et l’encéphalomyélite auto-immunitaire expérimentales. La protéine S100A4 n’est pas cruciale pour l’induction de ces deux pathologies, puisque son absence ne modifie pas leur développement.<p><p>Notre étude démontre clairement que la protéine S100A4 n’est pas requise pour la motilité des cellules T. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The goal of this thesis is the development of a tracking algorithm for populations of unmarked cancer cells that migrate in 3D in vitro gels. The tracking algorithm is intended to be a tool for analysing the motility of large population (i.e. hundreds) of cells in the context of the anti-migratory drug development and more specifically drug screening. In oncology, cancer cell migration plays pivotal roles in the spread of cancer cells from a primary tumor site to neighboring and secondary sites, i.e. the processes of tissue invasion and metastasis. Preventing such processes represents an important therapeutic approach to cancer treatment. Providing tools able to test potential anti-migratory drugs thus constitutes currently a real need in oncology therapy. The goal of drug screening in this context aims to rapidly and efficiently test the anti-migratory effects of many experimental conditions on cancer cell populations.<p>The focus in this thesis lies in two specific aspects that are important in anti-migratory drug screening: tracking cells inside an in vitro 3D environment and doing so using unmarked cells. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

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