Dans le but de mieux comprendre le mode d’action et de nous permettre de déterminer la cible de la tomatidine, nous avons dans un premier temps tenté de mieux circonscrire le spectre d’activité de la tomatidine. Grâce à ces travaux, nous sommes, en effet, maintenant en mesure de dire que la tomatidine possède une activité antibactérienne contre les espèces de la division des Firmicutes et plus précisément contre les bactéries de l’ordre des Bacillales dont font partie les genres Bacillus, Staphylococcus et Listeria. Nous avons également découvert, grâce à des expériences en collaboration avec le laboratoire d’Éric Marsault, qu’un analogue de la tomatidine (FC04-100) avait non seulement des propriétés similaires à la molécule naturelle, mais démontrait une activité par lui-même contre S. aureus à phénotype normal alors que la tomatidine possède uniquement une activité contre les « small colony variants ». De plus, alors que la tomatidine possède plutôt une activité bactériostatique contre la forme SCV de L. monocytogenes, le nouveau composé (FC04-100) démontre quant à lui, une forte activité bactéricide contre cette souche, tout comme contre la forme SCV des autres Bacillales. Parallèlement, et toujours dans le but de rechercher le mode d’action et la cible de la tomatidine, nous avons obtenu, par passages successifs dans un milieu avec antibiotiques, des mutants de S. aureus à phénotype normal et des SCV résistants à la tomatidine ou à la combinaison tomatidine et gentamicine. Après le séquençage de ces mutants, l’étude de la position de ces mutations, à l’aide de différents logiciels de bio-informatique, nous a permis d’émettre un modèle-hypothèse quant au mode d’action et à la cible de la tomatidine. Selon les résultats que nous avons à ce stade-ci, la cible de la tomatidine chez S. aureus serait la sous-unité c de l’ATP synthase. Cependant, son mode d’action serait également dépendant de la fonctionnalité de la chaine de transport des électrons et donc de la polarisation membranaire et de la production de ROS intracellulaire, ce qui expliquerait la différence d’activité entre les souches à phénotype normal et les SCV.

Membranes with a pore size rating of 0.2μm are recommended for the sterilisation of liquids by filtration and are validated for this purpose by a retention test with Pseudomonas diminuta. Practices for retention testing were found to vary among the membrane manufacturers and only one type of commercial 0.2μm rated membrane was found to reliably retain P. diminuta. The retention for P. diminuta given by experimental grafted membranes was studied and was sometimes higher than that given by non-grafted membranes due to obstruction of the pores by graft material. The dimensions for individual cells of P. diminuta was studied by scanning electron microscopy and a rapid electronic method. Bacteria of larger dimensions than the pore size rating of experimental membranes were found in test permeates. It was shown that cells from an aerated P. diminuta culture were larger than cells from a similar but stationary culture. A retention test procedure for 0.2 μm rated membranes using cross-flow filtration was developed. The procedure simulated process conditions and enabled tubular ceramic monolithic membranes and flat-sheet membranes to be retention tested with P. diminuta. It is feasible that a standard retention test using cross-flow filtration can be developed. The time needed for results from current retention test procedures to become available is a consequence of using traditional cultural techniques for permeate analysis. Test procedure were developed using three popular methods for the rapid detection and enumeration of bacteria (ATP luminescence, impedance microbiology and the DEFT) for the detection and enumeration of P. diminuta in retention test permeates. The method using ATP luminescence was found to be the most applicable. The development of a bioluminescent strain of P. diminuta through genetic engineering will enable the rapid, sensitive and straightforward retention testing of 0.2 μm rated membranes. Retention tests using a bioluminescent strain of Escherichia coli containing the structural genes for bacterial luciferase indicated that the proposed test is feasible. Developments were made towards cloning the same genes into P. diminuta. The use of all bioluminescent micro-organisms for membrane retention testing is the subject of a patent application and a proposal for a three year SERC research grant.

Multidrug resistance (MDR) is a major obstacle limiting chemotherapeutic efficacy. The purpose of these studies was to investigate the potential application of injectable paclitaxel (PX) and doxorubicin (Dox)-loaded nanoparticles (NPs) engineered from oil-in-water microemulsion precursors for overcoming P-glycoprotein (P-gp)- mediated drug resistance in solid tumors. An in-vitro study was performed to test whether the oil (stearyl alcohol and cetyl alcohol) used to make lipid nanoparticles could be metabolized. The results showed that the concentrations of the fatty alcohols within nanoparticles, which were quantitatively determined over time by gas chromatography, decreased to only 10-20% of the initial concentration after 15-24 h of incubation with horse liver dehydrogenase (HLADH) and NAD+ at 37ºC. Moreover, the surfactant Brij 78 (polyoxyethylene 20-sterayl ether) in the nanoparticles influenced the activity of the enzyme. Novel Cremophor EL-free paclitaxel-loaded nanoparticles were developed using experimental design combining Taguchi array and sequential simplex optimization. The resulting PX G78 and PX BTM NPs were stable at 4ºC over five months and in PBS at 37ºC over 102 h. Release of PX from PX NPs was slow and sustained without initial burst release. Interestingly, PX BTM NPs could be lyophilized without cryoprotectants and without changing any physiochemical properties and bioactivities. Cytotoxicity studies in breast cancer MDA-MB-231 cells showed that PX NPs have similar anti-cancer activities compared to Taxol. Optimized Dox-loaded NPs were prepared using an ion-pair agent, sodium tetradecyl sulfate (STS), to mask Dox charge and to enhance its entrapment in NPs. In-vitro cytotoxicity studies were carried out in both sensitive and resistant human cancer cells treated with PX and Dox-loaded NPs. All of drug-loaded NPs decreased IC50 values by 6-13-fold in resistant cells compared to free drugs. A series of in-vitro assays were used to understand the underlying mechanisms. The results, in part, showed that the NPs inhibited P-gp and transiently depleted ATP, leading to enhanced uptake and prolonged retention of the drugs in P-gp-overexpressing cancer cells. Finally, in-vivo anti-cancer efficacy studies were performed using pegylated PX BTM NPs after intravenous (i.v.) injection and showed marked anti-cancer efficacy in nude mice bearing resistant NCI/ADR-RES tumors versus all control groups. These results suggest that NPs may be used to both target drug and biological mechanisms to overcome MDR.

Cette thèse s’inscrit dans le cadre de l’étude de la relation structure-activité d’analogues de ligands naturels de récepteurs nicotiniques et purinergiques. Ce travail se divise en deux parties. Dans la première partie de cette thèse, nous avons réalisé la synthèse d’analogues de la 11-homosédinone, alcaloïde isolé de la plante Sedum acre, qui présente une activité agoniste sur différents récepteurs nicotiniques du système nerveux central. Les différents analogues ont été synthétisé par application de la méthoxylation anodique pour introduire succesivement deux substituants en postion 2 et 6 d’un noyau pipéridinique. Les analogues synthétisés se différencient par la nature du noyau aromatique, la présence d’un groupement méthyle sur l’atome d’azote de la pipéridine et l’oxydation du sustituant en position 2. Ce travail a notamment permis de montré l’importance du groupement N-méthyle vis-à-vis de l’activité des analogues. Nous avons également pu mettre en évidence que l’introduction d’un halogène sur le noyau aromatique diminuait l’activité de l’analogue sur le récepteur a7 tout en augmentant l’acitivité sur le récepteur a4b2 et que l’introduction d’un noyau furanique permettait d’augmenter la sélectivité vis-à-vis du récepteur a4b2 tandis que l’introduction sur le noyau aromatique d’un groupement nitro ou méthoxy conduit à une perte totale de l’activité. Dans la seconde partie de cette thèse, nous avons réalisé la synthèse d’analogues de la dATP, afin d’évaluer leur effet agoniste sur le récepteur P2Y11, impliqué dans différents mécanismes de différentiation cellulaire, dont notamment celui de la maturation des cellules leucémiques HL60 en cellules de type neutrophile. Les analogues synthétisés se différencient de la dATP par la présence d’un groupement méthylène ou dichlorométhylène entre les phosphores b et g de la chaîne polyphosphate, ainsi que par l’estérification de l’alcool en position 3’ du sucre. Ce travail a pu confirmer que les analogues en série 2’-désoxy conduisent à de meilleures activités que ceux de la série 2’-OH. Nous avons également pu montrer que l’estérification de la position 3’ conduit à une diminution de l’activité agoniste, à l’exception du groupement a-naphtoyle qui conduit à une augmentation significative de l’activité sur P2Y11.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Chronic exposure to arsenic-contaminated drinking water is correlated with obstructive lung disease (i.e. chronic obstructive pulmonary disease (COPD), bronchiectasis), reduced lung function and other respiratory effects (e.g. chronic cough, chest sounds). Researchers have associated arsenic exposure with reduced airway immunity. The airway epithelial innate immune system protects underlying tissue from inhaled particulates and pathogens through a variety of mechanisms. Such defects in innate immunity are associated with chronic bacterial infections and development of obstructive airway diseases, including COPD and bronchiectasis. We hypothesize that arsenic exposure may lead to recurrent lung infection and eventual obstructive lung disease by compromising mechanisms essential in airway innate immunity. In the work presented herein we evaluated the effects of arsenic on airway epithelial barrier properties, wound repair capacity, and signaling pathways essential in innate immunity. We previously published that acute (24 hr) arsenic (0.4-3.9 μM as Naarsenite) slowed wound repair in a human bronchial epithelial cell line (16HBE14o-). In the first study we hypothesized arsenic may be affecting wound repair by altering Ca²⁺ signaling that is important in multiple aspects of wound repair, including cell migration. We found wound-induced Ca²⁺ signaling was largely mediated by paracrine ATP in 16HBE14o- cells, and acute (24 hr) arsenic (0.8, 3.9 μM) exposure reduced ATPmediated Ca²⁺ signaling. We identified functional reductions in the ATP receptors P2Y₂ and P2X₄ following arsenic exposure. Both of these receptors are essential in airway innate immunity (e.g. mucociliary clearance). In the second study we found similar reductions in wound repair capacity and ATP-mediated Ca²⁺ signaling in 16HBE14o cells using a chronic (4-5 week) low-dose (0.13, 0.33 μM) arsenic exposure representative of U.S. drinking water standards. Further, wound-induced Ca²⁺ signaling was reduced in primary cultured tracheal cells derived from mice fed arsenic-free or arsenic-supplemented (50 ppb; 1μM=75 ppb) water for four weeks prior to experimentation. In the last study we demonstrated that the structure and function of the airway epithelial barrier was altered by a five-day exposure of arsenic (0.8, 3.9 μM). We conclude that arsenic at environmentally relevant levels compromises key functions in airway epithelial innate immunity that may underlie development of lung disease.

Hemocytes (blood cells) play an important role in the immune response in invertebrates, and thus the regulation of hemocyte homeostasis (hematopoiesis) is essential for the host survival against pathogens. Astakine 1, a homologue to vertebrate prokineticins, was first identified in the freshwater crayfish Pacifastacus leniusculus as a cytokine, and was found to be necessary for new hemocyte synthesis and release in vivo, and also to induce spreading and proliferation of Hematopoietic tissue cells (Hpt cells, precursor of hemocytes) in vitro. The work of this thesis is aimed to further our understanding of the molecular mechanisms involved in astakine 1 induced hematopoiesis. Crayfish transglutaminase (Tgase) has been identified in the hemocytes, and is essential for the coagulation reaction. Interestingly this enzyme is exceedingly abundant in the Hpt cells, and the spreading of Hpt cells induced by astakine 1 was accompanied by sequential loss of TGase activity from the surface of these cells. This loss of TGase activity may be an important effect of astakine 1, resulting in recruiting new hemocytes into the circulatory system. Although astakine 1 contain a prokineticin domain, it lacks the conserved N-terminal AVIT motif present in its vertebrate homologues. This motif is important for vertebrate prokineticins to interact with their receptors, indicating a different receptor interaction for crayfish astakine 1. Astakine 1 was indeed found to interact with a completely different receptor, the β-subunit of ATP synthase, on a portion of Hpt cells, and subsequently block its extracellular ATP formation. Surface ATP synthase has been reported on numerous mammalian cells, but now for the first time in an invertebrate. The activity of ATP synthase on the Hpt cells may be important for the survival and proliferation of Hpt cells, but the underlying mechanisms remain further study. With the finding of a second type of astakine in crayfish, invertebrate astakines can be divided into two groups: astakine 1 and astakine 2. The properties of astakine 2 are different from those of astakine 1 both in structure and function. In primary cell culture of Hpt cells, only astakine 1 can promote proliferation as well as differentiation into semigranular cells, whereas astakine 2 may play a potential role in the maturation of granular cells. Moreover, a novel cysteine rich protein, Pacifastacus hematopoiesis factor (PHF), was found to be one target gene of astakine 1 in Hpt cells. Down regulation of PHF results in increased apoptosis in Hpt cells in vitro, and in vivo silencing PHF leads to a severe loss of hemocytes in the animal. Therefore astakine 1 acquires the anti-apoptosis ability by inducing its downstream gene PHF in the Hpt cells. With its ability to promote the survival, proliferation and differentiation of Hpt cells, astakine 1 is proven to be an important hematopoietic growth factor.

Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.

An emerging trend towards non-laboratory based biological and microbiological marker analysis is occurring in multiple sectors of science and industry. In the medical sector, these trends have demonstrated that conducting sample analyses away from centralised laboratories not only makes analyses quicker and more convenient (e.g. a home pregnancy test), but can offer services that are otherwise impractical (e.g. mobile laboratories to diagnose disease in the developing world). In the environmental sector, similar benefits, plus the ability to develop and test hypotheses, protocols and sampling strategies within a field campaign, are possible with in-field analyses. Icy environments in particular would benefit from in situ or in-field life detection as they are typically remote, and hence impart high logistical costs for repeated field campaigns and associated sample return with the implication that the efficiency of scientific return is poor. Unfortunately, most equipment and protocols developed for microbiological analyses in other sectors of science and industry are unsuitable for direct application to in-field use in icy environments because of poor compatibility with icy environment sample matrices and frequently inappropriate microbiological targets. Hence within this work, two hypotheses were tested: that (i) microbiological detection infield in icy environments is possible and through this (ii) unique and more efficient scientific studies can be conducted. Cont/d.

Invadolysin (inv) is a member of the M8 class of zinc-metalloproteases and is conserved throughout metazoans. It is essential for development and invadolysin homozygous Drosophila mutants are third instar larval lethal. These larvae exhibit a reduced larval brain size and an absence of imaginal discs. Detailed analysis showed that inv mutants exhibit pleiotropic effects, including defects with chromosome architecture, cell cycle progression, spindle assembly, nuclear envelope dynamics, protein turnover and problems with germ cell migration. These findings indicated that Invadolysin must have a critical role in Drosophila. In order to better understand these roles, I set out to identify genetic interactors of invadolysin. I performed a genetic screen scoring for enhancer/suppressor modification of a ‘rough eye’ phenotype induced by invadolysin overexpression. Screening against the Drosdel ‘deficiency kit’ identified numerous genetic interactors including genes linked to energy regulation, glucose and fatty acid pathways. Immunofluorescence experiments in cultured cells showed that H. sapiens Invadolysin localises to the surface of lipid droplets (LD), and subcellular fractionation confirmed its enrichment to these structures. Lipid droplets are highly dynamic organelles involved not only in energy storage but also in protein sequestration, protein and membrane trafficking, and cell signaling. Drosophila fat bodies are enriched in LDs and therefore important energy stores. In addition, they are nutritional sensors and regulators, which are proposed to be the ortholog of vertebrate liver and adipose tissue. Mutant inv fat bodies appeared smaller and thinner than wild type fat body, and accumulated lower levels of triacylgylcerides. This indicated that the loss of invadolysin might be affecting lipid metabolism and storage, confirming the genetic data. However, it was not clear whether these effects were due to the direct action of Invadolysin. Hence, transgenic fly lines expressing either HA, RFP or FLAG tagged forms of Invadolysin were generated to identify physical interactors of Invadolysin. Subsequent mass spectrometry analysis detected ATP synthase-α, -β and -d as interactors. This result suggested that Invadolysin might play a role in regulating mitochondrial function, which might then be manifest in the fat body as the defects previously observed. Energy levels are known to affect the cell cycle, cell growth, lipid metabolism and inevitably development. Further in vivo and in vitro experiments confirmed this hypothesis. Genetic crosses confirmed the interaction of invadolysin with ATP-synthase subunit-α, whilst staining of mitochondria in mutant third instar larval fat bodies suggested decreased mitochondrial activity. Mutants also showed lower ATP levels and an accumulation of reactive oxygen species, hence indicating the possibility of a dysfunctional electron transport chain. Lipid droplets are known to interact with mitochondria, whilst ATP synthase has been found on lipid droplets by proteomic studies in Drosophila. Therefore, based on these data, we propose that Invadolysin is found, with ATP synthase, on lipid droplets, where Invadolysin (likely acting as a protease) could be aiding the normal processing or assembly of ATP synthase. This interaction is vital for the proper functioning of ATP synthase, and hence mitochondria. In this scenario, cellular ATP needs are not met, energy levels drop which results in an inhibition of fatty acid synthesis, cell and organismal growth defects.