Reproduction de : Thèse de doctorat : Etudes sur l'Asie et ses diasporas : Lyon 3 : 2004. / Titre provenant de l'écran-titre. Bibliogr.

The Ruv ABC and RecBCD protein complexes together can collapse and repair arrested replication forks. With their help a fork structure can be re-established on which replication can be restarted. ruv and recB mutants are therefore quite sensitive to UV light. Their survival is greatly decreased in the absence of the signalling molecules (p)ppGpp and increased when excess (p)ppGpp is present. (p)ppGpp are the effector molecules of the stringent response, regulating adaptation to starvation and other stressful environmental changes. Absence of (p)ppGpp can be compensated for by mutations in RNA polymerase that are called stringent mutations. Some of those, called rpo *, also - like excess (p)ppGpp - increase the survival of UV irradiated ruv and recB cells. A model proposed by McGlynn and Lloyd (Cell, Vol. 101, pp35-45, March 31, 2000) suggests that this is achieved by modulation of RNA polymerase, which decreases the incidence of replication fork blocks. In this work twenty-seven rpo * mutants were isolated, sequenced and mapped on the 3D structure of Thermus aquatic us RNA polymerase. I have found mutants in the ~ and ~' subunits of RNA polymerase. They lie mostly on the inner surface of the protein, well placed to make contact with the DNA substrate or the RNA product. A large number of rifampicin resistant mutations among rpo* mutations is explained by an overlap between the so-called Rif pocket and the "rpo* pocket". rpo * mutations, like stringent mutations, lead to a decrease in cell size, suppress filamentation and increase viability. For in vitro studies I purified wild type and two mutant RNA polymerases with help of a his-tagged a subunit. The experiments confirmed that rpo* mutant RNA polymerases form less stable open complexes than wild type, just like previously investigated stringent RNA polymerases. In addition I have shown here that (p)ppGpp leads to the destabilisation of RNA polymerase complexes stalled by nucleotide starvation or UV-induced lesions, though there is as yet no indication that rpo * mutations act in the same way.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 229-239). Also available in electronic version.

Spatiotemporal responses to extracellular signals have been documented in a wide variety of cells, such as neuronal synapses, cytotoxic T lymphocytes, germ cells and during embryo development. Selective release of a key molecule allows a cell to respond at a given moment, and cells ensure that the response can be initiated instantly by pre-producing and packaging the molecule, often storing the molecule as a granule. Differentially Expressed in FDCP6 is a guanine nucleotide exchange factor, primarily expressed in T cells, which has been previously shown to form cytoplasmic aggregates when phosphorylated by ITK. DEF6 also translocates to the immunological synapse following phosphorylation by LCK in response to antigenic presentation. As a result, DEF6 is a likely candidate in mediating a spatiotemporal response to an extracellular signal in T cells. Data presented in this work suggest that endogenous DEF6 forms cytoplasmic granules in a variety of T cell states and the DEF6 mutant Y210EY222E, which mimics ITK phosphorylation, interacts with mRNA. Moreover, DEF6 is hypothesised to have two unconventional RNA binding domains; a feature which has also been described in the literature within proteins that catalyse glycolysis. DEF6 is also shown to be in close proximity to PABP and eIF4E, both of which are translation factors, as well as active translation in resting Jurkat T cells and the immunological synapse. Furthermore, endogenous DEF6 co-localises with 4E-T, a P-body marker which is involved with miRNA mediated decay, in resting and stressed Jurkat T cells. These data corroborate that of Hey et al. (2012) and suggests that DEF6 does indeed interact with P-bodies. Finally, translocation of DEF6 appears to occur in response to an extracellular signal alternative to the T cell receptor during T-T communication and that the translocation may occur in vesicle-like structures in close proximity to LFA-1. Consequently, these data identify a novel link between DEF6 and active translation as well as mRNA turnover and that the extracellular signal required for this spatial response is not antigen presenting cell specific but rather a response to LFA-1 stimulation.

The Wharton’s Jelly (WJ) of human umbilical cord (HU) contains multipotent stem cells (WJ-MSC) which express mesenchymal markers but not hematopoietic markers. WJ-MSC are increasingly being tested for use in stem cell therapy, with intravenous delivery being the preferred route. Fetal stem cells from embryonic germ layers are present in maternal blood and can home to damaged maternal tissues. This study investigates how WJ-MSC cross the fetal and adult endothelial barriers; including the cellular and molecular mechanisms employed. WJ-MSC were isolated from HU (n=27) which were taken from normal term pregnancies after elective Caesarean section. Flow cytometry and immunofluorescence were used to check presence/absenc of mesenchymal versus haematopoietic markers. Cells were induced to become adipocytes, chondrocytes and osteocytes by using specific induction medium. Isolated WJ-MSC were added after labelling with PKH26 to confluent monolayers of isolated human umbilical vein endothelial cells (HUVEC) or commercially bought human uterine microvascular endothelial cells (HUtMEC) at a 1:5 ratio. Cell-cell interactions were monitored with real time microscopy for 24 to 40h. Fluorescence and confocal scanning microscopy, after vascular endothelial (VE) cadherin immunocytochemistry were used for detailed analysis of VE-cadherin junctional occupancy and spatio-temporal location of stem cells. Tyrosine phosphorylation status of VE-cadherin, whether at Tyr685 or Tyr731, at different time points were investigated by immunoblotting whilst levels of vascular endothelial growth factor (VEGF) in the conditional media (CM) were measured by ELISA. Three different isolates were tested, with 3 experimental repeats for all expermints. Statistical analyses were performed with ANOVA (One or Two way). Cells (>95%) from each passage were positive for the mesenchymal markers CD 29, CD 105, CD 90, CD 73 and CD 44. <2% cells showed positivity to the haematopoietic markers CD 34, HLA-DR, CD 14, CD 19 and CD 45. WJ-MSC differentiated into adipocytes, osteocytes and chondrocytes. WJ-MSC displayed exploratory behaviour for a minimum of 30 min on HUtMEC or 60 min on HUVEC with interrogation of paracellular openings before crossing rather than replacing endothelial cells. By 2h, half were found at sub-endothelial positions, with a majority reaching this within 16-22h. There was accompanying loss of junctional VE-cadherin (64.9 + 3.7 %; p<0.001 in HUVEC; 63 + 4.6%; p< 0.001 in HUtMEC) in the endothelial monolayers followed by a return at 16h and increased continuity by 22h (p<0.01 in HUVEC; p<0.001 in HUtMEC). Junctional disruptions were found close to overlying or migrating WJ-MSC. Confocal microscopy confirmed paracellular extravasation. VE-cadherin protein levels matched controls in the early hours (0-2h) and increased after 22h co-culture in both fetal and uterine endothelium. VE-cadherin showed a 2-fold increase in phosphorylation at Tyr685 from 30 min to 2h. P-Tyr731 remained unchanged, similar to untreated endothelial layers, then decreased at 2h and 22h. VEGF levels in WJ-MSC – HUtMEC co-culture supernatants was highest at 2h (88 + 3 pg/ml) and decreased by 22h, reaching negligible levels by 48h. Anti-VEGF blocked Tyr685 phosphorylation but did not affect the decrease in P-Tyr731; this was accompanied by a 25% decrease in transmigration of cells in the first two hours and a 43% decrease in total by 22h. in WJ-MSC – HUtMEC co-cultures. However, in HUVEC-WJ-MSC co-cultures, no VEGF were detected and anti-VEGF did not block Tyr685 phosphorylation and Tyr731 de-phosphorylation. WJ-MSC from term umbilical cords can be easily isolated and expanded in culture. They retain mesenchymal stem cell properties for the passages tested (up to P5) making them a valuable model for studies into mechanisms underlying extravasation. The data obtained suggest that WJ-MSC can influence expression of VE-cadherin, with perturbation during transmigration followed by upregulation and repair once the adlumenal side is reached. There was a similarity in the cellular and molecular mechanisms employed by WJ-MSC in their paracelluar migration across fetal and uterine endothelium, although VEGF may not be the key player in HUVEC interactions. For both endothelial types, WJ-MSC appear to induce phosphorylation events linked with paracellular permeability and de-phosphorylation events normally associated with leukocyte extravasation. The data from the uterine endothelial investigations suggests that fetal stem cells are able to influence paracellular junctional dynamics and strengthens the growing hypothesis that they may also play a role in re-modelling the uterine circulation for fetal advantage. The extra-embryonic WJ-MSC holds the promise of use in restoring junctional maturity and vascular repair in future therapeutic applications.

Placing our understanding of the function of the immune system into a more natural setting remains a fundamental challenge in biology, particularly how natural variation shapes the immune response and what the evolutionary consequences of such variation are. In this thesis, I use the three-spined stickleback as a model system for wild immunological studies. First, I developed a set of markers to measure the expression levels of key immune system genes using quantitative real-time PCR, representing the innate and adaptive immune response, and then used them to address a number of questions. I demonstrated that there are underlying differences in innate and adaptive gene expression levels between populations, as well as in innate immune response potential, which may reflect the contrasting challenges faced in these populations. By sampling individuals from multiple wild populations, I was able to demonstrate how a range of factors contribute to shaping immune system function, including sex, reproductive status, and infection with the common parasites Schistocephalus solidus and Gyrodactylus arcuatus. Next, I exposed laboratory raised fish to natural conditions and examined their response. Again, a range of factors where identified which appear to shape immune expression levels, particularly reproductive investment and infection with G. arcuatus. I also used this approach to demonstrate that immune system variation can be linked to Eda genotype, the gene which controls lateral plate phenotypic divergence during adaptive radiations. Finally, I performed a controlled infection experiment in the laboratory to show that both the innate and adaptive systems respond to Gyrodactylus infection. This thesis provides the basis for further immunological studies in stickleback, and adds to our growing understanding of the relevance of natural variation in shaping the immune response.

This thesis is divided into two main chapters. The first chapter relates to studies undertaken to gain insights into the mechanism of action on liver growth by the peroxisome proliferator (PP) ciprofibrate and the chemical cyproterone acetate (CPA) in rodents. Peroxisome proliferators are a class of chemicals that have diverse effects in rats and mice including increased DNA synthesis and peroxisome proliferation. Peroxisome proliferators include herbicides, plasticisers, hypolipidemic drugs and synthetic fatty acids. These chemicals act through ligand activation of nuclear membrane receptors termed ‘peroxisome-proliferator-activated receptors’ (PPARs), which ultimately activate nuclear transcription. PPs induce a cellular process in liver characterised by dramatic increases in the size and number of peroxisomes, correlated with both hepatocyte hypertrophy (i.e. an increase in the size of liver cells) and hyperplasia (i.e. an increase in the number of liver cells during replicative DNA synthesis and cell division). However, the mechanism of action of increased hepatocyte growth is not currently understood. Understanding the mechanism by which increased liver growth is induced by PPs in rodents will hopefully provide insights into how natural liver growth occurs and might have medical benefits for human health if the mechanism of PP toxicity can be overcome. Knowledge gained from the mechanism of PP activation might also then be applied to other chemical carcinogens. Therefore, firstly the mode of action of the peroxisome proliferator ciprofibrate was investigated. Previous work had indicated that two successive doses of ciprofibrate treatment separated by 24hr led to two rounds of liver cell replication, but it was not clear whether the same or different hepatocytes were involved in this growth response. To study this phenomenon, histochemical experimental work was undertaken to assess whether the same or different hepatocyte cells were stained during the two rounds of cell division following ciprofibrate treatment. The two histochemical stains used were EdU and BrdU, which are both base-pair analogues that stain nuclei undergoing DNA replication. It was hypothesized that if EdU was used to stain cells at 24 hr and then BrdU at 48 hr, that if the same cells were responding to ciprofibrate treatment then cells would be co-stained by both dyes, whereas if different cells were responding then there would be little or no double staining of hepatocyte cells. It was found that different cells were stained by the two dyes, indicating that ciprofibrate treatment was targeting different cells. Secondly, the mode of action of the carcinogen cyproterone acetate (CPA) on hepatocyte growth was investigated. Previous work had investigated the effects of CPA on hepatocyte growth in male and female rats and had suggested differences in response between the sexes. In the present study female rats were treated with CPA, to assess whether differences in labelling indices were present compared to previous male results. Female F-344/NHsd rats, aged 14-15 weeks, were treated with CPA and then injected with BrdU at 22 hr, and rats were killed 2 hr later. Results confirmed that the female rats had a considerably higher labelling index (50%) compared to male rats (6%). This suggested that upregulation of gene expression in female rats was much higher, which might provide an exciting opportunity to identify sets of genes involved in carcinogenic responses. To investigate whether there was any overlap between genes induced by ciprofibrate and CPA treatment a preliminary study was designed where female rats were gavaged with CPA and then killed 3 hr later. Real-time PCR analysis of a small number of target genes showed no consistent changes in expression between the present CPA and previous ciprofibrate treatment results, suggesting largely different modes of action of these chemicals. The second chapter of this thesis relates to studies undertaken to gain insights into neurodegenerative disorders. Neurodegeneration is a gradual loss of structure or function of neurons, which may lead to neuronal death. Neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s occur as a result of neurodegenerative changes. Several studies have suggested that PPARs have critical roles in reducing the brain inflammation which in turn might have a significant effect on reducing the fundamental processes involved. Work was performed using a mouse model of dementia with lewy body disease (Psmc1fl/fl; CaMKIIα-Cre) to represent neurodegenerative disorder, and involved parallel, in vivo and in vitro investigations to determine whether the development of neurodegenerative diseases occurs at the same rate in vitro and in vivo i.e. a comparison of rapidity of pathogenicity progression was made. Astrocytes were used to track the development of disease, given that these play a key role in neurological disorders, using an immunohistochemistry approach. A PPAR-γ analogue was used to investigate the role of PPARs in reducing astrocytes proliferation. To optimise the validity of the results, four controls were used including an antagonist T0070907 which abolished the effect of rosiglitazone treatment alone. The results on the effect of PPAR-γ agonist and rosiglitazone, after a week of treatment, showed that the PPAR-γ agonist inhibited astrocytes activation in both the cortex and hippocampus of the mutant mice organotypic slice culture. The number of GFAP +ve astrocytes was significantly decreased in mutant mice with 100 µM rosiglitazone in both areas, whereas 50 µM rosiglitazone showed a decrease in the number of astrocytes in the cortex, but the effect was less in the region of the hippocampus. This finding suggests that PPs such as rosiglitazone may have potential uses as therapeutic drugs to inhibit neurodegeneration.

GPCRs are the largest family of proteins in the human genome and a target for huge numbers of therapeutic drugs. However, the role of skeletal muscle in the action of these drugs is unclear. Given the unique importance of GPCR signalling in terms of glucose and fatty acid turnover in other tissues, it would be anticipated that GPCR identified to influence metabolism in these tissues might well be expressed in skeletal muscle. This study investigated the expression of genes encoding GPCRs in skeletal muscle and in cultured preparations thereof. In particular, this study focussed on the expression and signalling of adenosine receptors, a2-adrenoceptor, P2Y receptors and CBI cannabinoid receptors and the impact of CBI receptor modulation upon insulin signalling in rat primary skeletal muscle cells. All experiments in this work looked at GPCR expression and their signalling; with either tissues or cultured cells from rats. These experiments included: 1. Transcriptional profiling of skeletal muscle tissue in Wistar rats for GPCRs and proteins in associated signalling pathways. 2. Signalling of GPCRs (adenosine, a2A-adrenoceptor, P2y) in rat primary skeletal muscle cells. 3. Cannabinoid signalling pathways and cross-talk with insulin signalling. 4. CBI cannabinoid receptor antagonist/inverse agonist/agonist treatment of rat primary skeletal muscle cells. Expression of example members of the three major G protein coupling GPCR families was observed in rat skeletal muscle tissue. mRNA encoding Gs- (A2Aadenosine receptor, P2-adrenoceptor), Gi- (AI adenosine receptor, (l2A-adrenoceptor), and Gq-coupled (P2Y 1. P2Y2 and P2Y6 receptors) receptors were detected using gene microarray (Agilent, all ranked <10220 out of 41090). QRT-PCR (Taqman) identified (l2A-adrenoceptor and CBI cannabinoid receptor mRNA expression at low level similar across myoblasts, myotubes and skeletal muscle tissue. Functional responses to example members of the three major G protein coupling families of GPCR were also observed in rat primary skeletal muscle preparations. First, treatment of myotubes with the non-selective adenosine receptor agonist NECA elicited increases in cAMP, which were inhibited in the presence of the A2Badenosine receptorselective antagonist, PSB603. In contrast, the A2A-selective agonist, CGS21680 failed to evoke a significant cAMP elevation in myotubes. Second, neither basal nor forskolinevoked elevation of cAMP was altered in the presence of the Ar-selective agonist, SENBA. Third, the (l2-adrenoceptor agonist UK14304 inhibited forskolin-evoked cAMP levels, however, rauwolscine did not prevent this effect. Treatment with UK14304 also increased phosphorylation of ERK1/2; these responses, however, were inhibited by rauwolscine. In addition, rauwolscine in the absence of other ligands also inhibited ERK phosphorylation. Fourth, ATP and UTP, P2Y receptor agonists, elevated intracellular calcium ion levels in myoblasts. Although expression of mRNA for CBI cannabinoid receptors was detected in myoblasts, myotubes and skeletal muscle tissue, forskolin-evoked elevation of cAMP was unaltered in the presence of the CBI receptor-selective agonist ACEA or the antagonist/inverse agonist rimonabant in cultured myotubes. AICAR-stimulated AMPactivated protein kinase activity was also unaltered by ACEA. However, treatment with ACEA increased activation ofERK1I2 and p38 mitogen-activated protein kinases; these responses were significantly inhibited by rimonabant. Insulin treatment of myotubes increased the activation (phosphorylation) of AKT/protein kinase B, glycogen synthase kinase 3(1 and ~, ERK1I2 and p38 MAP kinases; however, pre-treatment with ACEA for 24 hours failed to alter these responses. In conclusion, these studies indicate expression and functional responses to select members of the three major G protein coupling families of GPCR in rat skeletal muscle preparations. These findings also provided evidence for expression of functionally active CB) cannabinoid receptors in skeletal muscle. However, they fail to support previous reports suggesting an interaction between insulin and CB) receptor signalling in these cells. The impact of CB) receptor function in skeletal muscle should be the subject of further investigation.

β-Arrestins are essential adaptors for G protein-coupled receptor (GPCR) trafficking. Evolutionary ancestors of the β-arrestins – dubbed α-arrestins – are present in yeast/fungi and, similar to β-arrestins, recognise cargo proteins and mediate their intracellular trafficking. Mammalian α-arrestins include five largely uncharacterised arrestin domain-containing (ARRDC1-5) proteins that display a predicted arrestin structure; the current study focuses on human ARRDC2. Confocal microscopy of exogenous, fluorescent protein-tagged ARRDC2 in U2OS cells in combination with compartment-specific markers indicated that ARRDC2 is dynamically distributed throughout the plasma membrane and endocytic system, predominantly to late endosomes/lysosomes. Anti-ARRDC2 immunostaining in several primary cell lines broadly supported this conclusion. ARRDC2 contains two proline-rich (PPxY) motifs that in other α-arrestins have been reported to mediate interactions with WW domain-containing NEDD4 family E3 ubiquitin ligases. Coimmunoprecipitation indicated that ARRDC2 is able to interact with several NEDD4 E3s via its PPxY motifs, and confocal microscopy suggested that this interaction may influence the subcellular targeting of the ligases. Ubiquitination of ARRDC2 was detected by coimmunoprecipitation, although this modification was independent of ARRDC2 interaction with NEDD4 E3s. ARRDC2 colocalised with agonist-stimulated, internalised GPCRs (β2-adrenergic receptor (β2AR) and δ-opioid receptor (δOR)) and colocalisation analysis indicated that this involved compartmental redistribution of ARRDC2 to receptor-containing early/recycling endosomes, suggesting a specific effect. Interaction of ARRDC2 with δOR was detected using coimmunoprecipitation, and confocal analysis suggested that ARRDC2 may influence δOR and β2AR intracellular trafficking. ARRDC2 was also found to oligomerise with itself and the β-arrestins. Confocal microscopy showed that ARRDC2 overexpression can induce the redistribution of β-arrestin1 to ARRDC2-positive vesicles, and a punctate bimolecular fluorescence complementation (BiFC) signal was detected between ARRDC2 and β-arrestin2. From this, it is speculated that α-/β-arrestins may function cooperatively or competitively to mediate discrete GPCR sorting events in the endocytic pathway.

In the absence of telomerase, Saccharomyces cerevisiae telomeres erode leading to senescence. Rare cells can survive after this stage as they can elongate their telomeres utilizing homologous recombination. Two different types of survivors can be easily distinguished by Southern blot. Type I survivor cells, elongate the telomere by amplifying Y elements and require RAD51, RAD54, RAD55 and RAD57 for establishment. Type II survivors elongate their telomere by amplifying TG1-3 repeats, however, they require the following genes to be established: RAD50, MRE11 and XRS2, RAD59, SGS1 and KU80 in some cases. Both types require the gene RAD52. In this study several candidate genes were deleted individually in diploid type II survivor strains. The main aim of this work was to see if these genes were required for type II telomere maintenance. Most of these genes are not required for type II telomere maintenance at least until ~150 generations after deleting these genes. The exceptions were KU80 and RPB9. Ku80Δ strains switched to a new survivor type that is similar to type I and continued for the long term. RPB9 was required for two independent type II survivor strains to survive, whereas the third type II strain did not require this gene at ~150 generations after deleting the gene. After many generations (~ 350), this strain switched to type I. At long term propagation (~500 generations) after deletion of the candidate genes, all type II strains displayed telomere shortening until the propagation was stopped. However, Rad50Δ strains switched to type I after long term. Finally, the absence of the candidate genes did not affect the sensitivity of type II survivor strains to temperature. On the other hand, type II survivor strains with some genes deleted displayed sensitivity to UV.