MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial.

BACKGROUND: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. METHODS: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. RESULTS: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7⁻ CD45RA⁺/⁻) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1⁻CD160⁻TIM3⁻LAG3⁻2B4⁺/⁻). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. CONCLUSIONS: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies.

Regulation of horizontal gene transfer of the catabolic island ICEclc

ICEclc is a mobile genetic element found in two copies on the chromosome of the bacterium Pseudomonas knackmussii B13. ICEclc harbors genes encoding metabolic pathways for the degradation of chlorocatechols (CLC) and 2-aminophenol (2AP). At low frequencies, ICEclc excises from the chromosome, closes into a circular DNA molecule which can transfer to another bacterium via conjugation. Once in the recipient cell, ICEclc can reintegrate into the chromosome by site-specific recombination. This thesis aimed at identifying the regulatory network underlying the decisions for ICEclc horizontal transfer (HGT). The first chapter is an introduction on integrative and conjugative elements (ICEs) more in general, of which ICEclc is one example. In particular I emphasized the current knowledge of regulation and conjugation machineries of the different classes of ICE. In the second chapter, I describe a transcriptional analysis using microarrays and other experiments to understand expression of ICEclc in exponential and stationary phase. By overlaying transcriptomic profiles with Northern hybridizations and RT- PCR data, we established a transcription map for the entire core region of ICEclc, a region assumed to encode the ICE conjugation process. We also demonstrated how transcription of the ICEclc core is maximal in stationary phase, which correlates to expression of reporter genes fused to key ICEclc promoters. In the third chapter, I present a transcriptome analysis of ICEclc in a variety of different host species, in order to explore whether there are species-specific differences. In the fourth chapter, I focus on the role of a curious ICEclc-encoded TetR-type transcriptional repressor. We find that this gene, which we name mfsR, not only controls its own expression but that of a set of genes for a putative multi-drug efflux pump (mfsABC) as well. By using a combination of biochemical and molecular biology techniques, I could show that MfsR specifically binds to operator boxes in two ICEclc promoters (PmfsR and PmfsA), inhibiting the transcription of both the mfsR and mfsABC-orf38184 operons. Although we could not detect a clear phenotype of an mfsABC deletion, we discuss the implications of pump gene reorganizations in ICEclc and close relatives. In the fifth chapter, we find that mfsR not only controls its own expression and that of the mfsABC operon, but is also indirectly controlling ICEclc transfer. Using gene deletions, microarrays, transfer assays and microscopy-based reporter fusions, we demonstrate that mfsR actually controls a small operon of three regulatory genes. The last gene of this mfsR operon, orf17162, encodes a LysR-type activator that when deleted strongly impairs ICEclc transfer. Interestingly, deletion of mfsR leads to transfer competence in almost all cells, thereby overruling the bistability process in the wild-type. In the final sixth chapter, I discuss the relevance of the present thesis and the resulting perspectives for future studies.

Phase Contrast X-Ray Tomographic Microscopy for Biological and Materials Science Applications

The application of two approaches for high-throughput, high-resolution X-ray phase contrast tomographic imaging being used at the tomographic microscopy and coherent radiology experiments (TOMCAT) beamline of the SLS is discussed and illustrated. Differential phase contrast (DPC) imaging, using a grating interferometer and a phase-stepping technique, is integrated into the beamline environment at TOMCAT in terms of the fast acquisition and reconstruction of data and the availability to scan samples within an aqueous environment. A second phase contrast method is a modified transfer of intensity approach that can yield the 3D distribution of the decrement of the refractive index of a weakly absorbing object from a single tomographic dataset. The two methods are complementary to one another: the DPC method is characterised by a higher sensitivity and by moderate resolution with larger samples; the modified transfer of intensity approach is particularly suited for small specimens when high resolution (around 1 mu m) is required. Both are being applied to investigations in the biological and materials science fields.

Host and invader impact of transfer of the clc genomic island into Pseudomonas aeruginosa PAO1

Genomic islands, large potentially mobile regions of bacterial chromosomes, are a major contributor to bacteria evolution. Here, we investigated the fitness cost and phenotypic differences between the bacterium Pseudomonas aeruginosa PAO1 and a derivative carrying one integrated copy of the clc element, a 103-kb genomic island [and integrative and conjugative element (ICE)] originating in Pseudomonas sp. strain B13 and a close relative of genomic islands found in clinical and environmental isolates of P. aeruginosa. By using a combination of whole genome transcriptome profiling, phenotypic arrays, competition experiments, and biofilm formation studies, only few differences became apparent, such as reduced biofilm growth and fourfold stationary phase repression of genes involved in acetoin metabolism in PAO1 containing the clc element. In contrast, PAO1 carrying the clc element acquired the capacity to grow on 3-chlorobenzoate and 2-aminophenol as sole carbon and energy substrates. No fitness loss >1% was detectable in competition experiments between PAO1 and PAO1 carrying the clc element. The genes from the clc element were not silent in PAO1, and excision was observed, although transfer of clc from PAO1 to other recipient bacteria was reduced by two orders of magnitude. Our results indicate that newly acquired mobile DNA not necessarily invoke an important fitness cost on their host. Absence of immediate detriment to the host may have contributed to the wide distribution of genomic islands like clc in bacterial genomes

Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study.

Huntington's disease (HD) is a monogenic neurodegenerative disease that affects the efferent neurons of the striatum. The protracted evolution of the pathology over 15 to 20 years, after clinical onset in adulthood, underscores the potential of therapeutic tools that would aim at protecting striatal neurons. Proteins with neuroprotective effects in the adult brain have been identified, among them ciliary neurotrophic factor (CNTF), which protected striatal neurons in animal models of HD. Accordingly, we have carried out a phase I study evaluating the safety of intracerebral administration of this protein in subjects with HD, using a device formed by a semipermeable membrane encapsulating a BHK cell line engineered to synthesize CNTF. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle; the capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed; however, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique.

Simultaneous determination of antidementia drugs in human plasma: Procedure transfer from HPLC-MS to UPLC-MS/MS.

A previously developed high performance liquid chromatography mass spectrometry (HPLC-MS) procedure for the simultaneous determination of antidementia drugs, including donepezil, galantamine, memantine, rivastigmine and its metabolite NAP 226-90, was transferred to an ultra performance liquid chromatography system coupled to a tandem mass spectrometer (UPLC-MS/MS). The drugs and their internal standards ([(2)H(7)]-donepezil, [(13)C,(2)H(3)]-galantamine, [(13)C(2),(2)H(6)]-memantine, [(2)H(6)]-rivastigmine) were extracted from 250μL human plasma by protein precipitation with acetonitrile. Chromatographic separation was achieved on a reverse phase column (BEH C18 2.1mm×50mm; 1.7μm) with a gradient elution of an ammonium acetate buffer at pH 9.3 and acetonitrile at a flow rate of 0.4mL/min and an overall run time of 4.5min. The analytes were detected on a tandem quadrupole mass spectrometer operated in positive electrospray ionization mode, and quantification was performed using multiple reaction monitoring. The method was validated according to the recommendations of international guidelines over a calibration range of 1-300ng/mL for donepezil, galantamine and memantine, and 0.2-50ng/mL for rivastimgine and NAP 226-90. The trueness (86-108%), repeatability (0.8-8.3%), intermediate precision (2.3-10.9%) and selectivity of the method were found to be satisfactory. Matrix effects variability was inferior to 15% for the analytes and inferior to 5% after correction by internal standards. A method comparison was performed with patients' samples showing similar results between the HPLC-MS and UPLC-MS/MS procedures. Thus, this validated UPLC-MS/MS method allows to reduce the required amount of plasma, to use a simplified sample preparation, and to obtain a higher sensitivity and specificity with a much shortened run-time.

Marker-free phase nanoscopy

We introduce a microscopic method that determines quantitative optical properties beyond the optical diffraction limit and allows direct imaging of unstained living biological specimens. In established holographic microscopy, complex fields are measured using interferometric detection, allowing diffraction-limited phase measurements. Here, we show that non-invasive optical nanoscopy can achieve a lateral resolution of 90 nm by using a quasi-2 pi-holographic detection scheme and complex deconvolution. We record holograms from different illumination directions on the sample plane and observe subwavelength tomographic variations of the specimen. Nanoscale apertures serve to calibrate the tomographic reconstruction and to characterize the imaging system by means of the coherent transfer function. This gives rise to realistic inverse filtering and guarantees true complex field reconstruction. The observations are shown for nanoscopic porous cell frustule (diatoms), for the direct study of bacteria (Escherichia coil), and for a time-lapse approach to explore the dynamics of living dendritic spines (neurones).

Intralesional adenovirus-mediated interleukin-2 gene transfer for advanced solid cancers and melanoma.

Numerous preclinical and clinical studies have shown that interleukin-2 (IL-2) induces regression of metastatic tumors. We have conducted a phase I/II, multicenter, open-label, dose-escalating study to evaluate the safety, efficacy, and biological effects of repeated intratumoral injections of adenovirus-IL-2 (TG1024) in patients with advanced solid tumors and melanoma. Thirty five patients (twenty-five with metastatic melanoma and ten with other solid tumors) were treated in eight successive cohorts at dose levels ranging from 3 x 10(8) to 3 x 10(11) viral particles (vp). Intratumoral TG1024 injections in combination with dacarbazine (DTIC) were tested in metastatic melanoma in one cohort. No clinical responses were observed at doses below 3 x 10(11) vp. Six local objective responses were recorded in patients receiving 3 x 10(11) vp per treatment [five in metastatic melanoma and one in metastatic squamous cell carcinoma (SCC) of the skin], of which two were complete responses (CRs). Most of the common side effects were injection site reactions and flu-like syndrome. TG1024 dose intensification across cohorts resulted in increased serum IL-2 levels after the injection. Intratumoral TG1024 injection induced pronounced inflammation of the treated lesion, with predominant CD8(+), TIA+ lymphocytic infiltrate. Our results show that intratumoral injections of TG1024 are safe and well tolerated. The clinical activity of TG1024 observed in this study warrants further investigations.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas.

Genomic islands (GEI) comprise a recently recognized large family of potentially mobile DNA elements and play an important role in the rapid differentiation and adaptation of bacteria. Most importantly, GEIs have been implicated in the acquisition of virulence factors, antibiotic resistances or toxic compound metabolism. Despite detailed information on coding capacities of GEIs, little is known about the regulatory decisions in individual cells controlling GEI transfer. Here, we show how self-transfer of ICEclc, a GEI in Pseudomonas knackmussii B13 is controlled by a series of stochastic processes, the result of which is that only a few percent of cells in a population will excise ICEclc and launch transfer. Stochastic processes have been implicated before in producing bistable phenotypic transitions, such as sporulation and competence development, but never before in horizontal gene transfer (HGT). Bistability is instigated during stationary phase at the level of expression of an activator protein InrR that lays encoded on ICEclc, and then faithfully propagated to a bistable expression of the IntB13 integrase, the enzyme responsible for excision and integration of the ICEclc. Our results demonstrate how GEI of a very widespread family are likely to control their transfer rates. Furthermore, they help to explain why HGT is typically confined to few members within a population of cells. The finding that, despite apparent stochasticity, HGT rates can be modulated by external environmental conditions provides an explanation as to why selective conditions can promote DNA exchange.

An operon of three transcriptional regulators controls horizontal gene transfer of the integrative and conjugative element ICEclc in Pseudomonas knackmussii B13.

The integrative and conjugative element ICEclc is a mobile genetic element in Pseudomonas knackmussii B13, and an experimental model for a widely distributed group of elements in Proteobacteria. ICEclc is transferred from specialized transfer competent cells, which arise at a frequency of 3-5% in a population at stationary phase. Very little is known about the different factors that control the transfer frequency of this ICE family. Here we report the discovery of a three-gene operon encoded by ICEclc, which exerts global control on transfer initiation. The operon consists of three consecutive regulatory genes, encoding a TetR-type repressor MfsR, a MarR-type regulator and a LysR-type activator TciR. We show that MfsR autoregulates expression of the operon, whereas TciR is a global activator of ICEclc gene expression, but no clear role was yet found for MarR. Deletion of mfsR increases expression of tciR and marR, causing the proportion of transfer competent cells to reach almost 100% and transfer frequencies to approach 1 per donor. mfsR deletion also caused a two orders of magnitude loss in population viability, individual cell growth arrest and loss of ICEclc. This indicates that autoregulation is an important feature maintaining ICE transfer but avoiding fitness loss. Bioinformatic analysis showed that the mfsR-marR-tciR operon is unique for ICEclc and a few highly related ICE, whereas tciR orthologues occur more widely in a large variety of suspected ICE among Proteobacteria.

Regulation of bistable ICEclc transfer in pseudomonas knackmussii B13

L'élément génétique intégratif et conjugatif auto-transférable de 103 kb qui se trouve dans le génome de Pseudomonas knackmussii B13 (ICEc/c) confère la capacité de dégrader le 3-chlorobenzoate et le 2-aminophénol. L'élément ICE c/c peut être transféré par conjugaison de la souche B13 à diverses bêta- et gamma- protéobactéries. Seule une sous-population de 3 à 5% des cellules transfère l'élément, les cellules dites "compétentes pour le transfert". L'acquisition de la compétence pour le transfert est vraisemblablement la conséquence d'une régulation bistable, conduisant une partie des cellules au transfert de l'élément ICE c/c tandis que, dans les autres, l'élément reste quiescent et ne se transfère pas. À ce jour, les mécanismes et les acteurs moléculaires qui régulent l'activation bistable de l'élément sont restés inconnus. Mon travail de doctorat visait à identifier les éléments bistables du régulon de la compétence pour le transfert et d'analyser les fondements moléculaires de la bistabilité de l'élément ICE c/c chez P. knackmussii. Le premier chapitre introduit le thème du transfert génétique horizontal avec un accent particulier sur les éléments intégratifs et conjugatifs (ICE) et ICEcIc. L'état actuel des connaissances sur l'organisation génétique, la régulation, l'intégration et le transfert de différents modèles de ICEs est exposé en détail. En outre, je m'étends sur les phénomènes d'hétérogénéité et de bistabilité phénotyplques, qu'on peut distinguer dans une population isogénique dans des conditions de culture homogènes, et qui sont susceptibles de jouer un rôle dans le transfert de l'élément ICE c/c, dans la mesure où il ne s'active et n'est transférable que dans une très petite sous-population de cellules. Dans le chapitre 2, je présente une analyse globale des régions promotrices minimales des gènes appartenant au régulon de la compétence pour le transfert de l'élément ICE c/c. Nous avons étudié les caractéristiques d'expression des promoteurs et, s'ils s'avéraient bistables, leur activation dans le temps par comparaison avec le mutant lntB13. Pour ce faire, nous avons utilisé des fusions de promoteurs avec des gènes rapporteurs et testé l'expression bistable chez P. knackmussii par microscopie à épifluorescence. Pour six promoteurs présentant une expression bistable, nous avons employé de la microscopie temporelle pour déterminer la chronologie de leur expression par rapport à Pint et PinR. Parmi eux, nous avons identifié deux gènes exprimés précocement et trois gènes exprimés tardivement dans le processus d'acquisition de la compétence de transfert. Dans le chapitre 3, j'expose une analyse d'expression génétique pour l'un des groupes de gènes dont la transcription est la plus élevée dans la région conservée de ICE c/c, les gènes orf81655-orf68241 contenus dans une région de 14 kb. Nous montrons d'abord que cet opéron fait partie du même régulon bistable que intB13 et inrR et analysons les caractéristiques génétiques qui conduisent à une transcription élevée. Nous étudions les fonctions biologiques de ce groupe de gènes par des délétlons ciblées et montrons que certaines d'entre elles empêchent le transfert de l'élément. Nous approfondissons la caractérlsatlon de I'orf8l655 en construisant une fusion transcrlptionnelle avec le gène codant pour la protéine fluorescente verte (egfp) (en utilisant le système minl-Tn5). L'expression de Vorf81655 dans des cellules individuelles est comparée au signal mesuré par hybridation in situ en fluorescence (FISH) sur le ARN messager du gène. En utilisant FISH, des délétlons du promoteur et de l'analyse directe de transcription, nous avons localisé la région promotrice du groupe de gènes. En outre, nous avons utilisé des mutations dirigées pour comprendre la bistabilité de cette région promotrice, caractérisée par une transcription très élevée et une traduction lente de l'ARN messager.  Dans le chapitre 4, nous nous efforçons de comprendre comment la bistabilité est générée au sein du régulon te de l'élément ICE c/c. Pour ce faire, nous avons tenté de reconstituer une expression bistable, dans un hôte qui ne présente pas de bistabilité naturellement, à partir d'éléments génétiques individuels. L'hôte choisi est Pseudomonas putida dans lequel nous avons introduit une copie unique de Pint, PinR ou PaipA fusionnés à la egfp, construits qui permettent d'observer l'apparition de bistabilité. Nous avons ensuite construit différents assemblages de composants génétiques de l'élément ICE c/c, en nous concentrant sur la région parA-inrR. En effet, nous avons pu démontrer qu'une expression bistable apparaît dans P. putida grâce à ces éléments en l'absence de l'élément ICE c/c complet. À noter que la plupart des construits génétiques activent PaipA ou P|,,R, mais qu'un seul recrée la bistabilité de Pint, ce qui suggère que la région parA-inrR permet à la fois d'engendrer la bistabilité et d'opérer la transition entre les promoteurs précoces et les promoteurs tardifs du régulon de la bistabilité. Dans le chapitre 5, nous concluons sur une discussion de la pertinence de nos résultats et sur de futures perspectives de recherche. -- The 103-kb self-transmissible integrative and conjugative element (ICE) of Pseudomonas knackmussii B13 (ICEc/c) confers the capacity to degrade 3- chlorobenzoate and 2-aminophenol. ICEc/c can be conjugated from strain B13 to a variety of Beta- and Gammaproteobacteria. Interestingly, ICE c/c transfer is observed in a subpopulatlon of cells (3-5%) only, the so-called 'transfer competent' cells. The formation of transfer competence (tc) is thought to be the consequence of a 'bistable' decision, which forces those cells to follow the developmental path which leads to ICEc/c transfer, whereas in others ICE c/c remains silent and does not transfer. So far, the mechanisms and molecular partners generating this bistable transfer activation in cells of P. knackmussii B13 remain mostly unidentified. This thesis aimed at understanding the extent of the tc bistability regulon and to dissect the molecular basis of bistabillty formation of ICEc/c in P. knackmussii. The first chapter is a general Introduction on horizontal gene transfer (HGT) with particular emphasis on ICEs and ICE c/c. The emphasis is made on the current knowledge about the HGT gene organization, regulation and specific integration and transfer aspects of the different ICEs models. Furthermore, I focus on the phenomena of phenotypic heterogeneity and bistability (the property of two distinguishable phenotypes existing within an isogenic population under homogeneous conditions), which may play a particular role in ICEc/c behaviour, since ICE activation and transfer only occurs in a very small subpopulation of cells. In Chapter Two, I focus on a global analysis of the different core promoters that might belong to the ICEc/c tc pathway regulon. We studied both expression patterns of ICEc/c promoters and, once being identified as "bistable", their temporal activation compared to that of intB13. In order to do this, we used promoter reporter fusions and tested blstability expression in P. knackmussii using epifluorescence microscopy. For the 6 promoters that showed bistable expression, we used time-lapse microscopy to study the timing of promoter expression in comparison to that of P,,,t or PlnR. We could establish two "early" and 3 "late" phase promoters in the process of transfer competence. In Chapter Three, I focused my attention on analysis of gene expression of one of the most highly transcribed gene clusters in the conserved core region of ICEc/c, a 14-kb gene cluster formed by the genes orf81655-orf68241. First we showed that this operon is part of the same bistability 'regulon' as intB13 and inrR, and analysed the genetic features that lead to high transcription. We studied the potential biological function of this cluster for ICE c/c by making specific gene deletions, showing that some interrupt ICEc/c transfer. We further analysed the orfdl655 promoter by constructing transcriptional egfp fusion reporter strains using the miniTn5 delivery system. Expression of the orf81655 promoter in single cells was compared to signals measured by Fluorescence In Situ Hybridization (FISH) on orfSl655 mRNA. We localized the promoter region of the gene cluster using FISH, promoter deletions, and by direct transcript analysis. We further used site-directed mutagenesis to understand the bistability character of the promoter region and the extremely high transcription but low translation from this mRNA. In Chapter Four, we set out to understand how bistability is generated in the tc pathway of ICEc/c. For this we tried rebuilding bistable expression from ICEc/c individual gene components in a host, which normally does not display bistability. As host we used P. putida without ICEc/c but with a single copy Pint-, PlnR- or PalpA- egfp fusion that enabled us to verify bistability formation. Subsequently, we built different assemblages of ICEc/c gene components, focusing on the parA-inrR region. Indeed, we found that bistable expression can be build from those components in P. putida without ICEc/c. Interestingly, most genetic constructs activated PaipA or PlnR, but only one resulted in bistable activation of PinT. This suggests that the parA-inrR region acts as a bistability "generator", but also as a bistability "relay" from early to late promoters in the tc pathway hierarchy. In the final fifth chapter, we conclude with a discussion of the relevance of the present thesis and the resulting perspectives for future studies.

CNDOL: A fast and reliable method for the calculation of electronic properties of very large systems. Applications to retinal binding pocket in rhodopsin and gas phase porphine.

Very large molecular systems can be calculated with the so called CNDOL approximate Hamiltonians that have been developed by avoiding oversimplifications and only using a priori parameters and formulas from the simpler NDO methods. A new diagonal monoelectronic term named CNDOL/21 shows great consistency and easier SCF convergence when used together with an appropriate function for charge repulsion energies that is derived from traditional formulas. It is possible to obtain a priori molecular orbitals and electron excitation properties after the configuration interaction of single excited determinants with reliability, maintaining interpretative possibilities even being a simplified Hamiltonian. Tests with some unequivocal gas phase maxima of simple molecules (benzene, furfural, acetaldehyde, hexyl alcohol, methyl amine, 2,5 dimethyl 2,4 hexadiene, and ethyl sulfide) ratify the general quality of this approach in comparison with other methods. The calculation of large systems as porphine in gas phase and a model of the complete retinal binding pocket in rhodopsin with 622 basis functions on 280 atoms at the quantum mechanical level show reliability leading to a resulting first allowed transition in 483 nm, very similar to the known experimental value of 500 nm of "dark state." In this very important case, our model gives a central role in this excitation to a charge transfer from the neighboring Glu(-) counterion to the retinaldehyde polyene chain. Tests with gas phase maxima of some important molecules corroborate the reliability of CNDOL/2 Hamiltonians.