No presente estudo, a diversidade bacteriana endofítica fixadora de nitrogênio foi pesquisada utilizando métodos microbiológicos e moleculares. Isolaram-se microrganismos de raiz, caule e folha de cana-de-açúcar. Análises por seqüenciamento do 16S rDNA identificaram 150 endófitos. No total, foram identificados 18 gêneros. Destes, apenas 4 estavam presentes em cana-de-açúcar submetida aos 3 tratamentos. A maior diversidade de gêneros foi encontrada em cana sob adubação orgânica: 10 gêneros, em cana sob adubação inorgânica foram 11 gêneros e 8 em cana sem adubação. A maior parte dos gêneros pertence à família Enterobacteriaceae, como Klebsiella, Pantoea e Enterobacter. A enzima endoglicanase foi produzida por 82% dos isolados de cana sob adubação orgânica, (54%) inorgânica e (48%) sem adubação. Quanto à atividade de pectinase: 42%, 60% e 36% foram apresentadas por isolados de cana orgânica, inorgânica e sem adubação, respectivamente. A capacidade de solubilizar fosfatos inorgânicos foi detectada em 76,6% dos isolados, sendo a maior capacidade de solubilização de fosfatos encontrada em bactérias isoladas de cana-de-açúcar sob adubação orgânica (71%), de cana submetida a adubação convencional (78%) e sem adubação (88%). Foram realizados estudos de colonização em plântulas de cana-de-açúcar com 4 endofitos geneticamente modificados (EGMs) capazes de expressar os genes gfp e dsred. A avaliação da colonização na cana pela microscopia de fluorescência, mostrou que os (EGMs) gfp e dsred colonizaram as raízes e caules das plantas inoculadas, sem causar qualquer sintoma de doença. / In the present study, endophytic bacterial diversity has been searched using both microbiologic and molecular methods. Microorganisms were isolated from sugarcane root, shoot and leaf. 150 isolates were identified by 16S rDNA sequencing. 18 genera were found and only 4 were present in sugarcane submitted to the three treatments. The greatest genera diversity was found in sugarcane submited to organic fertlization: 10 genera in sugarcane submitted to inorganic fertilization were found 11 genera and 8 genera in sugarcane without fertilization. Great part of the found genera belongs to the Enterobacteriaceae family: Klebsiella, Pantoea and Enterobacter. Some physiological characteristics were determined in the isolates. Endoglucanase was produced by 82% of the isolates from sugarcane submitted to organic fertilization. Lower activities were found in bacteria isolated from inorganic fertilization and no fertilization, respectively 54% and 48%. As far as pectinase activity is concerned, a percentage of 42%, 60% and 36% was presented by the isolates from organic fertilization, inorganic fertilization and no fertilization, respectively. The hability of phosphate solubilization was detected in 76.6% of the isolates. In sugarcane under organic fertilization a percentage of 71% was found, in bacteria from inorganic fertilization, 78%, and without fertilization, 88%. Plant colonization was determined using sugarcane plantlets inoculated with four genetically modified bactéria (GMEs), able to express the genes gfp and dsred. The colonization was evaluated by fluorescence microscopy, which showed that the endophytic bacteria expressing gfp and dsred genes had invaded roots and shoots from inoculated plants, without causing any disease symptom.

Os microrganismos endofíticos são aqueles, cultiváveis ou não, que habitam o interior da planta hospedeira sem causar danos aparentes ou estruturas externas visíveis. Essa interação microrganismos-planta é intrínseca a determinadas espécies de plantas e/ou bactérias. Nas últimas décadas os estudos de microrganismos endofíticos têm sido realizados em diversas plantas hospedeiras, sendo esses estudos direcionados principalmente para a diversidade e características benéficas induzidas, inclusive o controle biológico de doenças. A doença causada pelo fungo Ceratocystis fimbriata é considerada emergente no setor florestal. O Brasil está entre os maiores produtores mundiais de eucalipto e a expansão do setor juntamente com o cultivo clonal tem acarretado o aumento da incidência de patógenos. O surgimento de novas doenças exige estudos relacionados tanto a interação do agente patogênico com hospedeiro quanto de todos os componentes do patossistema. Neste contexto, os microrganismos endofíticos têm sido descritos como potenciais controladores biológicos de doenças. Dessa forma, o presente trabalho teve por objetivos avaliar a interação de C. fimbriata com a comunidade bacteriana associada à Eucalyptus sp. Adicionalmente, foi estudada a possível transferência desses endófitos via sementes e o padrão de colonização de Pantoea agglomerans em plântulas. Foi observado que plantas não infestadas por C. fimbriata apresentaram maior densidade bacteriana no rizoplano (20,66 x 104 UFC.cm2 -1 de raiz), enquanto que para a comunidade endofítica, a maior densidade foi observada em plantas infectadas pelo fungo (25,13 x 104 UFC.g-1 de raiz). As análises por ARDRA possibilitaram a obtenção de 8 e 13 ribotipos nas comunidades endofítica de raiz e do rizoplano, respectivamente. Os ribotipos mais freqüentes foram identificados como Bacillus cereus. As análises de diversidade por meio de DGGE das comunidades do rizoplano e endofítica de raiz mostraram que a infestação pelo fungo interfere na colonização de Eucalyptus. Foi observado também que bactérias endofíticas estão presentes no interior de sementes de Eucalyptus spp. em uma densidade de 0,33 a 1,83 X 102 UFC.g-1, para as espécies E. camandulensis e E. urophylla, respectivamente. A densidade bacteriana endofítica de plântulas obtidas de sementes desinfectadas superficialmente variaram entre 0,27 X 102 a 0,87 X 102 UFC.g-1, para E. citriodora e o híbrido E. robusta x E. grandis, respectivamente. Em algumas espécies de Eucalyptus não foram isoladas bactérias endofíticas das sementes e plântulas. Os resultados mostraram que algumas espécies de bactérias endofíticas podem ser transmitidas verticalmente por sementes. P. agglomerans inoculada nas sementes foi capaz de colonizar as plântulas após a germinação da semente, indicando que esta pode ser uma das formas utilizadas pelos microrganismos para colonizar e se estabelecer na planta hospedeira. Assim, os resultados obtidos neste trabalho mostram ainda que possa existir interação entre a presença de C. fimbriata e a comunidade bacteriana endofítica e do rizoplano de Eucalyptus. Foi possível observar também que estas bactérias endofíticas que são transmitidas por meio de sementes, permitindo que plântulas previamente inoculadas com bactérias benéficas possam ser produzidas antes de serem levadas a campo. / The endophytic microorganisms are those, cultivated or not, that inhabit the interior of the plant host without causing apparent damages or visible external structures. This interaction microorganisms-plant is specific to certain species of plants and/or bacteria. In the last few years studies of endophytic microorganisms have been carried out in several plant hosts, being these studies focused mainly to diversity and biotechnological potential, such as biological control of disease. The disease caused by the phytopathogenic fungi Ceratocystis fimbriata is considered emerging by the reforestation companies. Brazil is one of the largest world eucalyptus producers and the increasing of the eucalyptus production associated to clonal reproduction has allowed the increase in pathogen incidence. Studies that evaluate the interaction between pathogens and the microbial community associated to the host plant may allow understanding how disease symptoms come up. Endophytic microorganisms have been described as potential biological control of diseases and therefore, the aims of the present work were to i) study the interaction between C. fimbriata and the bacterial community associated to the Eucalyptus sp.; ii) evaluate the bacterial dissemination by seeds; iii) evaluate the colonization profile of Pantoea agglomerans in seedlings after seed inoculation. It was observed that the highest bacterial density on the rhizoplane (20.66 x 104 CFU.cm2 -1 of root) was observed in C. fimbriata uninfectedplants, while for endophytic community the highest density was observed in C. fimbriata infected plants (25.13 x 104 CFU.g-1 of root). The ARDRA analyses showed that the bacterial community of eucalyptus is composed by 8 and 13 ribotypes on rhizoplane and inside the roots (endophytic), respectively. The most frequent ribotypes were identified as Bacillus cereus. The DGGE analyses of diversity of endophytic and rhizoplane community showed that fungi infection shift the colonization of Eucalyptus associated bacteria. The bacterial community inside Eucalyptus spp. seeds ranged from 0.33 to 1.83 X 102 CFU.g-1, for E. camandulensis and E. urophylla, respectively. After seed germination the endophytic bacterial density in seedlings ranged from 0,27 X 102 to 0,87 X 102 CFU.g-1, for E. citriodora and the hybrid E. robusta x E. grandis, respectively. Although, endophytic bacteria have been isolated from seeds, for some plant species, bacteria were not isolated from seedlings. Also, some bacteria may be vertically transmitted from seed to seedlings, but some is specific for seeds. Seed inoculation of P. agglomerans resulted in seedlings colonized by these bacteria, suggesting that these bacteria could be seed transmitted. The results obtained in the present study show that the fungi C. fimbriata inside the Eucalyptus host can shift the endophytic and rhizoplane bacterial diversity. Also, these endophytic bacteria could be transmitted vertically by seeds, allowing that seeds previously inoculated with beneficial bacteria may result in protected plants before planting in the field.

Single-molecule localisation microscopy (SMLM), has allowed for optical microscopy to probe biological systems beyond the diffraction limit. The intrinsic 3D nature of biology has motivated the development of 3D-SMLM with novel techniques, including the double-helix point spread function (DHPSF). A bespoke microscope platform employing the DHPSF transformation was built, achieving ~10 nm lateral and ~20 nm axial localisation precision over a ~4 μm axial depth. Until recently, the DHPSF has been limited by spherical aberration present when imaging away from coverslip surfaces to the study of small volumes close to the coverslip. By matching the refractive index of the objective lens immersion liquid to that of the imaging media, this aberration can be minimised, facilitating large-volume imaging away from unphysiological flat surfaces. The work presented in this thesis illustrates the capabilities of the DHPSF for 3D-SMLM and single-particle tracking (SPT) in previously inaccessible areas of biological samples (e.g. in the nucleus and on the apical cell surface). Application of the DHPSF for SPT in eukaryotic cells are presented; tracking the motion of T-cell membrane proteins on the apical surface and components of the chromosome remodelling complex in the nucleus of embryonic stem cells. For these applications, meansquared displacement and jump distance diffusion analysis methodologies were extended into 3D and benchmarked against simulated datasets. A variety imaging applications that are facilitated by the extended depth of focus of the DHPSF are presented, focusing on quantification of T-cell membrane protein reorganisation upon immunological activation. Finally, the clustering distribution of the T-cell receptor is investigated by Ripley’s K analysis enabled by duel labelling of its position and the outer membrane in primary T cells.

Ca2+ is a universal intracellular messenger that regulates many cellular responses. Most cells express inositol 1,4,5-trisphosphate receptors (IP3R) that mediate Ca2+ release from the endoplasmic reticulum (ER) when they bind IP3 produced after activation of cell-surface receptors. Vertebrate genomes encode three closely related subtypes of IP3R (IP3R1-3). High-resolution optical analyses have revealed a hierarchy of IP3-evoked Ca2+ signals that are thought to arise from the co-regulation of IP3Rs by IP3 and Ca2+. The smallest events ('blips') report the opening of single IP3Rs, Ca2+ 'puffs' report the almost simultaneous opening of a few clustered IP3Rs, and as stimulus intensities increase further Ca2+ signals propagate regeneratively as Ca2+ waves. The aim of this study was to establish whether all three IP3R subtypes can generate Ca2+ puffs. I first used a haploid cell line (HAP1 cells) to generate, using CRISPR/Cas9, a line lacking all endogenous IP3Rs. However, for analyses of Ca2+ puffs, I used HEK cells that had been engineered, using CRISPR/Cas9 to disrupt endogenous genes, to express single IP3R subtypes. Local Ca2+ signals evoked by flash-photolysis of caged- IP3 were recorded using Cal520 and total internal reflection fluorescence (TIRF) microscopy in human embryonic kidney (HEK) cells. The Flika algorithm was used, and validated, for automated detection of Ca2+ puffs and to measure their properties. IP3 evoked Ca2+ puffs in wild-type HEK cells and in cells expressing single IP3R subtypes. In wild-type cells, the Ca2+ signals invariably propagated regeneratively to give global increases in cytosolic [Ca2+]. This occurred less frequently in cells expressing single IP3R subtypes, commensurate with their lower overall levels of IP3R expression. The properties of the Ca2+ puffs, including their rise and decay times, durations, the size of the unitary fluorescence steps as channels closed channel during the falling phase, and the estimated number of active IP3Rs in each Ca2+ puff, were broadly similar in each of the four cell lines. The latter observation suggests that despite lower overall levels of IP3R expression (~30%) in cells with single subtypes relative to WT cells, there is a mechanism that ensures formation of similarly sized IP3R clusters. The only significant differences between cell lines were the slower kinetics of the Ca2+ puffs evoked by IP3R2, which may suggest dissociation of IP3 from its receptor contributes to the termination of Ca2+ puffs. My results demonstrate, for the first time, that all three IP3R subtypes can generate Ca2+ puffs. I conclude that Ca2+ puffs are fundamental building blocks of all IP3-evoked Ca2+ signals.

Des techniques variées de visualisation de molécules d’intérêt sur cellules vivantes ou fixées ont permis de suivre leur synthèse, localisation, dégradation et autres activités. Dans cette étude, nous avons développé deux outils de fluorescence pour étudier la synthèse des protéines sur bactéries vivantes. Le premier décrit l’utilisation du système Spinach pour l’imagerie du ribosome. Cette approche diffère des méthodes conventionnelles qui utilisent des protéines fluorescentes puisque l’ARN ribosomal 16S contient un aptamère qui rend fluorescent un composé fluorogène. Une étude comparative de la performance de différents aptamères Spinach a été réalisée. Un deuxième outil se focalise sur l’accumulation d’un antibiotique de la famille des aminoglycosides (ligand du ribosome) conjugué à un fluorophore. Ce nouveau conjugué, qui a conservé son activité bactéricide permet pour la première fois de visualiser l’accumulation de l’antibiotique sur bactérie vivante. Cela permet une analyse au niveau de la cellule unique d’une population bactérienne exposée à l’antibiotique. Nous avons également obtenu des données sur la localisation de l’antibiotique une fois qu’il a pénétré dans la bactérie à une résolution inégalée par microscopie super-résolutive. Nous espérons que ces deux méthodes vont maintenant permettre une meilleure compréhension de la synthèse des protéines et fournir une vue nouvelle de la pénétration des antibiotiques dans les bactéries pour y produire leur action bactéricide. / Various visualizing techniques have previously enabled monitoring the fate of molecules of interest: their expression, localization, degradation and other activities in live or fixed cells. In this study, we have developed two fluorescent tools to study protein synthesis in live bacterial cell. The first one describes the application of Spinach system to ribosomes imaging. This is different from conventional methods (that use fluorescent proteins) in that 16S rRNA contains an inserted RNA aptamer that elicits fluorescence of a fluorogenic compound. A comparative study of the performance of different Spinach aptamers was performed here. A second system focuses on the uptake of a fluorescently labeled ligand of the ribosome, an antibiotic of the class of aminoglycosides. This novel conjugate, which kept its bactericidal activity allows for the first time imaging of aminoglycoside uptake on live bacteria. This opened the door to a single cell analysis of bacterial cell populations. We also obtained data about the localization of the antibiotic once inside the bacteria to an unprecedented resolution using super resolution microscopy. We hope that both of these methods will contribute to a better understanding of protein synthesis as well as provide a novel view on the way antibiotics penetrate into cells and perform their bactericidal action.

Fluorescence microscopy is a powerful imaging technique used in the biological sciences to identify labeled components of a sample with specificity. This is usually accomplished through labeling with fluorescent dyes, isolating these dyes by their spectral signatures with optical filters, and recording the intensity of the fluorescent response. Although these techniques are widely used, fluorescence intensity images can be negatively affected by a variety of factors that impact the fluorescence intensity. Fluorescence lifetime imaging microscopy (FLIM) is an imaging technique that is relatively immune to intensity fluctuations and also provides the unique ability to directly monitor the microenvironment surrounding a fluorophore. Despite the benefits associated with FLIM, the applications to which it is applied are fairly limited due to long image acquisition times and high cost of traditional hardware. Recent advances in complementary metal-oxide-semiconductor (CMOS) single-photon avalanche diodes (SPADs) have enabled the design of low-cost imaging arrays that are capable of recording lifetime images with acquisition times greater than one order of magnitude faster than existing systems. However, these SPAD arrays have yet to realize the full potential of the technology due to limitations in their ability to handle the vast amount of data generated during the commonly used time-correlated single-photon counting (TCSPC) lifetime imaging technique. This thesis presents the design, implementation, characterization, and demonstration of a high speed FLIM imaging system. The components of this design include a CMOS imager chip in a standard 0.13 μm technology containing a custom CMOS SPAD, a 64-by-64 array of these SPADs, pixel control circuitry, independent time-to-digital converters (TDCs), a FLIM specific datapath, and high bandwidth output buffers. In addition to the CMOS imaging array, a complete system was designed and implemented using a printed circuit board (PCB) for capturing data from the imager, creating histograms for the photon arrival data using field-programmable gate arrays, and transferring the data to a computer using a cabled PCIe interface. Finally, software is used to communicate between the imaging system and a computer.The dark count rate of the SPAD was measured to be only 231 Hz at room temperature while maintaining a photon detection probability of up to 30\%. TDCs included on the array have a 62.5 ps resolution and a 64 ns range, which is suitable for measuring the lifetime of most biological fluorophores. Additionally, the on-chip datapath was designed to handle continuous data transfers at rates capable of supporting TCSPC-based lifetime imaging at 100 frames per second. The system level implementation also provides sufficient data throughput for transferring up to 750 frames per second from the imaging system to a computer. The lifetime imaging system was characterized using standard techniques for evaluating SPAD performance and an electrical delay signal for measuring the TDC performance. This thesis concludes with a demonstration of TCSPC-FLIM imaging at 100 frames per second -- the fastest 64-by-64 TCSPC FLIM that has been demonstrated. This system overcomes some of the limitations of existing FLIM systems and has the potential to enable new application domains in dynamic FLIM imaging.

Chromosomal replication is a source of spontaneous DNA double-strand breaks (DSBs). In E. coli, DSBs are repaired by homologous recombination using an undamaged sister template. During repair, the RecA protein polymerizes on single-stranded DNA generated at the site of the DSB and catalyses the search for sequence homologies on the undamaged sister template. This study utilized fluorescence microscopy to investigate the spatial and temporal dynamics of the RecA protein at the site of a replication-dependent DSB generated at the lacZ locus of the E. coli chromosome. The DSB was generated by SbcCD-mediated cleavage of a hairpin DNA structure formed on the lagging strand template of the replication fork by a long palindromic sequence. The tandem insertion of a recA-mCherry gene with the endogenous recA gene at the natural chromosomal locus produced no detectable effect on cell viability in the presence of DSB formation. During repair, the fluorescently-labelled RecA protein formed a transient focus, which was inferred to be the RecA nucleoprotein filament at the site of the replication-dependent DSB. The duration of the RecA focus at the site of the DSB was modestly reduced in a ΔdinI mutant and modestly increased in a ΔuvrD or ΔrecX mutant. Most cells underwent a period of extended cohesion of the sister lacZ loci after disappearance of the RecA focus. Segregation of the sister lacZ loci was followed by cell division, with each daughter cell obtaining a copy of the fluorescently-labelled lacZ locus. The RecA focus at the site of the DSB was observed predominantly between the mid-cell and the 1⁄4 position. In the absence of DSB formation, the lacZ locus exhibited dynamic movement between the mid-cell and the 1⁄4 position until the onset of segregation. Formation of the DSB and initiation of repair occurred at the spatial localization for replication of the lacZ locus while the downstream repair events occurred very close to the mid-cell. Genomic analysis of RecA-DNA interactions by ChIP-seq was used to demonstrate that the RecA focus at the lacZ locus was generated by the repair of the palindrome-induced DSB and not the repair of one-ended DSBs emanating from stalled replication forks at the repressor-bound operator arrays. This study has shown that the repair of a replication-dependent DSB occurs exclusively during the period of cohesion of the sister loci and the repair is efficiently completed prior to segregation of the two sister loci.

Les cellules vivantes sont capables de réagir aux signaux mécaniques tels que la rigidité de la surface sur laquelle elles adhèrent, les forces de tractions ou compressions auxquelles elles sont soumises, le flux de liquide à la surface de leur membrane ou encore la géométrie de leurs adhésions ou de leur forme globale. Ces signaux influent sur des processus cellulaires tels que la prolifération, la différenciation, la migration et la mort cellulaire. Ces processus sont finement régulés par des réactions biochimiques qui forment un réseau de signalisation. La mécanotransduction est la traduction du signal mécanique en signal biochimique.C’est dans le but d’étudier la mécanotransduction que nous avons étudié l’utilisation d’ultrasons pour stimuler mécaniquement les cellules à des fréquences temporelles et spatiales relativement élevées. De nombreux montages expérimentaux et de nombreuses voies ont été considérées dans cette partie très exploratoire. Nous en retenons finalement des pistes prometteuses pour la continuation future de ce projet.Nous avons développé ce que nous nommons des substrats actifs, qui nous permettent de contrôler à la fois spatialement et temporellement la stimulation mécanique appliquée à des cellules vivantes. Ces substrats actifs consistent en des micropiliers de fer incrustés dans un élastomère peu rigide (PDMS) et manipulés par deux électroaimants. Nous pouvons contrôler dynamiquement le déplacement des piliers qui vont déformer localement et de manière continue la surface. Cette déformation va ensuite déformer en traction ou en compression les cellules vivantes étalées sur la surface à proximité. En employant des marqueurs fluorescents nous pouvons réaliser de la Microscopie de Forces de Traction et surveiller la contrainte appliquée par les piliers aux cellules à travers la surface de PDMS, et nous pouvons étudier la réponse mécanique des cellules. De plus, ces substrats sont compatibles avec la microscopie de fluorescence en cellule vivante, ce qui rend possible l’observation de la réponse cellulaire au niveau morphologique (forme des adhésions focales, activité protrusive, …) et surtout biochimique.En effet, pour étudier la réponse biochimique des cellules après une stimulation mécanique, nous observons par microscopie de fluorescence des biosenseurs portant des paires de fluorophores donneur/accepteur. Ces biosenseurs nous permettent d’observer l’activité de protéines impliquées dans la signalisation cellulaire en calculant l’efficacité de Transfert d’Énergie Résonnant de Förster (FRET) de ces biosenseurs. Pour ce faire, les échantillons sont illuminés alternativement aux longueurs d’ondes d’excitation des fluorophores donneurs puis accepteurs. Le signal de fluorescence est collecté simultanément dans un canal d’émission du donneur et un canal d’émission de l’accepteur. Une grande partie de ma thèse a été consacrée à la mise au point d’une méthode quantitative pour analyser les images de fluorescence afin de mesurer une efficacité de FRET qui ne dépende pas de facteurs expérimentaux ni de la quantité de biosenseurs présents dans les cellules. Nous évaluons alors les différentes méthodes pour déterminer les facteurs de correction répandus corrigeant le débordement de spectre du donneur dans le canal accepteur et l’excitation directe de l’accepteur à la longueur d’onde d’excitation du donneur. Pour obtenir des mesures plus quantitatives, nous avons mis au point une nouvelles méthode pour déterminer 2 facteurs de correction supplémentaires. Nous comparons cette méthode à la seule préexistante et évaluons l’influence des paramètres de traitement des images sur les valeurs d’efficacité de FRET mesurées. / Living cells can react to mechanical signals such as the rigidity of the surface they adhere on, the traction or compression forces applied on them, the liquid flow at their membrane surface or the geometry of their adhesions or of their overall shape. Those signals influence cellular processes such as proliferation, differentiation, migration or cell death. Those processes are tightly regulated by biochemical reactions that constitute a signaling network. Mechanotransduction is the translation of the mechanical signal into the biochemical one.In order to study mechanotransduction, we have considered the use of ultrasounds to mechanically stimulate cells at relatively high temporal and spatial frequencies. Numerous setups and options have been considered in this very exploratory project. Finally, we will retain some promising leads for the continuation of this project.We have developed what we call active substrates that allows us to control both spatially and temporally the mechanical stimulation on living cells. Those active substrates consist of iron micropillars embedded in a soft elastomer and actuated by 2 electromagnets. We can control dynamically the displacement of the pillar that will deform locally and continuously the surface. This deformation will then deform in traction or in compression the living cells spread on the surface nearby. Thanks to fluorescent trackers we can perform Traction Force Microscopy and monitor the stress applied by the pillars to the cells through the PDMS surface, and we can look at the mechanical response of the cells. Moreover, those substrates are compatible with live cell fluorescence microscopy, which makes possible the observation of the cellular response at the morphological level (focal adhesions, protrusive activity, …) and most importantly at the biochemical level.Indeed, in order to study the cellular biochemical response after a mechanical stimulation, we use fluorescence microscopy to observe biosensors containing pairs of donor/acceptor fluorophores. Those biosensors allow us to monitor the activity of proteins implied in cellular signaling by computing the Förster Resonance Energy Transfer (FRET) efficiency of those biosensors. To do so, samples are alternatively excited at donor and acceptor excitation wavelengths. The fluorescence signal is then simultaneously measured in donor and acceptor emission channels. A substantial part of my thesis has been dedicated to the development of a quantitative method to analyze fluorescence images in order to measure FRET efficiencies that do not depend on experimental factors or biosensors concentration in cells. We assess different methods to compute standard correction factors that account for spectral bleed-through and direct excitation of acceptors at donor excitation wavelength. To obtain more quantitative measurements, we have developed a new method to compute 2 additional correction factors. We compare this method with the only one preexisting, and we assess the influence of image processing parameters on FRET efficiency values.

Current single molecule localisation microscopy methods allow for multicolour imaging of macromolecules in cells, and for a degree quantification on molecule numbers in one colour. However, that has not yet been an attempt to develop tools capable of quantitative imaging with multiple colours in cells. This work addressed this challenge by designing linker peptides with chemospecific groups to allow attachment of activator and emitter dyes for STORM imaging, and a targeting module. The design ensured a stoichiometric ratio of targeting module to activator and emitter dyes. Peptides with HaloTag ligands attached were labelled with various activator and emitter pairs and used to label HaloTag fusions of S. pombe and mouse embryonic stem cells. These peptides were found to bind non-specifically to various areas of both cell types, and did not localise to HaloTag protein, whereas controls did. Another peptide was also labelled with activator-emitter pairs and attached to expressed anti-GFP and ant-mCherry nanobodies via native chemical ligation. The labelled anti-GFP nanobody was to demonstrate ensemble and single molecule imaging in S. pombe, as well as characterisation on single molecule surfaces in comparison to a conventional randomly labelled antibody. The stoichiometrically labelled nanobody had a more consistent number of photons detected per localisation, number of localisation per molecule and number of blinks per molecule, which implied that it could be more useful than randomly labelled nanobodies for counting experiments. It was also shown to be capable of specific laser activation for STORM imaging with both an Alexa405Cy5 and Cy3Cy5 pairs. These anti-GFP and anti-mCherry nanobodies and peptide linker are new tools for both counting and multicolour imaging in super-resolution, which could be widely applied to constructs that are already tagged with GFP or mCherry.

[pt] Defensinas de planta, componentes inatos do sistema imune das plantas, são peptídeos antifúngicos, catiônicos, com estrutura primária rica em cisteína. Evidência dada pela literatura demonstrou que trechos de esfingolipídios complexos na membrana dos fungos, contendo manosildiinositolfosforilceramida e glicosilceramida, são sítios de ligação seletivos para as defensinas de planta isoladas de Dahlia merckii e Raphanus sativus, respectivamente. Entretanto, desconhece-se se as defensinas de planta interagem direta ou indiretamente com alvos intracelulares dos fungos. A fim de identificar interações físicas e diretas do tipo proteína- proteína, um sistema de duplo-híbrido, em levedura, baseado no fator de transcrição GAL4, foi construído utilizando-se como isca, a defensina da planta Pisum sativum, Psd1 (Pisum sativum defensin 1). Proteínas alvos, capazes de interagirem com o peptídeo Psd1, foram detectadas através do rastreamento de uma biblioteca de cDNA do fungo Neurospora crassa. Do resultado deste rastreamento, nove dentre quinze candidatos, selecionados pelo método do duplo-híbrido, foram identificados como proteínas nucleares da N. crassa. Um clone, detectado com alta freqüência neste rastreamento, apresentou homologia de seqüência com a proteína ciclina F, relacionada com o controle do ciclo celular. O ensaio de co-purificação utilizando a proteína conjugada a glutationa S-transferase (GST) validou in vitro o resultado obtido pelo sistema duplohíbrido. Análise por microscopia de fluorescência da Psd1, conjugada a FITC, e, dos núcleos do fungo Fusarium solani, marcados com DAPI, demonstrou in vivo a co-localização da defensina de planta Psd1 com os núcleos do fungo. Para pesquisar o modo de ação da Psd1 ao nível do ciclo celular, utilizou-se o modelo multicelular da retina de ratos neonatais, em desenvolvimento. Neste modelo, a migração nuclear intercinética, correlacionada com as transições de fase de S para M do ciclo celular, foi observada na presença da Psd1. Verificouse que Psd1 impediu a migração nuclear em neuroblastos, parando o ciclo celular na transição de S para G2. Estes resultados revelaram modos de ação da defensina de planta Psd1 sobre a fisiologia nuclear. / [en] Plant defensins, innate components of the plant immune system, are cationic, antifungal peptides, with a cysteine- rich primary structure. Evidence from the literature demonstrated that fungus membrane patches containing complex sphingolipids, mannosyldiinositolphosphorylceramide and glucosylceramides, are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. However, whether the plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify direct physical protein-protein interactions, a GAL4-based yeast two-hybrid system was constructed, using the plant peptide, Pisum sativum defensin 1 (Psd1), as the bait protein. Target proteins, capable of interacting with the bait Psd1, were detected by screening a Neurospora crassa cDNA library. In this screening, nine out of fifteen two-hybrid candidates were identified as N. crassa nuclear proteins. One clone, detected with high frequency in the screening, presented sequence similarity to a N. crassa cyclin F, related to the cell cycle control. The GST pull- down co purification assay corroborated this two-hybrid result in vitro. Fluorescence microscopy analysis of FITC- conjugated Psd1 and DAPI-stained Fusarium solani nuclei demonstrated in vivo the co-localization of the plant peptide Psd1 and the fungus nuclei. We used the developing retina of neonatal rats as a multicellular model to study Psd1 mode of action at the cell cycle level. In this model, we observed in vivo the interkinetic nuclear migration, correlated to the transitions from S to M-phase of the cell cycle, in the presence of the Psd1 peptide. It was shown that Psd1 impaired nuclear migration of neuroblasts by arresting the cell cycle at the S to G2- phase transition. These results revealed modes of action of the plant defensin Psd1 upon the nuclear physiology.