Demographic data indicate that 60% of 6.7 million annual global cancer mortalities and 54% of 10.8 million new patients are in developing nations, unable or unwilling to avail of invasive screening tests that are the current norm. For most cancers, survival rate is strongly dependent on early detection, highlighting the need for improved screening methods. Studies have shown that cancers can be identified based on distinct sub-cellular morphological features and expression levels of specific molecular markers. Since 85% of cancers are known to originate in the epithelium, portable in vivo imaging techniques providing sub-cellular detail in tissue up to depths of 250 μm could help improve access to biopsy-free examination in low-infrastructure environments. The resultant early detection could dramatically improve patient prognosis, while reducing screening costs, treatment delay, and occurrences of unnecessary and potentially harmful medication. This dissertation investigates handheld instrumentation for laser-scanning confocal microscopy (LSCM) and its applicability to breast cancer detection and subsequent image-guided management. LSCM allows high-resolution mapping of spatial variations in refractive index or tumor marker expression within a single cell layer situated few hundred micrometers beneath the tissue surface. The main challenge facing miniaturization lies in the mechanism of beam deflection across the sample. The first part of the dissertation presents a fast, large-angle, high-reflectivity two-axis vertical comb driven silicon micromirror fabricated by a novel method compatible with complementary metal-oxide-semiconductor processing employed in the semiconductor industry. The process enables integration of rotation sensors on the chip to adaptively correct for aberrations in beam scanning while significantly reducing fabrication costs and barriers to market acceptance. The second part of the dissertation explores the integration of this micromirror with other optical and electronic components into a handheld laser-scanning confocal microscope. Applicability of the probe to epithelial breast cancer screening via reflectance and fluorescence imaging is investigated. Finally, enhanced imaging modalities based on the micromirror are presented. 3D cellular-level in vivo imaging via rapid swept-source optical coherence tomography is demonstrated. A method for “objective-less” microendoscopy, potentially resulting in substantially reduced probe dimensions, employing reflective binary-phase Fresnel zone plates monolithically integrated on the surface of the micromirror is presented. / text

Previous research into microbial colonies and biofilms shows a significant gap in our current understanding of how bacterial structures develop. Despite the huge body of research undertaken into the formation, genetic makeup, composition, and optimal growth conditions of colonies, no study has been successful in identifying all individual bacteria in a colony in three-dimensions as a function of time. This lack of bacterial cell lineage in such a simple class of organisms is conspicuous in the light of what is known about other organisms, such as Caenorhabditis elegans [1]. In this thesis I show that using laser scanning confocal microscopy in conjunction with developments in sample preparation and post acquisition image analysis, it is possible to fully reconstruct all individual bacteria within an Escherichia coli (E. coli ) microcolony grown in viscoelastic media. Additionally, I show that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information on protein structures inside bacteria at early stages of growth. This thesis is in three parts. The first part shows that by pushing the resolution of a commercial laser scanning confocal microscope system it is possible to achieve single cell resolution of a bacterial colony growing in three dimensions in a viscoelastic medium (agarose) from a seed bacterium. The growth of individual bacteria is examined as the concentration of agarose in the media is altered. Results show there is a nonlinear dependence between the rate of growth of a bacterium and the concentration of the agarose in the media with a peak in growth rate at 3% (weight) concentrations of agarose in M9 media. The second part of this work presents a study of how an initially two-dimensional colony growing between a glass slide and agarose gel suddenly invades the third spatial dimension by buckling. The results show that the cells within the centre of the colony flex and buckle, due to confinement by their neighbours, creating additional layers. Indeed, flexing is not limited to the buckling event but occurs throughout the early growth cycle of a colony. The final part of this thesis shows that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information about the temporal evolution of the spatial distribution of the FtsZ septation ring within the cell. As the bacterial colony grows from a seed bacterium to a microcolony, the error in placing the division accurately at the cell centre is seen to increase as the number of bacteria within the colony increases and spatial confinement occurs.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18587号 / 工博第3948号 / 新制||工||1607(附属図書館) / 31487 / 京都大学大学院工学研究科機械理工学専攻 / (主査)教授 北條 正樹, 教授 北村 隆行, 教授 琵琶 志朗 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Fluorescence microscopy has played a pivotal role in the realm of biological and biomedical research, allowing researchers to delve into the intricacies of living organisms at the cellular and molecular levels. By using fluorescent probes, one can visualize specific molecules and structures within cells, fundamentally transforming our comprehension of biology and medicine. However, fluorescence microscopy faces its own set of challenges, namely, photobleaching and photodamage. Photobleaching involves the irreversible loss of fluorescence signal during imaging, while photodamage results in harmful effects on cells. Both severely limit fluorescence signal and observation time. Although remedies exist to mitigate these problems, most of them rely on chemical approaches. In this dissertation, to address these issues, I investigated two optical approaches that exploit control of light either in space or time. Firstly, I developed multiline scanning confocal microscopy (mLS) with a digital micro-mirror device. This method provides programmable patterns of the illumination beam as well as the detection slit. Through experimental results and optical simulations, I assessed the depth discrimination of mLS under different optical parameters and compared it with a multipoint system such as spinning disk confocal microscopy (SDCM). Surprisingly, under the same illumination duty cycle, I found that mLS offers better optical sectioning than SDCM. Importantly, the parallelized line illumination showed a much lower photobleaching rate compared to single-line scanning microscopy, while their optical sectioning capabilities remained similar. I applied this technique to visualize heterogeneous mouse epiblast stem cells, a challenging task in imaging. Secondly, I delved into low photobleaching rate two-photon microscopy (2PM). 2PM inherently provides excellent optical sectioning due to its nonlinear effects, making it suitable for high-resolution imaging within biological tissues. However, the high peak power of ultrafast pulses has always been associated with severe photobleaching, posing a longstanding challenge. I found that controlling the repetition rates of ultrafast lasers is a potential strategy to enhance photostability. Specifically, I used repetition rates lower or higher than 80 MHz in 2PM and conducted systematic experiments to investigate how optical parameters such as wavelengths, excitation powers, and pulse schemes can influence the photobleaching kinetics of fluorescent proteins and organic fluorophores. This thesis embarks on a journey to explore innovative strategies and methodologies aimed at reducing photobleaching while maintaining high-quality imaging in the realm of fluorescence microscopy.

Yes / Confocal microscopy has been increasingly employed in the field of traceology to acquire metrological data of surface changes on a micro-scale. However, its advantages for a traditional visual inspection of use-wear are rarely highlighted. As traditional optical microscopy (OM) has proven unable to entirely fulfil the prerequisites for an ideal observation of highly reflective and irregular materials, alternative ways for providing better observation conditions must be sought. In this contribution, we explore the combination of laser scanning confocal (LSCM) and scanning electron microscopy (SEM) micro-graphs for the visual characterisation of wear on quartzite and evaluate the potential of both techniques. / AHRC Fragmented Heritage project (AH/L00688X/1) at the University of Bradford, and of the MICINN-FEDER (PGC2018-093925-B-C32), the AGAUR (SGR 2017-1040) and the URV (2018PFR-URV-B2-91) projects at IPHES-URV. One of the authors (A.P.) was beneficiary of a Catalan pre-doctoral grant (2014FI B 00539), at the Rovira i Virgili University (URV), the IPHES and the Muséum national d’Histoire naturelle of Paris.

O objetivo deste estudo foi comparar os ensaios de microtração (MI), ?push-out? (PS), ?push-out? modificado (PSM) e ?pull-out? (PL) na habilidade de mensurar a resistência de união (RU) entre pino de fibra e dentina radicular. Para tanto, além dos resultados de cada ensaio mecânico foram analisados fatores como: sensibilidade quanto às diferentes regiões radiculares, quantidade de falhas prematuras (FP), coeficiente de variação (CV), padrão de fratura (PF) pela microscopia confocal de varredura e distribuição das tensões pela análise de elementos finitos (AEF). Foram utilizados 40 dentes anteriores superiores endodonticamente tratados, divididos em quatro grupos de acordo com o tipo de ensaio realizado. Para os ensaios de PS e MI foi feita a cimentação do pino e posteriormente os cortes para a obtenção dos corpos-de-prova (cps). Para os outros ensaios (PSM e PL), a cimentação antecedeu aos cortes tanto das fatias de dentina como dos pinos. O sistema adesivo Scotbond Multi Uso Plus e o cimento Variolink II foram usados para a cimentação do pino de fibra de vidro FRC Postec Plus em todos os casos. Os cps do ensaio de PS foram obtidos pelos cortes seriados da raiz em fatias de 1±0,1mm, para os cps do ensaio de MI estas fatias sofreram ainda a confecção de entalhes laterais diametralmente opostos, que proporcionando um formato final de ampulheta. Para os testes de PL e PSM foram feitos cortes de fatias de dentina de 1±0,1mm e o pino foi dividido em três partes iguais para, então, cada fatia de uma região ser cimentada com o pino correspondente. Os cps do ensaio de PSM tinham 1mm de pino sobressalente para cima e para baixo da fatia de dentina, enquanto que os cps do PL o pino sobressalente estava apenas para cima da dentina. Todos os cps foram submetidos aos testes de RU, à velocidade de 0,5mm/min em máquina de ensaios universais. O PF foi avaliado em um aumento de cem vezes e foi feita uma distribuição de freqüência. Já a AEF obteve modelos tridimensionais de todos os ensaios, e estes foram analisados quanto à tensão de Von Mises. Para os resultados dos testes mecânicos foram feitas análises de variância de cada ensaio variando o fator região, com ou sem as FP, e análise comparando todos os ensaios. O teste Tukey foi feito quando necessário e as FP foram submetidas ao teste estatístico de Fisher. A inclusão ou não destas falhas mostra alteração dos resultados no ensaio de MI, uma vez que possui um maior número delas neste ensaio. O ensaio de PL mostrou as maiores médias de RU, seguido pelo ensaio de PSM, e ambos também tiveram o PF com maior quantidade de fraturas adesivas. O ensaio de PS conseguiu apontar diferença estatística entre as três regiões, mas mostrou a maior freqüência de falhas coesivas, e maior CV. O menor CV foi encontrado para o PSM que também mostrou uma adequada distribuição das tensões. A MI apresentou concentração de tensões nas bordas dos entalhes. Concluiu-se que o tipo de ensaio influencia diretamente nos resultados obtidos. / The aim of this study was to compare microtensile (MI), \"push-out\" (PS), modified \"push-out\" (PSM) and \"pull-out\" (PL) tests in the ability to accurately measure the bond strength of fiber posts luted inside root canals, at different root dentin levels, cervical, middle and apical. The evaluated parameters were: amount of premature failure (PF), coefficient of variation (CV), failure patterns (FP) and the stress distribution by finite elements analysis (FEA). Forty human intact single-rooted and endodontically treated teeth were divided into four groups according to the type test used. For PS and MI specimens, first the post was luted and then the cuts were done, for PSM and PL, first was done the cuts and then fiber post?s pices were luted. Scotbond Multi Use Plus adhesive system and Variolink II cement had been used for luting FRC Postec Plus glass fiber post, in all cases. PS samples were disc shaped of 1±0,1mm. Bond strength between post and dentin was measured in the root slice and the force applied on the center of the specimen. For the Mi test hourglass-shaped samples were obtained by trimming the proximal surfaces of each slice using a diamond bur until it touched the post. For PL and PSM tests cuts of 1±0,1mm of dentine slices were made and the fiber was divided into three parts, and then each slice were luted with a fiber post?s piece. Specimens for PSM had 1mm of post up and down the dentin slice and for PL all of the post was up the dentine slice. All specimens were submitted to a bond strength test at 0.5 mm/minute crosshead speed in a universal testing machine. For FP, each sample was examined under confocal microscopy at 100 times and a frequency distribution was made. For the AEF, three-dimensional models of each tests had been made and analyzed by Von Mises stress. Bond strength data were analyzed by ANOVA and when necessary was used Tukey?s test. For PF data the Fisher test was made. The inclusion of PF, only shows variation on MI, since these test showed the greater number of them. PL provided higher values of bond strength for root dentin and fiber post, followed by PSM. Both of them had also FP with greater amount of adhesive failures. The PS test was able to point statistically differences between root regions, but it showed the highest frequency of cohesive failure, and greater CV. Minor CV was found for the PSM that also showed adequate stress distribution. The MI showed concentration of tensions in the edges of the trimming area. One concluded that the type of test influences directly in the results obtained.

The waterborne pathogen Batrachochytrium dendrobatidis (Bd), amphibian chytrid, is implicated as being the causative agent for global amphibian declines. The fungus attacks the keratinized skin of adult and postmetamorphic animals and the keratinized mouthparts of tadpoles. Postmetamorphic animals seem to be more susceptible to Bd than tadpoles and adult frogs. Hypotheses exist that the origin of the fungus is in Africa. During the study different aspects of Bd infections in South African frogs were examined including the distribution of Bd, cultivation of Bd, preservation of cultures, the morphology of Bd as an infection as well as in culture and finally differences in host defense. Positive and negative localities for Bd were identified through surveys conducted in South Africa. These data will be contributed to the Bd Mapping Project and the African Bd Database in order to determine whether chytrid has any environmental preferences. Cultures obtained from the positive localities were maintained and cryopreserved for use in numerous experiments. In a future study, DNA extractions from the cultures will be analyzed using multilocus sequence typing in order to determine the sequence type of South African strains in comparison with global strains. This will provide important epidemiological information concerning the origin and control of Bd. The morphology of Bd was also examined using scanning electron microscopy and laser scanning confocal microscopy. Damage due to Bd infections was more severe on the larval mouthparts of Amietia vertebralis than that of Hadromophryne natalensis. The adverse effect of Bd is therefore not limited to postmetamorphic animals. Confocal microscopy uses fluorescent stains and lasers to examine specific structures within organisms. An especially effective stain used during confocal microscopy on Bd is Calcofluor White M2R. Due to its specificity this stain can be used as an effective screening tool for Bd in tissue. The role of antimicrobial skin peptides as a defense against Bd was also examined. A. vertebralis experiences die-offs due to chytrid, while H. natalensis does not experience the same effect in the presence of Bd. H. natalensis possess more antimicrobial skin peptides against Bd with a higher effectiveness than peptides extracted from A. vertebralis. This may explain the observed susceptibility of A. vertebralis to Bd. The relevance of this study is in order to identify areas in South Africa in which the probability of finding Bd is high. This will help in the surveillance of Bd and in the identification of susceptible species to be monitored and protected against the fungus. The effect of Bd on frog species can also be determined by means of exposure experiment using cultures isolated during this study. Through the identification of peptides effective against Bd, predictions can be made with regard to the susceptibility of different frogs to Bd, improving our ability to protect the amphibian biodiversity in South Africa. With the use of confocal microscopy in the examination of Bd, we became the first group to use the method. By the identification of a stain with a high potential as a screening tool, we also contributed to the more efficient identification of Bd in tissue. Keywords: Batrachochytrium dendrobatidis, Bd, amphibian chytrid, distribution, cultivation, antimicrobial skin peptides, laser scanning confocal microscopy, Amietia vertebralis, Hadromophryne natalensis, South Africa / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

The waterborne pathogen Batrachochytrium dendrobatidis (Bd), amphibian chytrid, is implicated as being the causative agent for global amphibian declines. The fungus attacks the keratinized skin of adult and postmetamorphic animals and the keratinized mouthparts of tadpoles. Postmetamorphic animals seem to be more susceptible to Bd than tadpoles and adult frogs. Hypotheses exist that the origin of the fungus is in Africa. During the study different aspects of Bd infections in South African frogs were examined including the distribution of Bd, cultivation of Bd, preservation of cultures, the morphology of Bd as an infection as well as in culture and finally differences in host defense. Positive and negative localities for Bd were identified through surveys conducted in South Africa. These data will be contributed to the Bd Mapping Project and the African Bd Database in order to determine whether chytrid has any environmental preferences. Cultures obtained from the positive localities were maintained and cryopreserved for use in numerous experiments. In a future study, DNA extractions from the cultures will be analyzed using multilocus sequence typing in order to determine the sequence type of South African strains in comparison with global strains. This will provide important epidemiological information concerning the origin and control of Bd. The morphology of Bd was also examined using scanning electron microscopy and laser scanning confocal microscopy. Damage due to Bd infections was more severe on the larval mouthparts of Amietia vertebralis than that of Hadromophryne natalensis. The adverse effect of Bd is therefore not limited to postmetamorphic animals. Confocal microscopy uses fluorescent stains and lasers to examine specific structures within organisms. An especially effective stain used during confocal microscopy on Bd is Calcofluor White M2R. Due to its specificity this stain can be used as an effective screening tool for Bd in tissue. The role of antimicrobial skin peptides as a defense against Bd was also examined. A. vertebralis experiences die-offs due to chytrid, while H. natalensis does not experience the same effect in the presence of Bd. H. natalensis possess more antimicrobial skin peptides against Bd with a higher effectiveness than peptides extracted from A. vertebralis. This may explain the observed susceptibility of A. vertebralis to Bd. The relevance of this study is in order to identify areas in South Africa in which the probability of finding Bd is high. This will help in the surveillance of Bd and in the identification of susceptible species to be monitored and protected against the fungus. The effect of Bd on frog species can also be determined by means of exposure experiment using cultures isolated during this study. Through the identification of peptides effective against Bd, predictions can be made with regard to the susceptibility of different frogs to Bd, improving our ability to protect the amphibian biodiversity in South Africa. With the use of confocal microscopy in the examination of Bd, we became the first group to use the method. By the identification of a stain with a high potential as a screening tool, we also contributed to the more efficient identification of Bd in tissue. Keywords: Batrachochytrium dendrobatidis, Bd, amphibian chytrid, distribution, cultivation, antimicrobial skin peptides, laser scanning confocal microscopy, Amietia vertebralis, Hadromophryne natalensis, South Africa / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

O objetivo deste estudo foi comparar os ensaios de microtração (MI), ?push-out? (PS), ?push-out? modificado (PSM) e ?pull-out? (PL) na habilidade de mensurar a resistência de união (RU) entre pino de fibra e dentina radicular. Para tanto, além dos resultados de cada ensaio mecânico foram analisados fatores como: sensibilidade quanto às diferentes regiões radiculares, quantidade de falhas prematuras (FP), coeficiente de variação (CV), padrão de fratura (PF) pela microscopia confocal de varredura e distribuição das tensões pela análise de elementos finitos (AEF). Foram utilizados 40 dentes anteriores superiores endodonticamente tratados, divididos em quatro grupos de acordo com o tipo de ensaio realizado. Para os ensaios de PS e MI foi feita a cimentação do pino e posteriormente os cortes para a obtenção dos corpos-de-prova (cps). Para os outros ensaios (PSM e PL), a cimentação antecedeu aos cortes tanto das fatias de dentina como dos pinos. O sistema adesivo Scotbond Multi Uso Plus e o cimento Variolink II foram usados para a cimentação do pino de fibra de vidro FRC Postec Plus em todos os casos. Os cps do ensaio de PS foram obtidos pelos cortes seriados da raiz em fatias de 1±0,1mm, para os cps do ensaio de MI estas fatias sofreram ainda a confecção de entalhes laterais diametralmente opostos, que proporcionando um formato final de ampulheta. Para os testes de PL e PSM foram feitos cortes de fatias de dentina de 1±0,1mm e o pino foi dividido em três partes iguais para, então, cada fatia de uma região ser cimentada com o pino correspondente. Os cps do ensaio de PSM tinham 1mm de pino sobressalente para cima e para baixo da fatia de dentina, enquanto que os cps do PL o pino sobressalente estava apenas para cima da dentina. Todos os cps foram submetidos aos testes de RU, à velocidade de 0,5mm/min em máquina de ensaios universais. O PF foi avaliado em um aumento de cem vezes e foi feita uma distribuição de freqüência. Já a AEF obteve modelos tridimensionais de todos os ensaios, e estes foram analisados quanto à tensão de Von Mises. Para os resultados dos testes mecânicos foram feitas análises de variância de cada ensaio variando o fator região, com ou sem as FP, e análise comparando todos os ensaios. O teste Tukey foi feito quando necessário e as FP foram submetidas ao teste estatístico de Fisher. A inclusão ou não destas falhas mostra alteração dos resultados no ensaio de MI, uma vez que possui um maior número delas neste ensaio. O ensaio de PL mostrou as maiores médias de RU, seguido pelo ensaio de PSM, e ambos também tiveram o PF com maior quantidade de fraturas adesivas. O ensaio de PS conseguiu apontar diferença estatística entre as três regiões, mas mostrou a maior freqüência de falhas coesivas, e maior CV. O menor CV foi encontrado para o PSM que também mostrou uma adequada distribuição das tensões. A MI apresentou concentração de tensões nas bordas dos entalhes. Concluiu-se que o tipo de ensaio influencia diretamente nos resultados obtidos. / The aim of this study was to compare microtensile (MI), \"push-out\" (PS), modified \"push-out\" (PSM) and \"pull-out\" (PL) tests in the ability to accurately measure the bond strength of fiber posts luted inside root canals, at different root dentin levels, cervical, middle and apical. The evaluated parameters were: amount of premature failure (PF), coefficient of variation (CV), failure patterns (FP) and the stress distribution by finite elements analysis (FEA). Forty human intact single-rooted and endodontically treated teeth were divided into four groups according to the type test used. For PS and MI specimens, first the post was luted and then the cuts were done, for PSM and PL, first was done the cuts and then fiber post?s pices were luted. Scotbond Multi Use Plus adhesive system and Variolink II cement had been used for luting FRC Postec Plus glass fiber post, in all cases. PS samples were disc shaped of 1±0,1mm. Bond strength between post and dentin was measured in the root slice and the force applied on the center of the specimen. For the Mi test hourglass-shaped samples were obtained by trimming the proximal surfaces of each slice using a diamond bur until it touched the post. For PL and PSM tests cuts of 1±0,1mm of dentine slices were made and the fiber was divided into three parts, and then each slice were luted with a fiber post?s piece. Specimens for PSM had 1mm of post up and down the dentin slice and for PL all of the post was up the dentine slice. All specimens were submitted to a bond strength test at 0.5 mm/minute crosshead speed in a universal testing machine. For FP, each sample was examined under confocal microscopy at 100 times and a frequency distribution was made. For the AEF, three-dimensional models of each tests had been made and analyzed by Von Mises stress. Bond strength data were analyzed by ANOVA and when necessary was used Tukey?s test. For PF data the Fisher test was made. The inclusion of PF, only shows variation on MI, since these test showed the greater number of them. PL provided higher values of bond strength for root dentin and fiber post, followed by PSM. Both of them had also FP with greater amount of adhesive failures. The PS test was able to point statistically differences between root regions, but it showed the highest frequency of cohesive failure, and greater CV. Minor CV was found for the PSM that also showed adequate stress distribution. The MI showed concentration of tensions in the edges of the trimming area. One concluded that the type of test influences directly in the results obtained.

L'exocytose régulée, est un processus qui permet la communication entre les cellules à travers la sécrétion des hormones et des neurotransmetteurs. Dans les neurones et les cellules neuroendocrines, l'exocytose est strictement contrôlée par des signaux extracellulaires tels que le potentiel trans-membranaire et la fixation des ligands sur des récepteurs. Des progrès substantiels ont été effectués afin de comprendre le mécanisme moléculaire de l'exocytose. Les composants majeurs de la machinerie de sécrétion ont été dévoilés. Maintenant, la question qui émerge concerne le rôle de la plateforme de protéines qui semble avoir une action coordonnée entre chaque protéine. Dans le cas de la famille des annexines, qui est bien connue pour son action dans l'exocytose, leurs modes d'interactions séquentielles ou concertées avec d'autres protéines ainsi que leurs effets régulateurs sur l'exocytose ne sont pas encore bien établis. Des résultats précédents indiquent que l'Annexine A6 (AnxA6) affecte l'homéostasie du calcium et la sécrétion de la dopamine à partir des cellules PC12, utilisées comme un modèle cellulaire de neurosécrétion (Podszywalow Bartnicka et al., 2010). Afin de déterminer l'effet inhibiteur de l'AnxA6 sur l'exocytose de la dopamine, nous cherchons des partenaires moléculaires de l'AnxA6 dans les cellules PC12. Nous faisons l'hypothèse que l'AnxA6 interagit avec la PLD1, une enzyme active dans l'étape de la fusion des vésicules avec la membrane plasmique. En utilisant la microscopie confocale et la microscopie à onde évanescente, nous avons trouvé que l'isoforme 1 de l'AnxA6 et la PLD1 sont tous les deux recrutés sur la surface des vésicules au cours de la stimulation des cellules PC12. AnxA6 inhibait l'activité de la PLD comme indiqué par notre méthode d'analyse enzymatique au moyen de la spectroscopie infrarouge. En conclusion, nous proposons que l'AnxA6 n'est pas seulement impliquée dans la réorganisation des membranes par ses capacités à se lier avec des phospholipides négativement chargés et avec le cholestérol, mais elle influence également l'activité de la PLD1, changeant la composition lipidique des membranes / The regulated exocytosis is a key process allowing cell-cell communication through the release of hormone and neurotransmitters. In neurons and neuroendocrine cells, it is strictly controlled by extracellular signal such as transmembrane potential and ligand bindings to receptors. Substantial progress has been made to understand the molecular mechanism of exocytosis. Major components of secretory machinery have been brought to light. Now the emergent question concerns the role of scaffolding proteins that are thought to coordinate the action of each other. In the case of annexin family well known to be involved in exocytosis, their modes of –sequential or concerted- interactions with other proteins, and their regulatory effects on exocytosis are not very well established. Previous findings indicated that Annexin A6 (AnxA6) affected calcium homeostasis and dopamine secretion from PC12 cells, used as cellular model of neurosecretion (Podszywalow-Bartnicka et al., 2010). To determine the inhibitory effect of AnxA6 on exocytosis of dopamine, we were looking for molecular partners of AnxA6 in PC12 cells. We hypothesized that AnxA6 interacts with phospholipase D1 (PLD1), an enzyme involved in the fusion step. By using confocal microscopy and total internal reflection fluorescence microscopy, we found that isoform 1 of AnxA6 and Phospholipase D1 are both recruited on the surface of vesicles upon stimulation of PC12 cells. AnxA6 inhibited phospholipase D activity as revealed by our enzymatic assay based on infrared spectroscopy. To conclude, we propose that AnxA6 is not only implicated in membrane organization by its capacity to bind to negative charged phospholipids and to cholesterol, but AnxA6 is also affecting PLD1 activity, changing membrane lipids composition