Les dynamiques de l’activité photosynthétique du phytoplancton ont été caractérisées dans un écosystème côtier macrotidal (le Détroit du Pas-de-Calais, Manche orientale) et associées aux conditions environnementales. La variabilité spatiale et temporelle des paramètres photosynthétiques a été étudiée à différentes échelles, entre Septembre 2008 et Août 2010. Le niveau de variabilité et les facteurs de contrôle des paramètres photosynthétiques dépendent de l’échelle considérée. Au niveau spatial, aucun gradient des paramètres photosynthétiques n’a été trouvé entre la côte et le large. A l’inverse, au sein de la colonne d’eau, des variations du rendement quantique maximum (Fv/Fm), du taux de transport maximum des électrons (ETRm) et du coefficient de saturation lumineuse (Ek), en lien avec l’histoire lumineuse des cellules, ont été observées. Aux courtes échelles de temps (de l’heure à l’échelle d’un cycle de marée morte-eau/vive-eau), des variations considérables des paramètres photosynthétiques ont été observées. Les conditions lumineuses et la disponibilité des nutriments étaient les principaux facteurs de contrôle. Aux plus grandes échelles de temps (de l’échelle semi-mensuelle à l’échelle pluriannuelle), Fv/Fm, α (l’efficacité maximale d’utilisation de la lumière) et ETRm variaient sans cycle saisonnier clair. Par opposition, Ek suivait le cycle saisonnier de la lumière. A ces échelles, les interactions entre les successions des communautés phytoplanctoniques et les changements de lumière, de température et de disponibilité en nutriments contrôlaient la variabilité de l’activité photosynthétique. / Dynamics of phytoplankton photosynthetic activity were characterised in a macrotidal coastal ecosystem (the Strait of Dover, eastern English Channel) and related to environmental conditions. Spatial and temporal variability of photosynthetic parameters were studied at different scales, between September 2008 and August 2010. The level of variability and controlling factors of photosynthetic parameters depend on the scale considered. In space, no gradient of photosynthetic parameters was found between coastal and offshore waters. By contrast, within the water column, variations of the maximum quantum yield (Fv/Fm), the maximum electron transport rate (ETRm) and the light saturation coefficient (Ek) in relation to the light history of cells were observed. At short time scale (from hour to the scale of a neap-spring tide cycle), considerable variations of photosynthetic parameters were observed. Light conditions, temperature and nutrient availability were the main controlling factors. At longer time scale (from fortnightly to inter-annual scales), Fv/Fm, α (the maximal light utilization efficiency) and ETRm varied without any clear seasonal cycle. By contrast, Ek followed the seasonal variations of light except during summer where its changes of Ek were small compared to the light variability. At these time scales, close interplays between shifts of phytoplankton communities and changes of light, temperature and nutrient availability controlled the variability of photosynthetic parameters.

Dans le cadre de l'application de la Directive Cadre sur l'Eau il serait intéressant de mettre au point de nouveaux outils de surveillance de la qualité des cours d'eau. Dans le cas d'une pollution ponctuelle, certains d'entre eux pourraient être basés sur l'évaluation de l'état physiologique des communautés de diatomées périphytiques qui constituent souvent le premier maillon des écosystèmes aquatiques continentaux. L'objectif de ce travail était d'étudier l'effet de polluants communément retrouvés dans les milieux aquatiques (Cu, Zn, Cd, atrazine, isoproturon) sur la réponse de communautés périphytiques naturelles et de mieux cerner l'influence de la lumière. Nous avons mis au point une démarche méthodologique originale, basée sur l'estimation de leur activité photosynthétique à l'aide de la technique de mesure de la fluorescence chlorophyllienne en lumière modulée (fluorescence PAM). Nos résultats ont mis en évidence une certaine résistance à court terme des communautés aux différents polluants testés, variable selon le mode d'action du polluant (herbicides vs métaux lourds) et la structure du biofilm (biomasse, composition spécifique). En combinant des expérimentations originales en conditions naturelles et au laboratoire, nous avons montré que l'application d'un stress supplémentaire comme la lumière amplifie de manière significative la toxicité de ces contaminants. Ce travail confirme tout l'intérêt de l'utilisation de la technique de mesure de la fluorescence chlorophyllienne en lumière modulée dans le cadre de bioessais écotoxicologiques. Il offre également des perspectives intéressantes en vue de la détection in situ de pollutions ponctuelles par les herbicides. / Ln the context of the European Water Framework Directive implementation, new tools for river toxicity assessment have to be developed. Periphytic communities, which play a fundamental role in the trophic web in lotic systems, could be regarded as early wamings for detection of acute toxicant exposure by monitoring its physiological state. Several studies were then carried out by means of the chlorophyll fluorescence measurement in modulated light technique (P AM fluorescence) which helps to estimate the photosynthetic activity in a non-intrusive way. The aim of the present work was to study the effects of several pollutants commonly found in aquatic ecosystems (Cu, Zn, Cd, atrazine, isoproturon) on stream periphyton physiology and to highlight the role of light as an addition al potentially stressful parameter. Our results showed a relative resistance of the natural biofilms to the pollutants on a short term scale (< 1 day) which depended both on the mechanism of action of the toxicant (herbicides vs heavy metals) and the biofilms architecture. By combining novel experimental designs both in field and laboratory conditions, we also demonstrated that periphyton can be more affected when another stress, such as light, is applied. This work confmns the usefulness of chlorophyll fluorescence-based methods in ecotoxicological studies, particularly in order to detect in situ herbicide toxic effects.

The objective of this project was to develop new controlled drug delivery systems using nanomeric particles and characterize the delivery of drugs into cells in real time by digital fluorescence imaging microscopy techniques. The project is based on the idea that it could be possible to improve efficacy of drug molecules when encapsulated in nanometer-sized particles. Due to their small dimensions the particles could permeate through cells and tissues and even through the blood brain barrier. The anti-cancer drug Doxorubicin was encapsulated into biodegradable Poly (DL-lactideco- glycolide) (PLGA) nanoparticles by simple nanoprecipitation method. The small size of these particles (<200nm) could be beneficial to realize passive tumor-targeted drug delivery through enhanced permeability and retention (EPR) effects. These drug-containing particles showed a sustained release profile. Fluorescence images indicated that these particles can be internalized by human breast cancer MCF-7 cells by non-specific endocytosis. The bioactivity of the drugs was also tested against cell culture. The results indicated that DXR-loaded PLGA nanoaprticles could be used to deliver Doxorubicin into breast cancer cells.

When developing new techniques to analyze neuro-chemical microenvironments, it is important to realize the incredible variability in the cellular content and the response to stimulation between cells and within a single cell. Conventional analysis techniques yield an average result to describe the content and function of cells. This approach often misses important information since the onset of pathological conditions is always initiated in a small number of cells. New minimally invasive single cell analysis techniques are required for single cell studies in order to gain new insights and understanding of cells' functions. The objective of my Ph.D. study was to fabricate, characterize, and apply submicrometric fluorescence sensors for the analysis of neuron cells. This dissertation will report the fabrication of miniaturized fluorescence sensors for Ca2+, pH and Zn2+ analysis. In the first approach, liposomes (phospholipid vesicles) were used as miniaturized containers for fluorescent sensing reagents. Liposome-based fluorescence sensing technology offers several advantages over commonly used fluorescence sensing techniques including high spatial resolution, protection of the sensing dye from quenchers and high biocompatibility. However, liposome based sensors were found to be unstable in the cellular environment. The second approach was to synthesize submicrometric particle-based fluorescence sensors named lipobeads to replace the fluorescent liposomes in cellular studies. Lipobeads are polystyrene particles that are coated with a phospholipid membrane. One unique advantage of fluorescent sensing lipobeads is the ability to immobilize hydrophobic indicator molecules in the phospholipid membrane. This enables the use of these indicators in aqueous media since the lipobeads are fully water miscible. The lipobeads also proved to be highly biocompatible in cellular studies. This is attributed to their phospholipid bilayer membrane, which is similar in structure to cell membranes. The dissertation will describe the analytical properties of fluorescence sensing lipobeads and their application in studying zinc ion release and pH changes near neuron cells under physiological conditions, conditions of neuronal injury and stress and acidic cortical spreading depression during stroke like conditions.

Le statut ganglionnaire a la valeur pronostique la plus importante chez les patientes atteintes d’un cancer du sein, le taux de survie étant corrélé au nombre de ganglions envahis. Dans le traitement des cancers du sein opérables d'une taille inférieure à 3 cm, la technique du ganglion sentinelle (GS) remplace le curage ganglionnaire de stadification. Toutefois, cette technique nécessite l’emploi simultané de radioisotopes et de colorants physiologiques présentant divers effets secondaires. Ainsi, notre stratégie a été de tester, sur modèle pré-clinique de nouveaux traceurs fluorescents, les Quantum Dots (QDs), pour la technique du GS. Une étude sur la détection in vivo du ganglion axillaire (GA) par spectrofluorimétrie fibrée après injection sous-cutanée (s.c.) de QDs émettant dans le rouge a été réalisée et leur quantification ex vivo a été effectuée par spectrofluorimétrie jusqu’à 24 h. Ce travail a montré l’accumulation rapide et sélective des QDs dans le GA chez la souris. Une étude sur la localisation in vivo de 2 ganglions régionaux par imagerie de fluorescence a été réalisée suite à l’administration s.c. de QDs émettant dans le proche infrarouge (PIR) ainsi qu’une étude de biodistribution ex vivo par spectrométrie de masse (ICP-MS). Les résultats obtenus ont montré que l’imagerie de fluorescence pouvait être utilisée après injection de QDs pour localiser et suivre leur accumulation dans les ganglions superficiels chez la souris saine. Pour se rapprocher de la clinique, le repérage in vivo du GS axillaire a été investi sur modèle tumoral murin de cancer du sein par imagerie de fluorescence après administration s.c. de QDs émettant dans le PIR. Deux techniques de détection des métastases ganglionnaires ont été utilisées puis comparées : l’histologie conventionnelle et la RT-PCR. Tous les GS axillaires ont été détectés in vivo par imagerie, sauf un GS qui était complètement envahi par les cellules tumorales et la meilleure incidence de métastases ganglionnaire a été observée avec la RT-PCR. Ainsi, les QDs pourraient être utilisés comme substitut des marqueurs actuellement employés dans la technique du GS pour les cancers du sein. / Lymph node (LN) status is the most important prognostic factor in breast cancer patients and a determinant predictor of recurrence and survival. In the treatment of operable breast cancers with a size inferior of 3 cm, the sentinel lymph node biopsy (SLNB) substitutes the axillary LN dissection. However, this technique requires the simultaneous use of a radiocolloid and a physiologic dye, which present several secondary effects. Thus, our strategy was to test in preclinical model the new fluorescent markers, Quantum Dots (QDs) for SLNB. A study on in vivo axillary LN (ALN) mapping by light induced fluorescence was performed after subcutaneous (s.c.) injection of red-emitting QDs and their ex vivo quantification was realized by spectrofluorimetry for up to 24 h. We showed a fast and selective accumulation of QDs in ALN in healthy mice. An in vivo study on localization of 2 regional LNs by fluorescence imaging was carried out after the s.c. administration of near-infrared (NIR) emitting QDs along with ex vivo biodistribution study by mass spectroscopy (ICP-MS). Obtained results shown that fluorescence imaging can be used after QDs injection to map and follow their accumulation in superficial LNs in healthy mice. To approach the human clinic in a best possible way, sentinel ALN mapping was investigated after s.c. delivery of NIR emitting QDs in murine breast cancer model by fluorescence imaging. Two techniques for LN metastasis detection were used and compared: conventional histology and RT-PCR. All sentinel ALNs were located in vivo by imaging except one ALNs which was completely invaded by cancer cells and best incidence of LN metastasis was registered by RT-PCR (60 %). Thus, QDs could be used as a substitute for the markers currently employed in SLNB in breast cancers.

David Lai. / Thesis -- Chinese University of Hong Kong. / Bibliography: l. 103-104. / Acknowledgement --- p.v / Abstract --- p.vi / Format of the Thesis --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Experimental Techniques / Chapter 2.1 --- Introduction --- p.10 / Chapter 2.2 --- Arrangement for Fluorescence Spectrum Measurements --- p.10 / Chapter 2.3 --- Fluorescence Spectrum Measurements by Photographic Method --- p.11 / Chapter 2.4 --- Arrangement for Fluorescence Decay Rate Measurements --- p.11 / Chapter Chapter 3 --- Soda-lime Glass Matrix / Chapter 3.1 --- Introduction --- p.17 / Chapter 3.2 --- Theory --- p.19 / Chapter 3.3 --- Samples --- p.23 / Chapter 3.4 --- Experiments and Results --- p.26 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Fluorescence Spectra --- p.35 / Chapter 3.5.2 --- Decay Rate Measurements --- p.39 / Table (3-1) --- p.43 / Table (3-3) --- p.43 / Table (3-2) --- p.44 / Chapter Chapter 4 --- Lithium Phosphate Glass / Chapter 4.1 --- Introduction --- p.45 / Chapter 4.2 --- Samples --- p.46 / Chapter 4.3 --- Experiments and Results --- p.47 / Chapter 4.4 --- Discussion --- p.54 / Table (4-1) --- p.64 / Table (4-2) --- p.65 / Table (4-3) --- p.66 / Table (4-4) --- p.66 / Chapter Chapter 5 --- Hydrogen Oxide (H20) and Deuterium Oxide (D20) / Chapter 5.1 --- Introduction --- p.67 / Chapter 5.2 --- Theory --- p.69 / Chapter 5.3 --- Experiments and Results --- p.71 / Chapter 5.4 --- Discussion --- p.71 / Table (5-1) --- p.78 / Table (5-2) --- p.79 / Chapter Chapter 6 --- Conclusion --- p.80 / Appendix I --- p.82 / Appendix II --- p.83 / Appendix III --- p.84 / Appendix IV --- p.85 / Appendix V --- p.86 / Appendix VI --- p.90 / Appendix VII --- p.91 / Appendix VIII --- p.95 / Appendix IX --- p.96 / Appendix X --- p.102 / References --- p.103

In recent years, fluorescence imaging has become an indispensable tool for the exploration of biological processes, demonstrating both molecular specificity and high spatial and temporal resolution. Despite the significant progress made in this field, a number of challenges still exist which, if addressed could potentially result in the transformative development of fluorescent imaging for a plethora of biological applications. This may include the development of new fluorescent probes for the detection of unknown analytes, or the improvement of existing probes in order to enhance their properties. In this research, a fluorescent probe for the detection of hydrogen sulphide was repurposed for use as a ‘first of its kind’ fluorescent probe for the detection of hydroxylamine. In addition, the known peroxynitrite-mediated oxidation of boronic acid to phenol has been exploited for the development of a range of reaction based fluorescent probes. Initially non-fluorescent, each probe is ‘turned on’ in the presence of peroxynitrite, resulting in the formation of a highly fluorescent phenol derivatives. Such probes have been successfully evaluated during cell imaging experiments; demonstrating clear potential in the field of medical diagnostics. Specific applications may include ‘oxidative stress’, neurodegenerative disease and the evaluation of drug efficacy in the treatment of Alzheimer’s disease.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 134-151). Also available in electronic version.

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Herein is presented the collective experimental record of research performed in the Laboratory for Biological Surface Science. These investigations are generally classified under the category of bioanalytical surface science and include the following projects. Chapters III and IV describe the creation of a microfluidic device capable of generating fixed arrays of concentration gradients. Experimental results were matched with computational fluid dynamics simulations to predict analyte distributions in these systems. Chapters V and VI demonstrate the discovery and utility of photobleaching fluorophores for micropatterning applications. Bleached fluorophores were found to rapidly attach to electron rich surfaces and this property was used to pattern enzymes inside microfluidic channels in situ. Finally, Chapter VII exhibits a method by which solid supported lipid bilayers can be dried and preserved by specifically bound proteins. The intrinsic property of lateral lipid mobility was maintained during this process and a mechanism by which the protein protects the bilayer was suggested.