Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients��� symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

FOOD CONCENTRATION AND TEMPERATURE EFFECTS ON LIFE CYCLE CHARACTERISTICS OF TROPICAL CLADOCERA (Daphnia gessneri Herbst, Diaphanosoma sarsi Richard, Moina reticulata (Daday)): I. DEVELOPMENT TIME

The effects of food concentration and temperature on embryonic and postem-bryonic duration of three tropical species, Daphnia gessneri(1.5mm), Diaphanosoma sarsi(1.2mm) and Moina reticulata(0.8mm), were investigated as part of life cycle studies which included growth, body size and reproduction. These are the very first experimental studies undertaken on these species. The long-term growth experiments were performed under controlled laboratory conditions at all combinations of temperature (22"C, 27"C and 32"C) and constant food concentration (0.03, 0.05, 0.10, 0.25, 0.50 and 1.00 mgC/L) of the unicellular green alga Scenedesmus acutus.Animals were examined twice daily throughout their life cycle from the neonate to third adult instar. In all three species, temperature exerted the most powerful influence on embryonic duration but there was also a smaller food effect. In D. gessneri,postembry-onic durations remained more or less the same at food levels 0.25 mgC/L but were influenced by temperature. At food concentrations of 0.1 mgC/L or lower, postembryonic durations became increasingly prolonged, particularly at high temperatures. This threshold concentration is affected by temperature: in D. gessneri,it was 0.1 mgC/L at 22oC and 27oC but higher at 32oC (between 0.25 and 0.50 mgC/L). At the same temperature of 27oC, the food threshold level varied between species: it was higher (0.25 mgC/L) for D. sarsiand lower (0.05 mgC/L) for M. reticulatacompared with D. gessneri(0.1 mgC/L). In both embryonic and postembryonic durations there is a body size effect as the absolute durations were longest in the largest species and shortest in the smallest species In all three species, prolongation of postembryonic duration at combinations of high temperature and lowered food levels was accompanied by increased number of juvenile instars.

The role of arl13b in cilia length regulation and in zebrafish development

RESUMO: Arl13b �� uma importante prote��na ciliar, presente em c��lios prim��rios e c��lios m��veis. Ratinhos mutantes para Arl13b t��m comprimento dos c��lios reduzido e defeitos nos B-t��bulos dos c��lios. Como consequ��ncia destes fen��tipos, defici��ncias na Arl13b originam, em modelos animais, v��rias doen��as cong��nitas, incluindo problemas no estabelecimento do eixo esquerda-direita, malforma����es cerebrais e deforma����es corporais. Nos seres humanos, defici��ncias na Arl13b levam a uma doen��a cr��nica cong��nita chamada S��ndrome de Joubert. Por outro lado, a sobreexpress��o de Arl13b origina c��lios mais longos, no entanto existe uma aus��ncia da caracteriza����o dos fen��tipos celulares e durante o desenvolvimento embrion��rio. Neste trabalho, quisemos explorar o efeito da sobre-express��o de Arl13b em embri��es de peixezebra. Descobrimos que, ao n��vel ciliar, a sobre-express��o de Arl13b nas c��lulas aumenta o comprimento ciliar em c��lios prim��rios e m��veis, no entanto, a esses c��lios falta adequada acetila����o da alfa-tubulina no citoesqueleto feito por microt��bulos. Os nossos resultados mostraram que esse efeito �� espec��fico de Arl13b sobre-express��o e quando se manipularam as enzimas respons��veis pela acetila����o (Mec17) e pela de-acetila����o (HDAC6) encontr��mos uma sinergia potencial com ambas. Test��mos ainda, que o aumento no comprimento ciliar n��o estava causalmente relacionado com a falta de acetila����o, ou seja, os c��lios com menos acetila����o n��o eram necessariamente os mais longos. Tamb��m mostr��mos que a sobre-express��o de Arl13b �� capaz de restaurar o comprimento dos c��lios em mutantes com c��lios curtos e como isso pode ser explorado para um futuro potencial papel terap��utico para Arl13b. Em seguida, foi avaliado o impacto do aumento da quantidade de Arl13b no desenvolvimento embrion��rio do peixe-zebra. Observou-se que a sobre-express��o de Arl13b apresentava fen��tipos muito fracos, quando comparados com a perda de fun����o dos mutantes de Arl13b. Focados no inesperado fen��tipo leve no estabelecimento do eixo esquerda-direita abord��mos a quest��o atrav��s do estabelecimento de uma colabora����o com matem��ticos, descobrimos que os c��lios mais longos que potencialmente t��m a capacidade de movimentar mais fluido s��o atenuados por amplitudes de batimento menores, e, como resultado, estes longos c��lios n��o prejudicam o movimento do fluido e consequentemente n��o afetam o estabelecimento dos padr��es de esquerda-direita. Sugerimos assim que a Arl13b �� um regulador chave, do comprimento ciliar. Descobrimos uma nova intera����o com as enzimas de acetila����o/de-acetila����o e levantamos novas hip��teses quanto aos mecanismos moleculares da fun����o da Arl13b. Propomos um novo modelo para o mecanismo molecular da Arl13b na regula����o do comprimento dos c��lios onde podemos integrar os nossos resultados com os relatados na literatura. Este trabalho adiciona mais conhecimento para o mecanismo de a����o da Arl13b e, portanto, fornece uma importante contribui����o para o campo da investiga����o em c��lios.---------------------------------------------------------------------------------------------------------------------- ABSTRACT: Arl13b is an important ciliary protein, present in primary and motile cilia. arl13b-/- mouse mutants have reduced cilia length and cilia B-tubule defects. As a consequence of these phenotypes, Arl13b loss of function animal models suffer from several congenital disorders including left-right problems, brain malformations and body deformations. In humans Arl13b depletion leads to a congenital chronic disease called Joubert Syndrome. On the other hand, overexpressing Arl13b leads to longer cilia but the characterization of the cellular and developmental phenotypes was missing. In this work we explore the effect of Arl13b overexpression in zebrafish embryos. We found that, at the ciliary level, Arl13b overexpression from 1 cell stage produces longer primary and motile cilia, but these cilia lack proper alpha tubulin acetylation of their microtubule cytoskeleton. Our results showed that this effect is specific from Arl13b overexpression and when we manipulated the enzymes responsible for acetylation, Mec17, and de-acetylation, HDAC6, we found a potential synergy of both mec17 knockdown and HDAC6 activity with Arl13b overexpression. We tested that the ciliary increase in length was not causally related to the lack of acetylation, meaning the more de-acetylated cilia were not necessarily the longer ones. We also showed that Arl13b overexpression is able to restore cilia length in short cilia mutants and how that may be explored to a potential future therapeutic role for Arl13b. Next, we evaluated the impact of increasing the amount of Arl13b in zebrafish embryonic development. We observed that Arl13b overexpression presented very mild phenotypes when compared to the loss of function mutants. We focused on the unexpected left-right mild phenotype and by establishing a mathematical modeling collaboration, we found out that the longer cilia generated force was attenuated by smaller beating amplitudes, and as a result, these long cilia were not impairing the cilia generated flow and the establishment of left-right patterning. We suggest that Arl13b is one key cilia length regulator. We disclosed a novel interaction with the acetylation / de-acetylation enzymes and raised new hypothesis as to the mechanisms of Arl13b function. We propose a new model for the Arl13b molecular mechanism of cilia length regulation where we integrate our findings with those reported in the literature. This work adds more knowledge to the Arl13b mechanism of action and therefore provides an important contribution to the cilia research field.

Cationic Liposomes as antigenic delivery systems: development of an immunoprotective strategy against Candida albicans

Tese de Doutoramento Biologia Molecular e Ambiental - Especialidade em Biologia Celular e Sa��de

Development of polymer wicks for the fabrication of bio-medical sensors

Polymer based wicking structures were fabricated by sintering powders of polycarbonate (PC), ultra-high molecular weight polyethylene and polyamide 12, aiming at selecting a suitable material for an innovative electroencephalography (EEG) bio-electrode. Preliminary experiments showed that PC based wicks displayed the best mechanical properties, therefore more detailed studies were carried out with PC to evaluate the influence of powder granulometry and processing parameters (pressure, temperature and time) on the mechanical properties, porosity, mean pore radius and permeability of the wicks. It was concluded that the mechanical properties are significantly enhanced by increasing the processing time and pressure, although at the expense of a significant decrease of porosity and mean pore diameter (and thus permeability), particularly for the highest applied pressures (74kPa). However, a good compromise between porosity/permeability and mechanical properties could be obtained by sintering PC powders of particle sizes below 500��m at 165��C for 5min, upon an applied pressure of 56kPa. Moreover, PC proved to be chemically stable in contact with an EEG common used disinfectant. Thus, wicking structures with appropriate properties for the fabrication of reusable bio-electrodes could be fabricated from the sintering of PC powders.

Development of hyaluronic acid, dextrin and extracellular matrix hydrogels for cell expansion

Disserta����o de mestrado integrado em Engenharia Biom��dica (��rea de especializa����o em Engenharia Cl��nica)

Molecular regulation of flowering induction in Quercus suber

Disserta����o de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepeneurship

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-Aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC50 10-28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 microM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas

Development of folate-targeted liposomes for rheumatoid arthritis therapy

Tese de Doutoramento em Biologia Molecular e Ambiental (��rea de especializa����o em Biologia Celular e Sa��de).

Development of Monoolein-based lipofection vectors for therapeutic siRNA delivery

Tese de Doutoramento em Biologia Molecular e Ambiental - Especialidade em Biologia Celular e Sa��de

Development of a poly(L-Lactic acid) based drug release system

Disserta����o de mestrado em Biof��sica e Bionanossistemas

Development of a water-in-oil-in-water multiple emulsion system integrating biomimetic aqueous-core lipid nanodroplets for protein entity stabilization. Part I: experimental factorial design

Lipid nanoballoons integrating multiple emulsions of the type water-in-oil-in-water enclose, at least in theory, a biomimetic aqueous-core suitable for housing hydrophilic biomolecules such as proteins, peptides and bacteriophage particles. The research effort entertained in this paper reports a full statistical 23x31 factorial design study (three variables at two levels and one variable at three levels) to optimize biomimetic aqueous-core lipid nanoballoons for housing hydrophilic protein entities. The concentrations of protein, lipophilic and hydrophilic emulsifiers, and homogenization speed were set as the four independent variables, whereas the mean particle hydrodynamic size (HS), zeta potential (ZP) and polydispersity index (PI) were set as the dependent variables. The V23x31 factorial design constructed led to optimization of the higher (+1) and lower (-1) levels, with triplicate testing for the central (0) level, thus producing thirty three experiments and leading to selection of the optimized processing parameters as 0.015% (w/w) protein entity, 0.75% (w/w) lipophilic emulsifier (soybean lecithin) and 0.50% (w/w) hydrophilic emulsifier (poloxamer 188). In the present research effort, statistical optimization and production of protein derivatives encompassing full stabilization of their three-dimensional structure, has been attempted via housing said molecular entities within biomimetic aqueous-core lipid nanoballoons integrating a multiple (W/O/W) emulsion.

Aspectos bioqu��micos y celulares de la reproducci��n de los vectores de la enfermedad de Chagas: regulaci��n e importancia fisiol��gica. Biochemical and cellular aspects of the reproduction of Chagas��� disease vectors: regulation and physiologycal impact

En la hip��tesis de trabajo del presente proyecto se considera la importancia del metabolismo de l��pidos y prote��nas en los insectos hemat��fagos, en particular en los vectores de la enfermedad de Chagas, para afrontar exitosamente la demanda energ��tica de la reproducci��n. Las hembras de estas especies pueden ingerir una comida de sangre abundante en l��pidos y prote��nas, los que son modificados en el intestino para su utilizaci��n y posterior almacenamiento en estructuras organizadas en el tejido ov��rico, sustentando as�� el r��pido crecimiento de los ovocitos. Estos aspectos resultan cr��ticos para el ciclo de vida del insecto y para el mantenimiento de la cadena epidemiol��gica de la enfermedad. En estas especies, recientemente hemos caracterizado a nivel bioqu��mico y celular la interacci��n entre lipoprote��nas y tejidos [Fruttero y col., Insect Biochem. Mol. Biol. 39: 322-331 (2009); Fruttero y col. Biocel 33 (3): 260 (2009)] y las fases del ciclo reproductivo [Aguirre y col., J. Insect Physiol. 54: 393-402 (2008)]. No obstante, los factores que participan en su regulaci��n son a��n escasamente conocidos. En este contexto, el estudio propone emplear dos especies de triatominos con el objeto de: (1) caracterizar los factores involucrados en la formaci��n y regulaci��n de reservas nutricionales en los ovocitos; (2) analizar los eventos que participan en la regresi��n del tejido ov��rico: atresia folicular y mecanismos de muerte celular. (3) evaluar el impacto de productos naturales (ureasas vegetales y p��ptidos derivados) en el desarrollo del tejido ov��rico. Para la ejecuci��n de los objetivos se llevar��n a cabo ensayos in vivo e in vitro con trazadores fluorescentes, fraccionamiento subcelular, estudios de expresi��n de prote��nas (mRNA y prote��na), estudios histo-morfol��gicos, ultraestructurales e inmunocitoqu��micos, microscop��a l��ser confocalizada, ensayos de actividad enzim��tica, ELISA, western-blot, electroforesis bidimensional, espectrometria de masas en t��ndem, etc. Tambi��n se evaluar��n los mecanismos de muerte celular (apoptosis/autofagia) mediante microscop��a electr��nica, detecci��n de apoptosis in situ (TUNEL), inmunofluorescencia, etc. Los resultados obtenidos permitir��n un mejor conocimiento sobre la fisiolog��a y bioqu��mica de estos vectores, los que resultan indispensables en el dise��o de nuevas estrategias para su control. Debido a la carencia de un tratamiento espec��fico para la enfermedad y a la falta de m��todos preventivos (vacuna), el control del vector es una de las v��as m��s importantes para reducir la incidencia de la enfermedad. Actualmente, la situaci��n socio-econ��mica que sufren amplios n��cleos de nuestra poblaci��n propicia condiciones de vida que facilitan la reproducci��n de los vectores y la transmisi��n vectorial del par��sito. El estudio permitir�� adem��s explorar aspectos bioqu��micos y celulares b��sicos, generando conocimientos que podr��an ser extensivos a otros insectos de importancia econ��mica en la ganader��a y/o agricultura. The aim of this project is to analyze the biochemical and cellular events involved in the lipid and protein metabolism in Chagas' disease vectors, and to evaluate their impact on the physiology of reproduction, particularly in the formation of nutritional resources in developing oocytes. At present, little is known about these critical aspects for the life cycle of the insect and for the epidemiology of the disease. The experimental approaches, which will be carried out using two species of triatomines, were designed: (1) to characterize factors involved in the formation and regulation of nutritional resources in developing oocytes; (2) to analyze the biochemical and cellular events that play a role during the regression of ovarian tissue, including the processes of oocyte resorption and programmed cell death. (3) to evaluate the impact of natural products (ureases from jackbean and related peptides) in the development of ovarian tissue. Methods and techniques involved in the project are: in vivo and in vitro assays with fluorescent tracers, ELISA, chemical assays, enzyme activities, western-blot; protein expression (mRNA), histological techniques, immunohistochemical and ultrastructural studies. Cell death will be analyzed by detection of apoptosis in situ (TUNEL), immunofluorescence (for autophagy), among others. The results obtained from the study will offer the opportunity to explore important aspects in the biology and physiology of Chagas' disease vectors that could be of potential utility in designing alternative strategies for the control of the insect.

Desarrollo de sistemas nano/micro-particulados con aplicaci��n en la fotodegradaci��n selectiva de contaminantes org��nicos en soluci��n. Development of nano / micro-particles systems with application to selective photodegradation of organic pollutants in solution.

IDENTIFICACI��N DEL PROBLEMA DE ESTUDIO. Las sustancias org��nicas solubles en agua no biodegradables tales como ciertos herbicidas, colorantes industriales y metabolitos de f��rmacos de uso masivo son una de las principales fuentes de contaminaci��n en aguas subterr��neas de zonas agr��colas y en efluentes industriales y dom��sticos. Las reacciones fotocatalizadas por irradiaci��n UV-visible y sensitizadores org��nicos e inorg��nicos son uno de los m��todos m��s econ��micos y convenientes para la descomposici��n de contaminantes en subproductos inocuos y/o biodegradables. En muchas aplicaciones es deseable un alto grado de especificidad, efectividad y velocidad de degradaci��n de un dado agente contaminante que se encuentra presente en una mezcla compleja de sustancias org��nicas en soluci��n. En particular son altamente deseables sistemas nano/micro -particulados que formen suspensiones acuosas estables debido a que estas permiten una f��cil aplicaci��n y una eficaz acci��n descontaminante en grandes vol��menes de fluidos. HIP��TESIS Y PLANTEO DE LOS OBJETIVOS. El objetivo general de este proyecto es desarrollar sistemas nano/micro particulados formados por pol��meros de impresi��n molecular (PIMs) y foto-sensibilizadores (FS). Un PIMs es un pol��mero especialmente sintetizado para que sea capaz de reconocer espec��ficamente un analito (mol��cula plantilla) determinado. La actividad de uni��n espec��fica de los PIMs en conjunto con la capacidad fotocatalizadora de los sensibilizadores pueden ser usadas para lograr la fotodescomposici��n espec��fica de mol��culas ���plantilla��� (en este caso un dado contaminante) en soluciones conteniendo mezclas complejas de sustancias org��nicas. MATERIALES Y M��TODOS A UTILIZAR. Se utilizaran t��cnicas de polimerizaci��n en mini-emulsi��n para sintetizar los sistemas nano/micro PIM-FS para buscar la degradaci��n de ciertos compuestos de inter��s. Para caracterizar eficiencias, mecanismos y especificidad de foto-degradaci��n en dichos sistemas se utilizan diversas t��cnicas espectrosc��picas (estacionarias y resueltas en el tiempo) y de cromatograf��a (HPLC y GC). As�� mismo, para medir directamente distribuciones de afinidades de uni��n y eficiencia de foto-degradaci��n se utilizaran t��cnicas de fluorescencia de mol��cula/part��cula individual. Estas determinaciones permitir��n obtener resultados importantes al momento de analizar los factores que afectan la eficiencia de foto-degradaci��n (nano/micro escala), tales como cantidad y ubicaci��n de foto- sensibilizadores en las matrices polim��ricas y eficiencia de uni��n de la plantilla y los productos de degradaci��n al PIM. RESULTADOS ESPERADOS. Los estudios propuestos apuntan a un mejor entendimiento de procesos foto-iniciados en entornos nano/micro-particulados para aplicar dichos conocimientos al dise��o de sistemas optimizados para la foto-destrucci��n selectiva de contaminantes acuosos de relevancia social; tales como herbicidas, residuos industriales, metabolitos de f��rmacos de uso masivo, etc. IMPORTANCIA DEL PROYECTO. Los sistemas nano/micro-particulados PIM-FS que se propone desarrollar en este proyecto se presentan como candidatos ideales para tratamientos espec��ficos de efluentes industriales y dom��sticos en los cuales se desea lograr la degradaci��n selectiva de compuestos org��nicos. Los conocimientos adquiridos ser��n indispensables para construir una plataforma vers��til de sistemas foto-catal��ticos espec��ficos para la degradaci��n de diversos contaminantes org��nicos de inter��s social. En lo referente a la formaci��n de recursos humanos, el proyecto propuesto contribuir�� en forma directa a la formaci��n de 3 estudiantes de postgrado y 2 estudiantes de grado. En las capacidades institucionales se contribuir�� al acondicionamiento del Laboratorio para Microscop��a ��ptica Avanzada (LMOA) en el Dpto. de Qu��mica de la UNRC y al montaje de un sistema de microscopio de fluorescencia que permitir�� la aplicaci��n de t��cnicas avanzadas de espectroscopia de fluorescencia de molecula individual. Water-soluble organic molecules such as certain non-biodegradable herbicides, industrial dyes and metabolites of widespread use drugs are a major source of pollution in groundwater from agricultural areas and in industrial and domestic effluents. Photo-catalytic reactions by UV-visible irradiation and organic sensitizers are one of the most economical and convenient methods for the decomposition of pollutants into harmless byproducts. In many applications it is highly desirable a high degree of specificity, effectiveness and speed of degradation of specific pollutants present in a complex mixture. In particular nano/micro-particles systems that form stable aqueous suspensions are highly desirable because they allow for easy application and effective decontamination of large volumes of fluids. Herein we propose the development of nano/micro particles composed by molecularly imprinted polymers (MIP) and photo-sensitizers (PS). The specific binding of MIP and the photo-catalytic ability of the sensitizers are used to achieve the photo-decomposition of specific "template" molecules in complex mixtures. Mini-emulsion polymerization techniques will be used to synthesize nano/micro MIP-FS systems. Spectroscopy (steady-state and time resolved) and chromatography (GC and HPLC) will be used to characterize efficiency, mechanisms and specificity of photo-degradation in these systems. In addition single molecule/particle fluorescence spectroscopy techniques will be used to directly measure distributions of binding affinities and photo-degradation efficiency in individual particles. The proposed studies point to a more detailed understanding of the factors affecting the photo-degradation efficiency in nano/micro-particles and to apply that knowledge in the design of optimized systems for photo-selective destruction of socially relevant aqueous pollutants.