Yes / The aim of this study was to design a controlled release vehicle for insulin to preserve its stability and biological activity during fabrication and release. A modified, double emulsion, solvent evaporation, technique using homogenisation force optimised entrapment efficiency of insulin into biodegradable nanoparticles (NP) prepared from poly (dl-lactic-co-glycolic acid) (PLGA) and its PEGylated diblock copolymers. Formulation parameters (type of polymer and its concentration, stabiliser concentration and volume of internal aqueous phase) and physicochemical characteristics (size, zeta potential, encapsulation efficiency, in vitro release profiles and in vitro stability) were investigated. In vivo insulin sensitivity was tested by diet-induced type II diabetic mice. Bioactivity of insulin was studied using Swiss TO mice with streptozotocin-induced type I diabetic profile. Insulin-loaded NP were spherical and negatively charged with an average diameter of 200–400 nm. Insulin encapsulation efficiency increased significantly with increasing ratio of co-polymeric PEG. The internal aqueous phase volume had a significant impact on encapsulation efficiency, initial burst release and NP size. Optimised insulin NP formulated from 10% PEG–PLGA retained insulin integrity in vitro, insulin sensitivity in vivo and induced a sustained hypoglycaemic effect from 3 h to 6 days in type I diabetic mice.

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Background: Conjunctival pedicle flaps are one of the most frequently employed surgical interventions used to address a variety of sight threatening corneal diseases in veterinary ophthalmic practice. Securing the conjunctiva to the cornea is typically achieved through suturing, which is technically challenging and can result in prolonged surgical times, increased corneal edema, increased scar tissue, foreign body reaction, suture abscess and dehiscence. In human ophthalmology, a number of sutureless techniques to affix ocular tissues are being explored. Specifically, these approaches include synthetic tissue adhesives, bioadhesives, and hydrogel sealants. The proposed advantages of adhesives over suture, include reduced operative times, watertight seals, decreased foreign-body sensation and inflammation, faster healing times and tissue regeneration with original architecture restoration. Objective. To evaluate the maximum tensile force a conjunctival pedicle flap is able to withstand with respect to different fixation methods, i.e., Tisseel® fibrin glue, ethyl cyanoacrylate adhesive, ReSure® hydrogel sealant, or 8-0 VICRYL suture. Animals Studied. Ex-vivo porcine globes Procedures. Following a 500-micron restricted depth lamellar keratectomy, conjunctival pedicle flaps were dissected and secured to corneal defects with either the bioadhesive Tisseel®, or the synthetic adhesives ReSure®, ethyl cyanoacrylate, or 8-0 VICRYL® suture. Harvested corneoconjunctival flap interfaces were clamped to an accelerometer and potentiometer device, and loaded under video surveillance until the point of failure. Peak load at failure was determined for each test and used to compare between sample types. Results. 40 flaps underwent tensile force testing, with 6 being omitted for dehiscence prior to tensile testing. Of the 34 tests included in analysis, 10 conjunctival flaps were secured with suture, 10 with cyanoacrylate, 8 with ReSure® hydrogel sealant, and 6 with Tisseel® fibrin glue. A significant increase in maximum withstood tensile force was recorded between sutured flap fixation when compared with cyanoacrylate glue (p=0.02474), ReSure® hydrogel sealant (p= 0.00000), and Tisseel® fibrin glue (p= 0.00002). Cyanoacrylate fixation was significantly stronger when compared with ReSure® hydrogel sealant and Tisseel fibrin glue (p=0.01194 and 0.01798 respectively). There was no significant difference in adhesion strength between ReSure® hydrogel sealant and Tisseel® fibrin glue (p=0.95675). Conclusions. Conjunctival pedicle flap fixation using 8-0 VICRYL® suture fixation was able to withstand significantly greater maximum tensile force application in comparison with the ReSure®, Tisseel®, or cyanoacrylate adhesives. / Master of Science / Conjunctival pedicle flaps are one of the most frequently employed surgical interventions to address sight threatening corneal disorders in companion animals. Due to its redundant nature and close proximity to the corneal surface, conjunctival tissue is readily available for grafting to the cornea. It is surgically dissected to appropriate size and repositioned over the corneal defect where it effectively aids in healing through direct provision of structural support and indirectly via its rich blood supply. Securing the conjunctiva to cornea is typically achieved through suturing, which is technically challenging and can result in prolonged surgical times, increased corneal edema, increased scar tissue, foreign body reaction, abscess and dehiscence. In human ophthalmology, a number of sutureless techniques to affix ocular tissues are being explored. Specifically, these approaches include synthetic tissue adhesives, bioadhesives, and hydrogel sealants. The proposed advantages of tissue adhesives over suture, include reduced operation times, watertight closures, decreased foreign-body reaction and inflammatory response, faster healing times and increased ability to induce regeneration of the original tissue architecture. The purpose of this study was to evaluate the maximum tensile force a corneoconjunctial pedicle flap is able to withstand with respect to four different fixation methods, 40 ex-vivo porcine globes underwent conjunctival pedicle flap procedures. Each pedicle flap was secured to cornea with either 8-0 Vicryl® suture, Tisseel®, ethyl cyanoacrylate, or ReSure®. After harvesting from the globe, the corneoconjunctival unions were clamped to an accelerometer and potentiometer device, and loaded under video surveillance until the point of failure. The peak load was determined for each test and used to compare between sample types.

Uma das caracterÃsticas fisiolÃgicas mais importantes das halÃfitas pertencentes ao gÃnero Atriplex à a enorme habilidade de acumular solutos orgÃnicos e inorgÃnicos com o intuito de se ajustar osmoticamente em condiÃÃes de elevada salinidade e deficiÃncia hÃdrica. Contudo estudos compreensivos sobre a contribuiÃÃo relativa desses solutos inorgÃnicos e orgÃnicos no ajustamento osmÃtico de folhas dessas halÃfitas ainda sÃo poucos explorados na literatura. Esse trabalho pretende pela primeira vez na literatura fazer um estudo detalhado, da participaÃÃo e da contribuiÃÃo relativa da maioria dos solutos orgÃnicos e inorgÃnicos no potencial osmÃtico de folhas de Atriplex nummularia, submetidas a concentraÃÃes crescentes de NaCl, ciclos sucessivos de seca e ao polietileno glicol (PEG). Para responder as questÃes acima mencionadas foram conduzidos trÃs grandes experimentos seqÃenciais. No primeiro experimento, mudas de Atriplex nummularia com quatro meses de idade, foram cultivadas em vasos de argila contendo como substrato areia, sendo irrigadas a cada dois dias com soluÃÃo contendo concentraÃÃes crescentes de NaCl (0, 100, 200, 300 e 400 mmol L-1) durante 120 dias. O experimento foi realizado em condiÃÃes de casa de vegetaÃÃo. No segundo experimento, mudas de A. nummularia, com idade de 18 meses foram transplantadas para recipientes de polietileno contendo 5.0 kg de vermiculita, em condiÃÃes de casa de vegetaÃÃo, onde foram submetidas aos seguintes tratamentos (Controle; seca; NaCl + seca; NaCl) por trÃs ciclos sucessivos de seca-reidrataÃÃo (3 semanas) seguido de uma recuperaÃÃo no final do Ãltimo ciclo (apÃs 48 h). No terceiro experimento, plÃntulas de A.nummularia, com idade de dois meses foram transplantadas para cultivo hidropÃnico em vasos plÃsticos com capacidade para 0,8 em sala de crescimento sob condiÃÃes controladas onde sofrerÃo um prÃ-condicionamento com ou sem NaCl por duas semanas. ApÃs foram submetidas aos seguintes tratamentos (controle; PEG; NaCl; NaCl + PEG). Em conclusÃo, os resultados da presente investigaÃÃo demonstram que as plantas de Atriplex nummularia foram capazes de manter o seu crescimento mesmo em condiÃÃes de alta salinidade, principalmente pela capacidade de manutenÃÃo de seu status hÃdrico. Os resultados tambÃm sugerem que a GB à o maior soluto orgÃnico envolvido com o ajustamento osmÃtico (AO) do citoplasma e que sua acumulaÃÃo parece ser uma resposta adaptativa ao estresse provocado pelo NaCl. Em adiÃÃo, o Ãons salinos Na+ e Cl- contribuÃram para a maioria do AO das folhas submetidas ao NaCl. O impacto relativo do K+ sobre o AO das folhas foi reduzido consideravelmente com o aumento da dose externa de NaCl. Os resultados evidenciam tambÃm que o prÃ-condicionamento com NaCl influenciou positivamente no crescimento de Atriplex nummularia, em condiÃÃes de seca, evidenciado pelo maior acÃmulo de massa seca e melhor status hÃdrico das folhas. Dentre os solutos inorgÃnicos analisados, o Na+ e o Cl- foram os que mais contribuÃram para a osmorregulaÃÃo das folhas em condiÃÃes de deficiÃncia hÃdrica. Enquanto, GB foi o soluto orgÃnico mostrou o maior percentual de contribuiÃÃo para o potencial osmÃtico. Entretanto os resultados mostraram que o prÃ-condicionamento das plÃntulas de Atriplex nummularia com NaCl nÃo foram capazes de atenuar os efeitos induzidos pelo PEG.

Actuellement les traitements thérapeutiques pour soigner la schizophrénie, par voie intraveineuse ou orale, ne sont qu’en partie efficaces et associés généralement à des effets extrapyramidaux souvent dangereux et très gênants pour les patients. Afin d’augmenter l’efficacité du traitement toute en neutralisant les effets indésirables, ce travail a eu comme objectif de concevoir des capsules composites (PLGA-PEG / alginate) destinées à être administrées par voie orale et capables de libérer localement, de façon spécifique et contrôlée, le neuroleptique halopéridol dans le cerveau. L’optimisation du protocole de synthèse a permis d’obtenir de façon reproductible des nanocapsules de PLGA poreuses monodisperses et peu agrégées, possédant un diamètre hydrodynamique moyen inférieur à 80 nm et une bonne stabilité en solution aqueuse. Une fois fonctionnalisées avec le Poly (éthylène glycol) diamine, des études in vitro ont montré la faible toxicité de ces nanoparticules furtives ainsi que leur capacité à encapsuler une quantité satisfaisante d’halopéridol et de libérer ce principe actif sur une durée d’un mois avec un faible effet « burst ». L’incorporation des nanoparticules pégylées dans des matrices préparées à haute concentration d’alginate et de 100 % CaCl2 a permis d’obtenir des billes nanocomposites possédants une meilleure stabilité à la sortie du milieu gastrique simulé et persistent environ 30 minutes en milieu intestinal simulé. Enfin des études in vivo préliminaires sur des souris adultes utilisant des nanoparticules injectées et des billes nanocomposites ingérées ont démontré l’efficacité de ces systèmes à délivrer l’halopéridol au cerveau. / Currently therapeutic treatments for schizophrenia, intravenously or orally, are only partially effective and generally associated with extrapyramidal effects often dangerous and very troublesome for patients. In order, to increase the treatment efficiency by neutralizing any side effects the aim of this work was to design composite capsules (PLGA-PEG / alginate) intended to be administered by way oral and able to release locally, in a specific and controlled way, the neuroleptic “haloperidol” in the brain. The optimization of the protocol of synthesis allowed to obtain in a reproducible way of the nanocapsules of monodisperse and not very aggregate porous PLGA, having an average hydrodynamic diameter lower than 80 Nm and a good stability in aqueous solution. Once functionalized with Poly (ethylene glycol) diamine, in vitro studies showed the low toxicity of these furtive nanoparticles as well as their ability to encapsulate a satisfactory amount of haloperidol and release this active principle over a period of one month with a low burst effect. The incorporation of the PEGylated nanoparticles in matrices prepared with a high concentration of alginate and 100% CaCl2 made it possible to obtain nanocomposite beads having a better stability at the exit from the simulated gastric medium and persist approximately 30 minutes in simulated intestinal medium. Finally, preliminary in vivo studies on adult mice using injected nanoparticles and ingested nanocomposite balls showed the effectiveness of these systems to deliver haloperidol in the brain.

Le système immunitaire est un ensemble de mécanismes impliquant différents types de cellules qui produisent des facteurs solubles (cytokines, chimiokines ou molécules cytotoxiques) qui contribuent à la régulation et aux réponses immunitaires. La caractérisation à l'échelle cellulaire de la production de ces facteurs solubles présente un grand intérêt d'une part sur le plan fondamental pour comprendre les cascades d'événements des régulations cellulaires, et d'autre part dans le suivi de la réponse immunitaire (infections, cancers, auto-immunités, greffes, vaccins, etc.). Cependant, la plupart des techniques actuellement disponibles (ELISpot, cytométrie en flux, microarrays, etc.) ne permettent pas d'étudier plusieurs cytokines en rapport avec le phénotype des cellules sécrétrices et/ou sans marquage et d'analyser les secrétions de cytokines en temps réel par des cellules individuelles. Dans cette étude, une « biopuce à cellules » a été développée pour analyser les secrétions de cytokines par les cellules individuelles (lymphocytes T) in vitro. La biopuce est fonctionnalisée par greffage électrochimique des motifs d'anticorps spécifiques aux protéines membranaires de cellules et/ou d'anticorps spécifiques aux cytokines, tous couplés au pyrrole. Ensuite, un traitement de surface est effectué avec du poly (éthylène glycol) thiol (Thiol-PEG) pour empêcher la fixation non spécifique de cellules sur la surface de la biopuce. Un dispositif microfluidique en polydimethyllsiloxane (PDMS) et maintenu à 37°C a aussi été développé afin d'intégrer toutes les opérations d'analyse et de détection dans un seul système. La biopuce développée dans cette étude permet la capture spécifique et stable de lymphocytes T individuels viables et la détection ultérieure de cytokines sécrétées par chaque cellule individuelle. Dans ce travail, la détection des cytokines sécrétées (IL-2 et IFN-γ) a été effectuée par fluorescence dans un format en sandwich. Cette «biopuce à cellules» est également compatible avec l'imagerie par résonance plasmonique de surface (SPRi), ce qui pourrait permettre de réaliser des analyses en temps réel et la détection sans marquage de plusieurs cytokines sécrétées par des cellules individuelles. Cette technique fournit un outil très prometteur pour l'analyse de marqueurs biologiques et de l'activité de cellules et l'étude des réponses immunitaires. / The immune system is a set of mechanisms involving many different cell types which communicate through downstream signals mediated principally by soluble factors (i.e. cytokines) to protect the host against invading organisms and to control adequate immune responses. The identification and characterization at the cellular level of cytokine production has a huge interest for both fundamental research and clinical studies. However, the majority of techniques currently available (ELISpot, flow cytometry, microarrays, etc.) have several shortcomings including notably the assessment of multiple cytokines in relation to secreting cell phenotypic classification and/or label-free and real-time analysis of cytokine secretions at individual cell level. Hence, in this study, we developed a « cell biochip » to analyze the secretion of cytokines by individual cells (T lymphocytes) activated and cultured in vitro. The biochip is functionalized by electrochemically grafting patterns of pyrrole-conjugated cell membrane-specific and cytokine-specific antibodies and treated with Poly(ethylene glycol)thiol (Thiol-PEG) self-assembled monolayers (SAMs) to stably avoid non-specific binding of cells on the surface. A polydimethyllsiloxane (PDMS)-based microfluidic device maintained at 37°C was also developed in order to integrate all the detection assay operations in a single system. The biochip developed here allows specific and stable capture of viable and bioactive individual T cells and subsequent detection of secreted cytokines in the close vicinity of each individual cell. In this work, the detection of secreted cytokines (IL-2 and IFN-γ) was performed by fluorescence in an immunosandwich assay format. This « cell biochip » is also compatible with surface plasmon resonance imaging (SPRi), which could therefore allow expanding its functionality to enable real-time and label-free detection of multiple cytokines from individual cells. Such technique provides a very promising tool for the analysis of biomarkers and cell activity and the monitoring of immune responses.

Syftet med denna systematiska litteraturstudie som inkluderar 18 artiklar, har varit att belysa vilka nutritionsstöd som kan förebygga och behandla malnutrition hos barn med cancer samt vilka omvårdnadsbehov som finns i samband med nutritionsbehandling. Resultatet visade att barn med cancer som behandlas med strålning och/eller cytostatika ofta drabbas av olika biverkningar som påverkar nutritionen. Cirka 46 % av barnen utvecklade malnutrition. Näringsintaget försämrades ofta på grund av biverkningarna vilket fick till följd att behandlingen försämrades samt tillväxt och utveckling påverkades negativt. Olika sätt fanns för att upprätthålla gott nutritionsstatus. Oralt intag förordades för att upprätthålla mag- tarmkanalens struktur och funktion. Alternativ för kostbehandling när barnet inte klarade att inta oral kost var nasogastrisk sond (NS), Perkutan Endoskopisk Gastrostomi (PEG) och Total Parenteral Nutrition (TPN). NS visade sig vara relativt biverkningsfritt och ett alternativ att tillgå under kortare nutritionsbehandlingar. PEG var ett gott alternativ med relativt få biverkningar då barnet behöver nutritionsstöd under längre tid. TPN var ett alternativ enbart då barnet inte kunde äta relaterat till bristande upptag i mag- tarmkanalen. Försämrat näringsintag påverkade barnets livskvalitet. Familjen påverkades genom att ätandet var en stark källa till konflikt mellan barnet och föräldrarna. Det var viktigt att barnet och familjen fick stöd i barnets förmåga att kunna äta samt att erbjudas näringsrik kost. Bedömning av nutritionsstatus och dokumentation av oralt intag, parenteralt intag och effekter av nutritionsbehandlingen var av stor vikt för att kunna ge barnet optimal omvårdnad.

Les nanotechnologies sont devenues depuis plusieurs années un axe majeur de développement dans les domaines du diagnostic, de l’imagerie, de la délivrance de médicament, du suivi thérapeutique et de l’ingénierie tissulaire. L’administration de produits non injectables sous leur forme libre ou bien possédant une toxicité élevée, peut être facilitée par l’utilisation de nanovecteurs, modifiant leur distribution. Ils permettent donc de réduire les doses administrées, limiter les effets secondaires et diriger le contenu du vecteur (agent de contraste, drogue) vers un organe cible ou une tumeur, en présentant à la surface de celui-ci des molécules de ciblage de ces zones spécifiques.Cette thèse s'inscrit dans cette thématique : nous avons exploré les possibilités de former des gels chimiques à partir des nanoparticules lipidiques qui sont constituées d'un cœur huileux pouvant encapsuler de petites molécules hydrophobes et d'une couche de surfactants permettant la stabilisation des gouttelettes en phase aqueuse.Nous avons pu synthétiser plusieurs surfactants PEGylés fonctionnalisés (thiol, maléimides, amine, oxyamines et ONB-maléimide). Les protocoles de synthèse de ces surfactants sont affinés et reproductibles.Ces surfactants ont été incorporés à la surface des nanoparticules lipidiques. Les nanoparticules fonctionnalisées ainsi obtenues ont été caractérisées et les fonctions présentes à leur surface sont mises en évidence.Différentes sortes de gels chimiques stables, résistant à la dilution, rapides à fabriquer et contrôlables ont été élaborés.Ces gels chimiques peuvent être utilisés dans l’avenir pour encapsuler des protéines ou des drogues. / Nanotechnology became for several years a major development in the areas of diagnostics, imaging, drug delivery, therapeutic monitoring, and tissue engineering. The administration of non-injectable products in their free form or with high toxicity, can be facilitated by the use of nanocarriers, changing their distribution. They therefore reduce the doses administered, limit side effects and direct the contents of the vector (contrast agent, drug) to a target organ or tumor, by presenting to the surface thereof targeting molecules of these areas specific.This thesis fits into this theme : we have explored the possibilities of forming chemical gels based on lipid nanoparticles which are composed of an oily heart can encapsulate small hydrophobic molecules and a layer of surfactants allowing stabilization of the droplets in the aqueous phase.We have synthesized several PEGylated functionalized surfactants (thiol, maleimide, amine, and oxyamines ONB-maleimide). synthesis protocols of these surfactants are refined and repeatable.These surfactants were incorporated into the surface of lipid nanoparticles. The functionalized nanoparticles thus obtained have been characterized and the functions present at their surface are highlighted.Different kinds of chemical gels stable, resistant to dilution, fast and controllable manufacturing have been developed.These chemical gels can be used in the future for encapsulating proteins or drugs.