Thesis (doctoral)--Uppsala University, 1982. / Includes bibliographical references (p. 35-39).

Orientador: Luiz Carlos da Silva Filho / Resumo: A reação de acilação de Friedel-Crafts é uma das mais importantes reações de formação de ligações carbono-carbono em compostos aromáticos. As reações de acilação de Friedel-Crafts ocorrem basicamente por meio da reação entre compostos aromáticos com haletos de acila ou anidridos na presença de um ácido de Lewis. Assim, neste trabalho foi estudado o uso do pentacloreto de nióbio como ácido de Lewis nas reações de acilação de Friedel-Crafts visando à síntese de corantes sensibilizadores. Os derivados de fluoresceína foram sintetizados a partir de derivados fenólicos e derivados de anidrido, na presença de NbCl5 e sob aquecimento. Os produtos de interesse foram obtidos com rendimentos de 54 a 90% e em curtos tempos reacionais que variaram de 50 a 200 minutos. As fluoresceínas apresentaram absorção e emissão de luz dentro do comprimento de onda do visível, com alta intensidade e altos valores de rendimento quântico de fluorescência (0,60 a 0,93) quando dissolvidos em soluções alcalinas. Os derivados de fluoresceínas foram aplicados na confecção de células solares de Gratzel apresentando eficiências de conversão que variaram de 0,11 a 0,23%. Também foi estudado a aplicação dos derivados de fluoresceínas como corantes iniciadores em reações de fotopolimerização de resinas de dimetacrilato. Estas resinas tem sido largamente utilizadas na odontologia, devido as suas propriedades como baixa toxicidade, baixa solubilidade em água e alta resistência mecânica. As fluoresceínas promoveram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Friedel-Crafts acylation reaction is one of the most important reactions for the formation of carbon-carbon bonds. The Fridel-Crafts acylation reaction occurs basically by a reaction between aromatic compounds with acyl halides or anhydrides in the presence of a Lewis acid. Thus, in this work it was studied the use of niobium pentachloride as Lewis acid in Friedel-Crafts acylation reaction, aiming the synthesis of sensitizing dyes. The fluorescein derivatives were synthesized from phenolic derivatives and anhydride derivatives, in the presence of NbCl5 and under heating. The products of interest where obtained in yields of 54 to 90% and in short reaction times ranging from 50 to 200 minutes. The fluorescein derivatives presented absorption and emission within the range of visible and with high intensity and high fluorescence quantum yield (0,60 to 0,93) when dissolved in alkaline pH. Due to these good results obtained in the photophysical study, the fluorescein derivatives were applied in Gratzel solar cells, presenting convertion efficiencies that ranging from 0.11 to 0.23%. It has also been studied the application of fluorescein derivatives as initiator dyes in photopolymerization reactions of dimethacrylated resins. This resins have been widely used in dentistry, due this properties as, low toxicity, low solubility in water and high mechanical resistance. The fluoresceins promoted the photopolymerization of dimethacrylated resins with conversion values ranging from 1.2... (Complete abstract click electronic access below) / Doutor

Abstract Cardiac surgery with cardiopulmonary bypass (CPB) results in hemodilution, systemic inflammatory response, activation of coagulation and fibrinolysis, and microembolisation, which may all contribute to postoperative organ dysfunction. As an attempt to attenuate these side effects, the use of minimized cardiopulmonary bypass (MCPB) systems has increased. Compared to conventional CPB (CCPB), they are characterized with reduced artificial surface area and blood-air interface. The goal of these alterations has been to reduce systemic inflammation, preserve coagulation function and minimize the need for blood tranfusions. This study was aimed at determining whether or not MCPB attenuates the adverse effects of CPB. In study I, the safety, feasibility and effect on transfusion requirements of MCPB was investigated in unselected coronary artery bypass surgery (CABG) patients. In studies II and III, the incidence of retinal microembolism after CABG and aortic valve replacement (AVR) surgery with MCPB was compared to that of CCPB by means of fluorescein angiography. Furthermore, in studies II and III, the effect of MCPB on systemic inflammation, coagulation, endothelial activation and injury, as well as on platelet activity, was compared to those of CCPB. In study IV, the effect of MCPB on intestinal mucosal damage following CPB was compared to CCPB in a porcine model of prolonged CPB. MCPB appeared as safe and feasible as CCPB in unselected CABG patients (Study I). MCPB was associated with decreased retinal microembolism compared to CCPB in CABG patients (Study II). Conversely, the difference in retinal microembolism in AVR patients was not statistically significant (Study III). MCPB was associated with a decrease in neutrophil activation in CABG and AVR patients as compared to CCPB. However, there were no differences in coagulation, endothelial activation and injury, or in platelet activity (Studies II, III). There were no differences in markers of intestinal mucosal damage between MCPB and CCPB following prolonged CPB in the experimental model (Study IV). The results of this study suggest that MCPB may be used safely with CABG patients, with beneficial effects on hematocrit, and attenuated neutrophil activation. In CABG patients, MCPB is associated with reduced retinal microembolism, suggesting a decreased embolic load to the brain. The clinical feasibility of MCBP requires further technical evolution in the management of valve surgery. The results of the animal model support previous concerns regarding intestinal mucosal damage during CPB. / Tiivistelmä Sydänkeuhkokoneen käyttö aiheuttaa elimistössä hemodiluution, yleistyneen tulehdusvasteen ja hyytymisjärjestelmän aktivoitumisen sekä mikroembolisaatiota. Ilmiöt ovat yleensä lieviä ja ohimeneviä, mutta voivat johtaa elintoimintahäiriöihin ja pitkittyneeseen toipumiseen sydänleikkauksen jälkeen. Haittojen lievittämiseksi sydänkeuhkokonetta on pyritty kehittämään fysiologisemmaksi. Miniperfuusiolaitteistoissa kiertävän veren kontakti pintamateriaalien ja ilman kanssa jää pienemmäksi ja veren laimenemista tapahtuu vähemmän. Tutkimuksen tavoitteena oli selvittää voidaanko miniperfuusiolla lievittää sydänkeuhkokoneen haittoja. Ensimmäisessä osatyössä selvitettiin miniperfuusion käyttökelpoisuutta ja vaikutusta verensiirtotarpeeseen ohitusleikkauspotilailla valikoimattomassa aineistossa. Toisessa ja kolmannessa osatyössä selvitettiin silmänpohjan mikroembolioiden ilmaantuvuutta miniperfuusion ja perinteisen sydänkeuhkokoneen käytön jälkeen ohitusleikkauspotilailla ja aorttaläppäleikkauspotilailla. Toisessa ja kolmannessa osatyössä selvitettiin lisäksi miniperfuusion vaikutuksia yleistyneen tulehdusvasteen voimakkuuteen, hyytymisjärjestelmään sekä endoteelin aktivaatioon perinteiseen sydänkeuhkokoneeseen verrattuna. Neljännessä osatyössä verrattiin kokeellisessa mallissa miniperfuusion ja perinteisen sydänkeuhkokoneen vaikutuksia suoliston limakalvon eheyteen. Miniperfuusio ilmeni yhtä käyttökelpoiseksi kuin perinteinen sydänkeuhkokone ohitusleikkauspotilaiden hoidossa. Ohitusleikkauspotilailla ilmeni vähemmän silmänpohjan mikroembolioita miniperfuusion jälkeen, mutta aorttaläppäleikkauspotilailla ero ei ollut tilastollisesti merkitsevä. Miniperfuusion käyttöön liittyi vähemmän neutrofiilien aktivaatiota. Tekniikoiden välillä ei ilmennyt eroa hyytymisjärjestelmän eikä endoteelin aktivaatiota osoittavissa merkkiaineissa. Sydänkeuhkokoneen käyttö aiheutti saman tasoisen suoliston limakalvon vaurion miniperfuusiolla ja perinteisellä sydänkeuhkokoneella. Tutkimuksen perusteella miniperfuusiotekniikkaa voidaan käyttää turvallisesti ohitusleikkauspotilaiden hoidossa ja sen käyttö vähentää hemodiluutiota ja neutrofiilien aktivaatiota verrattuna perinteiseen sydänkeuhkokoneeseen. Miniperfuusiolla voidaan vähentää sydänkeuhkokoneen käytön aiheuttamaa silmänpohjan mikroembolisaatiota, joka saattaa viitata vähäisempään aivoverenkierron mikroembolisaatioon. Miniperfuusiotekniikoiden tulee edelleen kehittyä hyödyttämään enemmän myös aorttaläppäleikkauspotilaita. Löydökset koskien sydänkeuhkokoneen aiheuttamia suoliston limakalvovaurioita vahvistavat aiempaa olettamusta suoliston haavoittuvuudesta sydänleikkauksen jälkeen.

Depuis plusieurs années, la vectorisation de molécules est considérée comme la stratégie la plus prometteuse pour améliorer la pénétration cutanée des principes actifs et pour cibler et contrôler leur libération, augmentant ainsi l'efficacité thérapeutique des traitements tout en limitant leurs effets secondaires. Les nanoparticules polymériques ont fait l'objet de nombreuses études car elles possèdent une très grande stabilité et une capacité supérieure aux autres vecteurs à libérer les principes actifs de façon prolongée. Récemment, le laboratoire de Pharmacie Galénique de Besançon a montré, in vivo, que les nanoparticules polymériques de diamètre inférieure ou égale à 100 nm pénétraient spécifiquement dans la peau inflammée de la souris, alors qu'aucune pénétration n'était observée dans la peau saine. Le premier objectif de cette thèse était de confirmer ces hypothèses dans la peau inflammée d'un autre animal, le porc. Les résultats de notre étude in vivo confirment l'absence de pénétration des nanoparticules polymériques dans la peau saine du porc et montrent une pénétration taille dépendante dans la peau inflammée. Le second objectif poursuivi était de vérifier ces résultats chez l'Homme. Pour cela, une preuve de concept a été mise en place dans le service de Dermatologie du CHRU de Besançon. Les premiers résultats de cette étude clinique semblent confirmer l'absence de pénétration des nanoparticules polymérique de I 00 nm dans la peau des volontaires sains et dans la peau non lésée des patients souffrant de dermatite atopique. A l'inverse, une forte pénétration des nanoparticules est observée au niveau des plaques d'eczéma des patients. Si les résultats de l'étude clinique se confirment, nous prévoyons d'encapsuler un anti­inflammatoire ou un immunosuppresseur pour vérifier l'intérêt thérapeutique de ces vecteurs en médecine humaine dans le traitement de la dermatite atopique. Parallèlement, une évaluation pourrait être réalisée en médecine vétérinaire pour le traitement de cette pathologie fréquente chez le chien, et dont le traitement actuel repose sur l'administration per os de corticoïdes à l'origine de nombreux effets secondaires. / For several years now, nanocarriers have been considered as the most promising strategy to improve skin penetration of active ingredients. Moreover, these carriers are more efficient at targeting and controlling drug release into the skin, which leads to increased treatment efficiency and reduced side effects. Polymeric nanoparticles have been the object of an increasing number of studies due to their good physicochemical stability and prolonged release of active ingredients which is superior to any other carriers. Recently, the Laboratory of Pharmaceutical Engineering at Besançon proved in vivo, that polymeric nanoparticles with a diameter smaller or equivalent to I 00 nm specifically penetrated in inflamed skin of mice whilst no penetration was observed in healthy skin. The first aim ofthis thesis was to confirm this hypothesis on another animal's inflamed skin, in instance the pig. Our results confirm the poor penetration of polymeric nanoparticles in healthy skin ofpigs and show various degree ofpenetration depending on the size of the nanoparticles into the inflamed skin area. The second objective ofthis work was to evaluate the skin penetration of our polymeric nanoparticles in humans. A proof of concept has been developed in the Department of Dermatology at Besancon University Hospital. The first results ofthis clinical trial tend to confim1 the greater penetration ofour carrier, specifically in inflamed skin. In fact, no penetration of polymeric nanoparticles with a size close to 100 nm was observed in healthy skin ofvolunteers or in the non-inflamed skin of patients suffering from atopic dennatitis. Conversely, a high penetration ofthese carriers was observed in the skin lesions of patients with atopic dermatitis. If the results ofthis clinical trial are confirmed, we plan to load an anti-inflammatory or an immunosuppressive drug into the nanoparticles to evaluate the therapeutic value ofthese nanocarriers in human medicine in the treatment ofatopic dermatitis. Meanwhile, a similar study may be undertaken in veterinary medicine for the treatment of atopic dermatitis in dogs which is a common disease whose current treatment is based on the oral administration of corticosteroids and cause many undesirable side effects.

This diploma thesis is focused on the study of inverse fluorescence correlation spectroscopy, especially with the regard for the usage of different fluorescent probes and different sized analysed particles. At first, the proper concentration of fluorescent probes was determined. In this concentration is the probe considered as a medium surrounding the analysed particles. Based on this concentration, which was determined as 400 M, several sets of samples were prepared. This samples contained different concentration of polystyrene particles of 100 and 500 nm diameter and multilamellar liposomes. Then, the FCS curves of samples with different fluorescent probes were measured. Fluorescein, rhodamine 6G and Atto 488 were used as fluorescent probes. As a result from experiments, it was found, that particles with 100 nm diameter cannot be analysed with none of the fluorescent probes. Inverse FCS method can be applied to systems, that contains particles with 500 nm diameter and fluorescein. Systems with rhodamine 6G have the same behaviour as typical FCS measurement. It is caused by dimerization of this probe and it cannot be used for 500 nm particles. Liposome samples can be established with iFCS method, but the results are biased by random distribution of liposomes size.

The thesis studies detection of fluorescein for coloration experiments in surface and underground water. The first part of the work deals with the adsorption of fluorescein on active charcoal from water and desorption by means of different desorption solutions. The results of measurements are used for practice of coloration experiments in karst research. In the second part of thesis it is researched the option of automated record of fluorescence concentration and its detection by means of laser induced fluorescence with confocal microscope.

Several experiments were designed for utilization of fluorescence spectroscopy as a method of aerosol particle detection in a model of lungs. One of the experiments was to arranged use luminescent properties of DEHS (bis(2-ethylhexyl)decandioate) for calculating aerosol deposition. The outcome of this experiment was confirmation of clusters existence, which causes luminescence of DEHS. But the luminescence is not enough dependent on concentration and as such is not suitable for calculation of aerosol deposition. As the next experiment DEHS-fluorescein particles were generated by condensation monodisperse aerosol generator (CMAG), where water was used instead of isopropyl alcohol as a solvent. By this alteration the negative influence of DEHS was eliminated, which caused results refinements of aerosol deposition. Generation of fluorescein sodium salt particles by small-scale powder disperser (SSPD) was designed as a last experiment. The lower deposition efficiency measured by this method was caused by particles polydispersion. Photo records were used for documentation of Hot-spots. Outcomes of this study are new knowledge of fluorescence spectroscopy utilization for study of aerosol deposition and possibilities of fluorescent aerosol particles generation. Acquired data can serve for knowledge extension of possible detection methods for aerosol particles in the model of lung and can serve for validation of numerical simulations.

Development of methods for studying drug delivery systems is of great significance for the improvement of topical formulations. Active compounds for topical drug delivery are often formulated into gels and creams, that can be applied onto skin surfaces. It is important to know the extent of the permeability of the active compounds, in order to determine the medical effect. This study examines the possibilities of using an animal-free skin equivalent for penetration and permeation experiments, i.e. a silk scaffold integrated with viable human dermaland epidermal cells. Mammalian cell culturing together with silkconstruct formation, constituted the upstream bioprocess and acquisition of the skin equivalents. Permeability of fluorescently labelled Bovine Serum Albumin and Sodium Fluorescein salt was assessed, using a Franz- cell setup incorporated with the skin equivalents. Furthermore, fluorescence analysis and SDS-PAGE was performed on the collected samples, along with cryosectioning and image analysis of the skin equivalents. The results indicate variations in tissue integrity, leading to both high and low permeability. Fluorescence intensity can be correlated with the amount of sample liquid passing through. The model is still under development, hence more research is needed to draw a conclusion regarding the cellular composition of the skin equivalents, and how it influences permeability. / NextBioForm

Fluorescence-based imaging techniques provide a simple, highly sensitive method of studying live cells and whole organisms in real time. Without question, fluorophores such as GFP, fluorescein, and rhodamines have contributed vastly to our understanding of both cell biology and biochemistry. However, most of the fluorescent molecules currently utilized suffer from one major drawback, the use of visible light. Due to cellular autofluorescence and the absorbance of incident light by cellular components, fluorescence imaging with visible wavelength fluorophores often results in high background noise and thus a low signal-to-noise ratio. Fortunately, this situation can be ameliorated by altering the wavelength of light used during imaging. Near-infrared (NIR) light (650-900 nm) is poorly absorbed by cells; therefore, fluorophores excited by this light provide a high signal-to-noise ratio and low background in cellular systems. While these properties make NIR fluorophores ideal for cellular imaging, most currently available NIR molecules cannot be used in live cells. The first half of this thesis addresses the synthetic difficulties associated with preparing NIR fluorophores that can be used within living systems. Small molecule NIR fluorophores are inherently hydrophobic which makes them unsuitable for use in the aqueous environment of the cell. Water-solubility is imparted to these dyes through highly polar sulfonates, which subsequently prevents the dyes from entering the cell. The novel work presented here details vii synthetic routes to aid in the development of sulfonated NIR fluorophores, which can be delivered into live cells through the inclusion of an esterase-labile sulfonate protecting group. Application of these synthetic techniques should allow for the development of novel NIR fluorophores with intracellular applications. The second half of this thesis addresses the need for novel NIR imaging reagents. Although several classes of NIR scaffolds do exist, most NIR probes are derivatives of a single class, heptamethine indocyanines. The work described here increases this palette by displaying the ability of NIR oxazines to function as an imaging reagent in live cells and in vivo and as a molecular sensor of biologically-relevant environmental conditions. Combined, the work contained herein has the capacity to not only advance the current NIR toolkit, but to expand it so that fluorescence imaging can move out of the dark and into the NIR light.

Background: Cancer is a common disease, and the choice of treatment becomes more difficult over time due to chemotherapy resistant in cancer cells. To improve the in vitroassay and the individual cancer treatment, a luminescence-based endpoint assay, CellTiter Glo 2.0 was compared with the currently in use fluorescence endpoint assay, fluorometric microculture cytotoxic assay.  Aim: The aim of this study was to validate and compare the CellTiter Glo 2.0 assay with awell-established method (FMCA) and MTT [3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2Htetrazolium bromide] assay. Moreover, investigate whether the generated data can be used as a reference database for validation of patient samples in the future.   Materials and methods: The validation was performed on peripheral blood mononuclear cells from different healthy donors and two cell lines (HCT116-wt and HT-29) of colorectal cancer carcinoma were ordered frozen from American Type Culture Collection. Analysis was also done in solid samples (ovarian and kidney cancer cells). To get as correct evaluation as possible all materials were analyzed in parallel between the two methods.  Results and conclusion: A clear trend was observed when using CellTiter Glo 2.0 assay,post FMCA directly on tumor cells. This setup, makes it possible to collect reference data in the future. In addition, a high spread of the survival index data was noted between the two methods. The reason is still unknown but could be due to the low number of tested tumor cells, therefore more tumor cells need to be tested in future studies