Dissertations / Theses on the topic 'Biolabel'

Nanoparticles (nPs) demonstrate significant advantages over other sensor and marker technologies. The most useful optical nanosensor and label platform for biological samples would be non-toxic, hydrophilic, resistant to non-specific protein interactions and degradation over time or under harsh conditions, highly retentive of entrapped components, and easily functionalized for target specificity. The work described here is part of an investigation into the fabrication and application of polyacrylamide, polyacrylamide/silica hybrid, and polystyrene-core silica-shell nPs. Polyacrylamide (PA) nP nitric oxide (NO) sensors were made by co-entrapping 4, 5-diaminofluorescein (DAF-2) and Texas Red dextran in 60 nm PAnPs. Sensors were used to measure NO produced by a diazeniumdiolate NO donor in solution, and have a response time of 30 seconds or less. Entrapped DAF-2 was protected from non-specific interactions with bovine serum albumin (BSA). Sensor response to NO in FBS solutions was reduced compared to buffer, although improvement over free dyes was observed. The sensors were applied to J477A.1 macrophages as well as a HT1080 cell line (HTRiNOS) in preliminary studies for measuring intracellular NO production. Polyacrylamide/silica hybrid nPs were fabricated and nP architecture was evaluated by transmission electron microscopy. Isopycnic centrifugation of nP samples indicates that the hybrid nPs have a density between 1.70 and 1.76 g/cm³. Silica in the hybrid nPs was covalently labeled with Texas Red, suggesting that the hybrid nPs may be used as ratiometric or possibly multiplexed sensors. Hybrid nPs coated with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibit reduced adsorption of TRITC-BSA compared to uncoated hybrid nPs. Hybrid nP pH sensors were prepared and responded reproducibly and reversibly to changes in pH, nominally from pH 6.0 to 8.0. Core-shell nPs for scintillation proximity assay (SPA) were fabricated by entrapping the scintillants p-terphenyl and 4-bis(4-methyl-5-phenyl-2oxyzolyl)benzene in polystyrene, onto which silica shells were subsequently added. Core-shell nPs were found to have a scintillation response similar to that of shell-less polystyrene cores, indicating that the presence of the silica shells does not reduce scintillation efficiency. Preliminary studies using core-shell nPS for biotin-streptavidin binding SPA do not indicate an enhancement in scintillation efficiency, although this may be due to high nP:radiolabeled analyte ratios.

Thesis (Ph. D.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains v, 137 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 126-137).

A study to optimise an IgG based immunosensor is presented, that has been carried out by absorbing monolayers to a gold transducer surface at varying immersion times and temperatures. The theory and kinetics of monolayer adsorption are analysed and discussed. Existing mathematical models are reviewed and experimentally researched, to highlight gaps in knowledge that would facilitate high quality, cost effective immunosensor production. The creation of two mathematical models to predict monolayer adsorption kinetics and optimal immersion times are discussed. Details are provided of how the new mathematical models may be advanced, and how the production of immunosensors may be further improved. The first novel mathematical model (PTCS) has been created to model the presence of two sequentially forming structures on the surface of a substrate. It gives an insight into the percentages of each structure on the surface, along with the actual adsorption process. This model provides a good fit to all applicable experimental data and has allowed the deduction of optimum immersion times. The second novel model (PIF) provides a greater insight than existing models into the individual contributions to surface coverage by both random and island growth. This allows an insight into how the monolayer surface is covered, which is critical to determine the optimum conditions for adsorption. This model also provides a good fit to the isotherm data it has been applied to. To provide a thorough understanding of the bulk properties of monolayer formation over the gold transducer, and how these properties vary with immersion time and temperature, various measurement techniques have been employed. Electrochemical Impedance Spectroscopy (EIS) has been the principle measurement technique used to measure the bulk properties, but confirmation studies have also been carried out including, Contact angle measurements, FTIR microscopy with BSA molecular labels, Fluorescence microscopy for small adsorbed molecules and AFM for layers assembled from macromolecules. The data generated from the different techniques show consistency with the arguments discussed in each instance. Two different IgG adsorption processes have been compared. These include direct IgG addition and a multilayered streptavidin-based process. The results indicate that IgG molecules adsorbed via the streptavidin based multilayer process are more vertically orientated and have a higher packing density of IgG molecules. Keywords: Self Assembled Monolayer, impedance-based immunoassay, Streptavidin, biotinylated IgG, mathematical adsorption modelling.

In 2015, the United nations presented the 17 Global Goals that would put an end to extreme poverty, inequality and climate change by 2030. One of these goals was clean water and sanitation. In 2015 1.8 billion people did not have access to clean water. Because of the contaminated water, one million people die every year worldwide. Africa, and especially Ghana, has had a high development in the recent years. The population has grown and more resources are needed. Clean water in Ghana is not a given matter, three million people live without access to clean water. To work towards the Global Goal water can be clean locally. A simple and cheap way is to build slow sand filters, which also are the purpose of this project. These filters purify the water mechanically, chemically and biologically. The biologically purification takes place in the biolayer that grows on the sand inside the filter and it consumes contaminants in the water. It takes about a month for the biolayer to be fully developed and clean the water to its full potential. The positive aspects with sand filters are that people get healthier and can save money that can be invested in education or business. It can also reduce the need for water in plastic bags or bottles and would reduce littering. The companies that produce this water could end their business and air pollutions would be reduced as well.   During this project, slow sand filters have been tested and evaluated in Sweden and Ghana with the purpose to develop a theoretical filter that benefits the biolayer under local conditions in Ghana, this was of the one aims. Experiments in Sweden showed that the flow decreased with increased sand height and decreased hydraulic head. In Ghana three filters were built with the sand heights 30, 50 and 80 cm to clean 7 litres of drinking water for a family of four. None of these produced drinkable water by WHO’s and EU’s standards.   The next aim was to understand which chemical and physical factors that effected the development of the biolayer. The detected relations were absolute conductivity, total alkalinity, coliform bacteria and oxidantial reduction potential which were between the biolayer in the 30 and 50 filters.   The flow rate in Ghana was too high and to lower it, a new diffuser with smaller holes would be built to get the recommended flow of 0,4 m3/m2/h. A too high flow broke the bound between the biolayer and made an uncomfortable environment. A sedimentation should be installed before the sand filter to reduce the variations of the incoming water such as turbidity, suspended solids etc., so the biolayer would flourish. It was not enough dissolved oxygen in the water so the pause period would be decreased to 12 hours to get more oxygen in the filter each day. For a sand filter to work as planned a lot of attention should be given to the filter. It is a system that should be used all the time for the best purification. To build a filter takes a lot of time and it also takes time for the biolayer to develop. If it is not going to be used much, another treatment method should be used.   The last aim was to evaluate the cost of the materials that could be bought locally to the filter. One filter cost about 130 GHS.
2015 tog Förenta nationerna fram de 17 globala målen för att få ett slut på extrem fattigdom, ojämlikhet och klimatförändringen till år 2030. Ett av dessa mål handlar om rent vatten och sanitet. 2015 var det 1,8 miljarder människor som inte hade tillgång till rent vatten. På grund av det förorenade vattnet dör en miljon människor i hela världen varje år. Afrika, och speciellt Ghana, har haft en snabb utveckling de senaste åren. Folkmängden har ökat och mer naturresurser behövs. Rent vatten i Ghana är inte en självklarhet, tre miljoner människor lever idag utan tillgång till rent vatten i Ghana. Ett sätt för att jobba mot det globala målet är rening av vatten lokalt. Ett enkelt och billigt sätt är att bygga långsamsandfilter, vilket även var syftet med denna studien. Dessa filter renar vattnet mekaniskt, kemiskt och biologiskt. Den biologiska reningen sker av en biofilm som växer på sanden inuti filtret som konsumerar föroreningar i vattnet. Det tar ungefär en månad för biofilmen att bli färdigutvecklad och rena vattnet till sin fulla potential. Det positiva med sandfilter är att människorna skulle bli friskare och spara pengar som kan investeras på utbildning eller företag. Ur miljöpunkt skulle reduktionen av köpt vatten i plastpåsar och flaskor minska nedskräpningen och företagen som producerar dessa kan avsluta produktionen och därmed minska luftföroreningar.    Under detta projekt har långsamsandfilter utvärderats både i Sverige och Ghana för att utveckla ett nytt teoretiskt filter som gynnar tillväxten av biofilm under lokala förhållanden i Ghana, vilket var ett mål. Experimenten i Sverige visade att flödet sjönk med ökad sandhöjd, men även med minskat hydrauliskt tryck. I Ghana byggdes tre filter med sand höjderna 30, 50 och 80 cm för att rena 7 liter dricksvatten till en familj på fyra. Ingen av dessa lyckades producera drickbart vatten enligt WHO:s och EU:s standarder.   Nästa mål var att förstå vilka av de kemiska och fysiska faktorer som påverkade biofilmstillväxten. Det förhållanden som upptäcktes var absolut konduktivitet, total alkalinitet, coliform bacteria och oxidential reduction potential vilket fanns i 30 och 50 filtret.   Flödet i Ghana var för högt, så för att minska det skulle en diffusör med mindre hål byggas för att få det rekommenderade flödet 0,4 m3/m2/h. Ett för högt flöde gjorde sönder bindingen mellan biofilmen och skapade en otrivsam miljö. En sedimentation skulle installeras innan sandfiltret för att minska variationer på ingående vatten i filtret för att få biofilmen att trivas bättre. Det fanns för lite löst syre i vattnet och om pausperioden minskas till 12 timmar skulle mer syre i filtret varje dag. För att ett sandfilter ska fungera som planerat måste mycket tid läggas på filtret. Sandfilter är ett system som bör används ofta för bästa rening. Att bygga ett filter kräver mycket tid, samt att det tar tid innan biofilmen har utvecklats. Om sandfiltret inte kommer används mycket föreslås att en annan metod används istället.   Det sista målet var att utvärdera kostnaden av materialen som kunde köpas lokalt till filtret. Ett filter kostade runt 130 GHS.

Les ARN interférents sont de puissants outils thérapeutiques et biologiques pour la mise en silence de l'expression des gènes. Afin d'améliorer leur stabilité enzymatique, leur biodistribution et leur pénétration cellulaire, nous proposons de développer une approche prodrogue d'ARN interférent. Ce manuscrit rapporte la synthèse et l'évaluation de pro-ARN masqués temporairement par des groupements biolabiles susceptibles d'être hydrolysés dans les cellules afin de libérer l'ARN naturel actif. Deux types de modifications sont présentés : des groupes acétalesters enlevés par des carboxyestérases et des groupes alkyldithiométhyles sensibles à un environnement réducteur. Dans une première partie, une nouvelle méthode de synthèse de pro-ARN partiellement modifiés en position 2' par des groupements acétalesters est décrite. Plusieurs groupements variant par leur caractère lipophile ou cationique sont évalués. Des résultats prometteurs d'études physico-chimiques, de stabilité enzymatique, de pénétration cellulaire et d'inhibition de gènes mettent en valeur l'intérêt d'utiliser certains pro-ARN modifiés en tant qu'outils thérapeutiques. Une deuxième partie présente une voie de synthèse originale de pro-ARN modifiés en position 2' par des groupements alkyldithiométyles. Les propriétés physico-chimiques, la stabilité enzymatique et le démasquage de ces pro-ARN sont décrits. Parallèlement, l'étude d'une réaction d'échange thiol-disulfure permettant l'incorporation de liens disulfures intrabrin au sein de duplex d'ARN et de constructions tige-boucles est détaillée dans ce manuscrit
SiRNA are powerful therapeutic and biological tools for gene silencing. In the aim of improving their stability, their biodistribution and their cellular delivery, we propose to develop a siRNA prodrug-like approach.This manuscript reports the synthesis and the study of pro-RNA temporarily masked by biolabile groups which could be hydrolyzed inside cells in order to release the active unmodified RNA. Two types of modifications are presented: acetalester groups removed by carboxyesterases and alkyldithiomethyl groups cleaved in a reducing environment within cells.In a first part, a new synthesis strategy of partially modified 2'-O-acetalester pro-RNA is described. Several acetalester groups varying in their lipophilicity and their charge are evaluated. Promising results obtained in physical-chemical studies, enzymatic stability and gene inhibition highlight the use of these modified pro-RNA as therapeutic drugs. A second part introduces an original approach for the synthesis of 2'-O-alkyldithiomethyl pro-RNA. The physical-chemical properties, the enzymatic stability and the unmasking of this pro-RNA are described.Moreover, the study of a thiol-disulfide exchange reaction allowing the incorporation of intrastrand disulfide bond into secondary structure duplex and hairpin is reported in this manuscript

The relatively young discipline of astronautics represents one of the scientifically most fascinating and technologically advanced achievements of our time. The human exploration in space does not offer only extraordinary research possibilities but also demands high requirements from man and technology. The space environment provides a lot of attractive experimental tools towards the understanding of fundamental mechanism in natural sciences. It has been shown that especially reduced gravity and elevated radiation, two distinctive factors in space, influence the behavior of biological systems significantly. For this reason one of the key objectives on board of an earth orbiting laboratory is the research in the field of life sciences, covering the broad range from botany, human physiology and crew health up to biotechnology. The Columbus Module is the only European low gravity platform that allows researchers to perform ambitious experiments in a continuous time frame up to several months. Biolab is part of the initial outfitting of the Columbus Laboratory; it is a multi-user facility supporting research in the field of biology, e.g. effect of microgravity and space radiation on cell cultures, micro-organisms, small plants and small invertebrates. The Biolab IEC are projects designed to work in the automatic part of Biolab. In this moment in the TO-53 department of Airbus Defence & Space (formerly Astrium) there are two experiments that are in phase C/D of the development and they are the subject of this thesis: CELLRAD and CYTOSKELETON. They will be launched in soft configuration, that means packed inside a block of foam that has the task to reduce the launch loads on the payload. Until 10 years ago the payloads which were launched in soft configuration were supposed to be structural safe by themselves and a specific structural analysis could be waived on them; with the opening of the launchers market to private companies (that are not under the direct control of the international space agencies), the requirements on the verifications of payloads are changed and they have become much more conservative. In 2012 a new random environment has been introduced due to the new Space-X launch specification that results to be particularly challenging for the soft launched payloads. The last ESA specification requires to perform structural analysis on the payload for combined loads (random vibration, quasi-steady acceleration and pressure). The aim of this thesis is to create FEM models able to reproduce the launch configuration and to verify that all the margins of safety are positive and to show how they change because of the new Space-X random environment. In case the results are negative, improved design solution are implemented. Based on the FEM result a study of the joins has been carried out and, when needed, a crack growth analysis has been performed.

A presença de Salmonella ssp torna os alimentos impróprios para consumo humano, sendo necessário métodos de detecção confiáveis para a salmonelose, nos laboratórios de controle de qualidade e de diagnóstico. A detecção de Salmonella spp. em alimentos pelo método tradicional é demorada, o que explica a abundância de sistemas automatizados e kits disponíveis comercialmente para detecção rápida deste patógeno. Muitos desses métodos alternativos são validados por organizações internacionalmente reconhecidas, mas podem apresentar resultados diferentes quando utilizados com alimentos naturalmente contaminados. Principalmente pelo fato de que a maioria das validações é realizada com amostras contaminadas artificialmente, não representando a realidade da microbiota normal das amostras. O objetivo deste trabalho é a cavaliação dos métodos rápidos alternativos VIDAS® Salmonella (SLM), BAX® System e método tradicional (IN 62, MAPA) na detecção de Salmonella spp. em alimentos. Os métodos foram testados com amostras naturalmente contaminadas de carne de frango, suína e bovina. O método tradicional detectou 20 amostras (2,1 %) positivas para Salmonella spp. enquanto o método VIDAS® encontrou 87 amostras positivas (9,2 %), demonstrando uma especificidade de 93,0 %. No método BAX® foram 741 (12,7 %) resultados positivos para Salmonella spp. sendo que a metodologia tradicional detectou 230 (4,0 %) amostras positivas indicando 90,5 % de especificidade. Do total de 221 amostras de Salmonella spp. sorotipadas, o sorovar mais freqüente foi S. Minnesota (n=28; 15,7 %), seguida de S. Mbandaka (n=17; 9,5 %), S. Schwarzengrund (n=15; 8,4 %), S. Saintpaul (n=14; 7,8 %) e S. Enteritidis (n=13; 7,3 %). A diminuição no isolamento de S. Enteritidis (SE) pode ser uma conseqüência do Programa Nacional de Redução de Patógenos implantado pelo Ministério da Agricultura, Pecuária e Abastecimento, em 2003 e também pela vacinação para SE das matrizes de corte. Não foi possível saber com segurança se essa diminuição no isolamento de SE levou a um impacto positivo na saúde pública. É muito importante o contínuo monitoramento e a melhoria do diagnóstico em casos esporádicos e de surtos de infecção humana. Embora a freqüência do isolamento de S. Minnesota tenha aumentado, esse sorovar não foi associado a casos esporádicos e surtos de salmonelose ocorridos em 2009 e 2010 no Paraná. No entanto, é essencial que as autoridades sanitárias avaliem a repercussão na saúde Pública do aumento do isolamento deste sorovar em carne de frango.
The presence of Salmonella ssp makes food unfit for human consumption, requiring reliable detection methods for Salmonella in laboratories for quality control and diagnostics. The detection of Salmonella spp. in food by the traditional method is time consuming, which explains the abundance of automated systems and commercially available kits for rapid detection of this pathogen. Many of these alternative methods are validated by internationally recognized organizations, but may have different results when used with naturally contaminated food. Especially the fact that most of the validations is performed with artificially contaminated samples do not represent the reality of the normal microbiota of the samples. The objective of this work is the quick alternative methods cavaliação VIDAS ® Salmonella (SLM), BAX ® System and the traditional method (62 IN, MAP) in the detection of Salmonella spp. in foods. The methods were tested with naturally contaminated samples of chicken, pork and beef. The traditional method detected 20 samples (2.1%) tested positive for Salmonella spp. VIDAS ® while the method found 87 positive samples (9.2%), demonstrating a specificity of 93.0%. In the BAX ® method were 741 (12.7%) tested positive for Salmonella spp. being that the traditional method detected 230 (4.0%) positive samples indicating 90.5% specificity. Of the total 221 samples of Salmonella spp. serotyped, the most frequent serovar was S.Minnesota (n = 28, 15.7%), followed by S. Mbandaka (n = 17, 9.5%), S. Schwarzengrund (n = 15, 8.4%), S. Saintpaul (n = 14, 7.8%) and S. Enteritidis (n = 13, 7.3%). The decrease in the isolation of S. Enteritidis (SE) may be a consequence of the National Pathogen Reduction implemented by the Ministry of Agriculture, Livestock and Supply, in 2003 and also for SE by vaccination of cutting dies. It was not possible to know with certainty whether this decrease in the isolation of SE led to a positive impact on public health. It is very important to the continuous monitoring and improvement of diagnosis in sporadic cases and outbreaks of human infection. Although the frequency of isolation of S. Minnesota has increased, this serovar was not associated with sporadic cases and outbreaks of salmonellosis occurred in 2009 and 2010 in Paraná. However, it is essential that health officials to assess the public health impact of increased isolation of this serovar in poultry meat.

Le travail décrit dans cette thèse se concentre sur le développement de matériaux « durs et mous » ainsi que leur interaction avec les cellules biologiques pour une application finale dans le domaine de la théranostique couvrant l'imagerie, la détection, la thérapie génique et la thérapie du cancer. Dans ce contexte, nous avons tout d'abord étudié l'utilisation de complexes (II) de platine phosphorescents auto-assemblés comme sonde cellulaire. Nous avons étendu l'idée de bio-imagerie en introduisant un concept d’imagerie basée sur l’émission stimulée où nous étions en mesure de générer un laser provenant d'une cellule biologique unique sans utiliser de cavité optique conventionnelle. En outre, des nano-transporteurs multifonctionnels à base de matières poreuses dures à savoir des zéolithes L et des nanoparticules de silice mésoporeuse pour de la « drug delivery » (relargage de médicaments et d’oligonucléotides) in vitro ide ont été développés avec succès et testés pour le traitement du glioblastome. Un autre nano-vecteur, qui est construit à partir de silice biodégradable, a également été synthétisé et sa capacité d'encapsuler des protéines et de les libérer dans les cellules vivantes lors de la dégradation de la structure dans un environnement réducteur a été démontrée. Enfin, l'utilisation de nouveaux matériaux plasmonique sur la base de nanoparticules d'argent enrobées de silice cassable pour la détection d'agents réducteurs a été mise en valeur
The work described in this thesis focuses on the development of soft- and hard-materials as well as their interaction with biological cells for applications in the field of theranostics covering imaging, sensing, and gene, and cancer therapy. In this context, we first investigated the use of phosphorescent self-assembled platinum(II) complexes as cellular probes. We extended the concept stimulated emission-based bioimaging by generating a laser-like emission coming from a single biological cell without using any conventional optical cavity. In addition, we successfully developed multifunctional nanocarriers based on porous hard materials, namely zeolites-L and mesoporous silica nanoparticles for drug and oligonucleotide delivery in vitro and they were tested to treat glioblastoma. Another nanovector, which is constructed from biodegradable silica, was also synthesized and its ability to encapsulate proteins and release them in living cells upon degradation of the structure in reductive environment was demonstrated. Finally, the use of novel plasmonic structures based on breakable silica-coated silver nanoparticles for detection of reducing agents was successfully investigated

En dépit d’importants progrès dans le domaine de la thérapie anti-cancéreuse, les traitements actuels restent controversés, notamment en raison de la quantité importante d'effets secondaires induits. L'immunothérapie ciblée a récemment émergée en tant qu'alternative, afin d'améliorer les modalités de traitement des patients atteints du cancer. Malgré tout, seul un nombre limité d’approches sont aujourd’hui disponibles, et une grande partie des problèmes demeurent actuellement sans solution. C'est dans ce contexte que nous nous sommes intéressés à la conception de structures biomoléculaires innovantes et bifonctionnelles, capables de rediriger des anticorps endogènes, présents naturellement dans la circulation sanguine de l'homme, contre les tumeurs et, ce, sans immunisation préalable. Les anticorps naturels circulant étant polyspécifiques et ayant la capacité d’interagir avec des antigènes glycosylés, nous nous sommes plus particulièrement concentrés sur la conception de glycoconjugués multivalents, ligands d’anticorps endogènes. Une première partie de notre étude a consisté à synthétiser différents glycodendrimères multivalents, reposant sur des châssis peptidiques et obtenus par ligations chimiosélectives, tout en variant la nature du motif glycosylé et des plateformes, ainsi que la valence du conjugué. Puis, dans un second temps, des tests d’interaction par biopuce ont été mis en place avec une lectine modèle, la lectine Helix Pomatia Agglutinin (HPA). Des protocoles expérimentaux visant à calculer des constantes de dissociation de surface, ainsi que des IC50 ont été mis en place, permettant d’identifier de bons ligands de HPA avec des affinités de l’ordre du nanomolaire. Les tests par biopuce ont ensuite été confirmés avec d’autres méthodes d’analyses (BLI, ELLA). Finalement, afin d'identifier des architectures tri-dimensionnelles permettant une affinité optimale avec des anticorps, les tests d’interaction ont été adaptés au criblage de séra humains. Un large panel de glycoconjugués a alors été criblé par biopuce avec une vingtaine de séra, permettant la détermination de structures glycosylés prometteuses, qui pourront par la suite être utilisées dans le cadre de notre approche anti-cancéreuse
Despite significant progress in anti-cancer therapy, current treatments are still controversial due to numerous side effects. Targeted immunotherapy recently emerged as an ideal alternative to improve treatment modalities for cancer patients. However, very limited approaches are available today and major issues remain to be addressed. In this context, we are interested in the design of biomolecular structures, innovative and bifunctional, able to hijack endogenous antibodies - which are naturally present in the human blood stream - toward cancer cells without pre-immunisation. Since natural circulating antibodies are polyspecific and have the ability to interact with multiple carbohydrate antigens, we focused on the design of multivalent glycodendrimers, as ligands for endogenous antibodies. The first part of our study consisted in synthesizing several multivalent glycoconjugates, based on peptide scaffolds and obtained by chemoselective ligations. To evaluate their influence on antibodies, the nature of both the carbohydrate and the scaffold, and the valency were varied. Then, in a second part of the study, microarray assays were developed with a model lectin, the Helix Pomatia Agglutinin (HPA). Experimental procedures were designed to determine surface dissociation constant and IC50 values, leading to the identification of high affinity ligands for HPA in the nanomolar range. Microarray assays were confirmed by other analytical methods (BLI, ELLA). Finally, the assays on slides were adapted to human sera screening, in order to identify tridimensional architectures highly affine to sera antibodies. A large panel of glycoconjugates were screened by microarray with around twenty sera, leading to the determination of promising glycosylated structures, as antibody ligands. The latter could be subsequently used for our anti-cancer approach

Orientador: Gilberto de Nucci
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-21T11:09:12Z (GMT). No. of bitstreams: 1 Modolo_FabianaDuarteMendese_D.pdf: 4445677 bytes, checksum: 5d3f6fadf0884c24742741c6059792dc (MD5) Previous issue date: 2012
Resumo: Método rápido, sensível e específico foi desenvolvido para quantificar ciprofibrato no plasma humano, utilizando bezafibrato como padrão interno (SI). O analito e o padrão interno foram extraídos do plasma, por extração líquido-líquido, usando um solvente orgânico (éter etílico/diclorometano 70/30 (v/v)). Os extratos foram analisados por cromatografia líquida de alta eficiência acoplada à espectrometria de massa (HPLC-MS/MS), do tipo eletrospray. A cromatografia foi realizada com coluna Gênesis C18 4?m analítica (4,6 x 150mm id) em fase móvel composta de acetonitrila/água (70/30, v/v) e 1 mM de ácido acético. O método teve um tempo de corrida cromatográfica de 2,7min e curva de calibração linear no intervalo de 0,1 - 60 ?g/mL (R2> 0,99). O limite de quantificação foi de 0,1?g/mL. Os valores de acurácia e precisão intra-corrida e inter-corrida foram inferiores a 13,5%. Os testes de estabilidade não indicaram degradação significativa. A recuperação do ciprofibrato foi de 81,2%, 73,3% e 76,2% para as concentrações de 0,3, 5,0 e 48,0 ?g/mL, respectivamente. Para o ciprofibrato, os parâmetros otimizados da energia declustering, energia de colisão e energia de saída foram -51 (V), -16 (eV) e -5 (V), respectivamente. O método também foi validado sem o uso do padrão interno. Este procedimento HPLC-MS/MS foi usado para avaliar a bioequivalência de duas formulações de comprimidos ciprofibrato 100mg, em voluntários sadios de ambos os sexos. Os seguintes parâmetros farmacocinéticos foram obtidos das curvas de concentração plasmática de cipofibrato versus tempo: ASCULTIMO, ASC0-168h, CMAX e TMAX. A média geométrica com intervalo de confiança correspondente a 90% (CI) para a razão de Teste / Referência foram: 93,80% (IC 90%= 88,16 - 99,79%) para CMAX, 98,31% (90% CI = 94,91-101,83%) para ASCULTIMO e 97,67% (90% CI = 94,45-101,01%) para ASC0-168h. Uma vez que o intervalo de confiança de 90% para a razão geométrica de CMAX , ASCÚLTIMO e ASC 0 -168h estavam dentro do intervalo de 80% -125% proposto pelo FDA dos EUA, concluiu-se que ciprofibrato (Lipless® 100mg comprimido) formulação fabricado pela Biolab Sanus Farmacêutica Ltda. é bioequivalente à formulação Oroxadin® (100mg comprimido) para a velocidade e a extensão da absorção
Abstract: A rapid, sensitive and specific method for quantifying ciprofibrate in human plasma, using bezafibrate as the internal standard (IS) is described. The analyte and the IS were extracted from plasma, by liquid-liquid extraction using an organic solvent (diethyl ether / dichloromethane 70/30 (v/v)). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed using Genesis C18 4?m analytical column (4.6mm x 150mm i.d.) and a mobile phase consisting of acetonitrile/water (70/30, v/v) and 1mM acetic acid. The method had a chromatographic run time of 2.7 min and a linear calibration curve over the range 0.1-60 ?g/ml (R2>0.99). The limit of quantification was 0.1?g/ml. The intra and interday accuracy and precision values of the assay were less than 13.5%. The stability tests indicated no significant degradation. The recovery of ciprofibrate was 81.2%, 73.3% and 76.2% for the 0.3, 5.0 and 48.0?g/mL standard concentrations, respectively. For ciprofibrate, the optimized parameters of the declustering potential, collision energy and collision exit potential were -51 (V), -16 (eV) and -5 (V), respectively. The method was also validated without the use of the internal standard. This HPLC-MS/MS procedure was used to assess the bioequivalence of two ciprofibrate 100mg tablet formulations, in healthy volunteers of both sexes. The following pharmacokinetic parameters were obtained from the ciprofibrate plasma concentration vs. time curves: AUCLAST, AUC0-168h, CMAX and TMAX. The geometric mean with corresponding 90% confidence interval (CI) for Test/Reference percent ratios were 93.80% (90% CI= 88.16 - 99.79%) for CMAX, 98.31% (90% CI= 94.91 - 101.83%) for AUCLAST and 97.67% (90% CI= 94.45 - 101.01%) for AUC0-168h. Since the 90% CI for AUCLAST, AUC0-168h and CMAX ratios were within the 80-125% interval proposed by the US FDA, it was concluded that ciprofibrate (Lipless® 100 mg tablet) formulation manufactured by Biolab Sanus Farmacêutica Ltda. is bioequivalent to the Oroxadin® (100mg tablet) formulation for both the rate and the extent of absorption
Doutorado
Clinica Medica
Doutora em Clínica Médica

I documenti cartacei vengono attualmente rimpiazzati dalle loro versioni elettroniche, che contengono anche alcune caratteristiche biometriche; questo ha permesso il controllo automatico, sia quando il documento viene rilasciato, sia quando l'identità della persona deve essere verificata. Per rendere questo possibile è necessario che la fotografia rispetti degli standard di qualità. Lo standard ISO/IEC 19794-5 fornisce alcune guide linea ed esempi di immagini di volto accettabili e non-accettabili. Negli ultimi anni, molte aziende hanno sviluppato SDK con lo scopo di implementare i test proposti dallo standard. La tesi si prefigura il compito di fornire un framework che fornisca buone prestazioni, sia per quanto riguarda i tempi sia per l'accuratezza dei risultati.

Nanotechnology is more and more present in our world today and different fields are taking advantage of its possibilities. Among others, microscopists have been interested in using nanoparticles in combination with available techniques, one of which is fluorescence microscopy. Lanthanide-doped nanoparticles for example have been studied for many years now for their interesting luminescence and upconversion characteristics. This research presents the development of upconverting biolabels operating in the near-infrared (NIR) to eventually allow scientists to probe deeper into tissues using fluorescence microscopy. Two distinct types of nanoparticles were fabricated using the lanthanide ions Yb3+ and Tm3+ for their upconversion capabilities (from 980 to 800 nm) within the biological window (700 to 1000 nm). The first one, an annealed silica-coated LaF3:Yb,Tm nanoparticle, could not be used as a biolabel due to its lack of dispersibility in aqueous environment. However, the second type, a silica-coated NaYF4:Yb,Tm nanoparticles proved to be very promising. Two surface modifications of these particles were successfully performed. The first introduced NH2 groups while the second incorporated polyethyleneglycol (PEG). The latter was achieved using two distinct methods: one through a reaction with the amino groups and one through a second silica coating involving PEGsilanes. Stable dispersions of these PEGylated nanoparticles were obtained and imaging of ovarian cancer cells grown in their presence showed that they interact with the cells although the nature of this interaction is still to be determined.

Relatório de estágio de mestrado, Análises Clínicas, Universidade de Lisboa, Faculdade de Farmácia, 2016
Este trabalho encontra-se dividido em duas partes distintas: relatório de estágio e monografia. Na primeira parte é apresentado o relatório de estágio profissional realizado no Biolabor, Laboratório de Análises Clínicas. Neste relatório são descritos os equipamentos e respectivas técnicas manuais utilizadas durante o estágio, nas áreas de bioquímica, microbiologia, hematologia e imunologia. Na fase pré-analítica abordou-se as secções de triagem e colheita de produtos biológicos. Na triagem dos produtos biológicos há que ter vários aspectos em conta, pois é a partir desta fase que os produtos são analisados e encaminhados para dar os resultados aos utentes. É muito importante verificar se os produtos estão em condições de serem analisados, ou seja, se estão na conformidade necessária à execução analítica. Caso isto não aconteça é necessário que haja uma rejeição da amostra e será pedido ao utente que venha repetir noutro dia, consoante a urgência. Existem situações em que a amostra pode vir a ser utilizada, mesmo não estando em conformidade, quando existe indicação médica para tal. Por outro lado, no que diz respeito à colheita dos produtos biológicos, também há que ter uma elevada atenção, de modo a que a colheita seja o mais eficaz possível, tanto no que diz respeito à satisfação do utente, como na obtenção de um produto conforme para o laboratório. No que diz respeito à fase analítica, ao longo desta primeira parte do trabalho, descrevem-se os vários equipamentos que foram utilizados durante o estágio profissional, tendo todos uma manipulação diferente e seguindo um controlo de qualidade muito rigoroso. Para além disso, foram também realizadas várias técnicas manuais ainda em utilização neste laboratório, imprescindíveis para uma boa obtenção de resultados analíticos. Para finalizar, aborda-se os princípios do controlo de qualidade posto em prática no laboratório, sendo o seu cumprimento de grande importância para a obtenção de resultados de elevada fiabilidade. A fase pós-analítica integra a validação biopatológica pela directora técnica do laboratório, sem a qual a entrega dos resultados aos utentes não é possível A segunda parte deste relatório consiste numa monografia sobre o tema “Plaquetas e as suas implicações nas doenças vasculares“, onde é feita uma revisão bibliográfica sobre o assunto, sendo um dos objectivos encontrar uma justificação para um caso clínico real. Inicialmente, é feita uma introdução ao tema das plaquetas, abordando não só a sua fisiologia (estrutura, formação e funções das plaquetas) como também o seu papel na inflamação e as alterações quantitativas e qualitativas que podem ocorrer nas plaquetas e que podem levar a distúrbios patogénicos no organismo humano. Para finalizar este grande capítulo, são abordadas algumas aplicações das análises clínicas no diagnóstico de patologias plaquetárias. Neste subcapítulo dá-se especial importância ao PFA-100, um novo método de avaliação da função plaquetária que apresenta vantagens significativas em comparação com o tempo de hemorragia. Tal como o título indica, as doenças vasculares também são um dos grandes capítulos deste trabalho, sendo essencial a sua compreensão para criar uma relação entre as plaquetas e as doenças vasculares. Assim, são abordadas as doenças arteriais (doença das artérias coronárias e doença das artérias periféricas) e as doenças venosas (trombose das veias profundas). Não são abordadas todas as doenças arteriais e venosas existentes, dando-se maior relevância às que são importantes para a compreensão da relação entre as plaquetas e as doenças vasculares. Sendo uma das principais motivações deste trabalho, é descrito um caso clínico real que relaciona a trombocitopenia como consequência de doenças vasculares, sendo feita uma descrição cronológica dos acontecimentos que têm ocorrido ao longo dos anos e da conclusão de cada um dos procedimentos aplicados até agora. Em forma de conclusão, são descritas algumas abordagens terapêuticas, como os tratamentos antiplaquetários aplicados na prevenção do AVC em pacientes com doenças vasculares, a monitorização/controlo laboratorial da terapêutica e os novos agentes antiplaquetários que estão a começar a ser utilizados no mercado farmacêutico.
This work is divided into two distinct parts: the internship report and the monograph. On the first part it's presented the professional internship report held at Biolabor, Clinical Analysis Laboratory. In this report, it's described the equipment and manual techniques used during the internship in biochemistry, microbiology, hematology and immunology. In the pre-analytical phase it was focused the sections of screening and harvest of biological products. Screening of biological products it's necessary to take various aspects into account, since it's from this stage on that the products are analyzed and forwarded to give the results to users. It's very important to check if the products are under the right conditions to be analyzed, i.e., whether they are under the conformity to the required analytical process. If this is not the case it's necessary to reject the sample and it will be requested the user to come again to repeat the sampling another day, depending on the urgency. There are situations where the sample might be used, even if it's not in conformity. Such cases occur when there is medical indication for such. On the other hand, regarding the harvest of the biological products, there should also be given high attention, so that the harvest is as effective as possible, both with regard to the satisfaction of the user, and with the obtainment of the product according to a laboratory analysis. Regarding the analytic phase, during this first part of the work, it's described the various equipment that were used during the professional internship, all with different handling and following a very rigorous quality control. Besides this, it was also performed various manual techniques still used in laboratory, essential for obtaining good analytical results. Finally, it’s discussed the principles of quality control implemented in the laboratory and its application of great importance to obtain highly reliable results. The post-analytical phase integrates biopathologic validation by the laboratory's technical director, without which the delivery of results to the users would not be possible. A monograph entitled "Platelets and its implications for vascular diseases" is presented in the second part of this document. A literature review about this subject was made aiming to find a justification for a real clinical case. An introduction about platelets was made, addressing not only their physiology (structure, formation and function of platelets) as well as its role in inflammation and the quantitative and qualitative alterations that may occur in platelets and that can lead to pathogenic disorders in the human organism. To end this big chapter, it’s discussed some applications of medical tests in the diagnosis of platelet disorders. In this sub-chapter it’s given great importance to PFA, a new method for the evaluation of platelet function that has significant advantages compared with the time of bleeding. As the title indicates, vascular diseases are also one of the biggest chapters of this work, being essential their understanding to create a relationship between platelets and vascular diseases. Thus, arterial diseases (coronary artery disease and peripheral artery disease) and venous disease (deep venous thrombosis) are addressed. Not all existing arterial and venous diseases are addressed, giving much more relevance to those that are important to understand the relationship mentioned above. Being one of the main objectives of this work, it’s described a real clinical case relating thrombocytopenia as the consequence of vascular diseases, with a chronological description of the events that have occurred over the years and the conclusions reached with each of the procedures until now. In conclusion, some therapies are described, such as antiplatelet treatments, applied as a prevention of an AVC on patients with vascular diseases, the laboratorial monitorization/control of the therapeutics and the new antiplatelet agents that are beginning to be used in the pharmaceutical market.

Which one of us never noticed the emerging trend of natural products? The presented case study aims to study the increasing demand for natural and organic cosmetics and how can Biolage R.A.W. leverage from it. Biolage R.A.W. is a natural professional haircare brand taking its first steps in the Portuguese market. The brand is already facing a lack of awareness which, alongside with the higher prices of its products can leave the brand more exposed. So, how can Biolage R.A.W. come up with a launch strategy to solve these main problems? To better understand the market and brand, an exhaustive qualitative and quantitative analysis was developed that allowed to draw some primary conclusions: Millennials are the healthy beauty products generation and are willing to pay more for natural products. Regarding the launch communication strategy, Biolage R.A.W should follow an integrated communication strategy, using different communication options, creating synergies, to obtain different outcomes in a more cost-efficient way. Taking into consideration millennials’ characteristics, online platforms and e-influencers should take a big part of the brands’ communication. Although recent in the market, Biolage R.A.W. can easily be aware of the growing demand for natural cosmetics and take it as an opportunity to develop and expand the brand. In this case study is clear that the brand has four large strategic core areas to develop, of which two are remarkable for their potential for expansion: distribution channels and innovation. The brand must in the future explore new distribution channels such as natural supermarkets and prioritize a brand extension at the product and category level by launching a line of natural dyes for the hair.
Quem de nós nunca notou a tendência emergente de produtos naturais? O estudo de caso apresentado visa estudar a crescente procura por cosméticos naturais e orgânicos e perceber como pode Biolage R.A.W. beneficiar com isso. Biolage R.A.W. é uma marca natural de cuidados capilares profissionais naturais que está atualmente a dar os primeiros passos no mercado português. Enfrenta já alguma falta de awereness que, juntamente com os preços altos praticados quando comparados com os seus concorrentes, a deixam mais suscetível a eventuais. Então, como pode Biolage R.A.W. encontrar uma estratégia de lançamento para resolver esses principais problemas? Para entender melhor o mercado e a marca, uma análise qualitativa e quantitativa exaustiva foi realizada de forma a retirar algumas conclusões principais: os millennials são uma geração de produtos de beleza saudáveis e estão dispostos a pagar mais por produtos naturais. Em relação à estratégia de comunicação de lançamento, Biolage R.A.W deve seguir uma comunicação integrada usando diferentes opções de comunicação, criando sinergias para obter diferentes resultados e de forma eficiente. Tendo em consideração as características dos millennials, as plataformas on-line e os influenciadores digitais devem ter parte fulcral da comunicação da marca. Embora recente no mercado, Biolage R.A.W. pode facilmente estar ciente da crescente procura por cosméticos naturais e encará-la como uma oportunidade para desenvolver e expandir a marca. Neste estudo de caso fica claro que a marca tem quatro grandes áreas estratégicas principais para desenvolver, das quais duas são notáveis pelo seu potencial de expansão: canais de distribuição e inovação. A marca deve, no futuro, explorar novos canais de distribuição, como os supermercados naturais, e priorizar a extensão da marca ao nível do produto e categoria, lançando uma linha de coloração natural para o cabelo

In this thesis, luminescent lanthanide-doped nanocrystals, and lead-based quantum dots nanocrystals are explored as alternative bioimaging agents to fluorescent proteins and organic fluorophores for deep-tissue imaging. The first chapter gives a brief introduction on the aforementioned nanocrystals and their special optical properties. In chapter 2 the simple changes in the drying and baking temperature of the Yb3+ and Ho3+ doped LaF3 nanocrystals-silica sol-gel mixture aid in the explanation of the formation of two types of silica. The difference in the phonon energies of the two types of silica is found to control effectively the ratio of red to green emissions obtained from the upconversion process. However, the nanocrystals do not disperse in water making them unsuitable for bioimaging. Chapter 3 describe the synthesis of NaYF4 nanocrystals doped with Yb3+/Er3+ or Yb3+/Tm3+ ions followed by two surface modification strategies (intercalation and crosslinking) to disperse them in physiological buffers and biological growth media. Of the two methods, the crosslinked polymer coating of the nanocrystals alone exhibits stability in aforementioned media. In chapter 4 the applicability of lanthanide-doped NaYF4 nanocrystals are studied as bioimaging agents in two-photon upconversion laser scanning microscopy for deep-tissue imaging. Their performance as bioimaging agents was not better than fluorescent proteins and organic molecules. On the other hand with two-photon upconversion wide field microscopy (TPUWFM), brain blood vessels over a depth of 100 µm could well be separated. Furthermore, with the 800 nm emission from Tm3+ ions one can image twice as deep as the green emission with TPUWFM. In chapter 5, probing the NaYF4 nanocrystals with energy-dependent XPS shows that, the Y3+ ions on the surface of the nanocrystals are different from the ones present inside the nanocrystals. This chapter is concluded with a preliminary investigation of Yb3+ and Tm3+ doped NaYF4 with resonant XPS. Chapter 6 examines four different types of surface modification strategies to transfer hydrophobic lead-based quantum dots to physiological buffers and biological growth media. Of the four methods, the crosslinked polymer coating of quantum dots alone exhibits colloidal stability and the QDs retain their luminescence in aforementioned media over several months. The conclusions and future outlook for the work are elucidated in chapter 7.
Graduate