This thesis describes the synthesis and application of a new series of amphiphilic graft copolymers with a hydrophobic polyolefin backbone and pendent hydrophilic poly(ethylene glycol) (PEG) grafts. These copolymers are synthesized by ruthenium benzylidene-catalyzed ring-opening metathesis polymerization (ROMP) of PEG-functionalized cyclic olefin macromonomers to afford polycyclooctene- graft -PEG (PCOE- g -PEG) copolymers with a number of tunable features, such as PEG graft density and length, crystallinity, and amphiphilicity. Macromonomers of this type were prepared first by coupling chemistry using commercially available PEG monomethyl ether derivatives and a carboxylic acid-functionalized cycloctene. In a second approach, macromonomers possessing a variety of PEG lengths were prepared by anionic polymerization of ethylene oxide initiated by cyclooctene alkoxide. This methodology affords a number of benefits compared to coupling chemistry including an expanded PEG molecular weight range, improved hydrolytic stability of the PEG-polycyclooctene linkage, and a reactive hydroxyl end-group functionality for optional attachment of biomolecules and probes. The amphiphilic nature of these graft copolymers was exploited in oil-water interfacial assembly, and the unsaturation present in the polycyclooctene backbone was utilized in covalent cross-linking reactions to afford hollow polymer capsules. In one approach, a bis -cyclooctene PEG derivative was synthesized and co-assembled with PCOE-g-PEG at the oil-water interface. Upon addition of a ruthenium benzylidene catalyst, a cross-linked polymer shell is formed through ring-opening cross-metathesis between the bis -cyclooctene cross-linker and the residual olefins in the graft copolymer. By incorporating a fluorescent-labeled cyclooctene into the graft copolymer, both oil-water interfacial segregation and effective cross-linking were confirmed using confocal laser scanning microscopy (CLSM). In a second approach, reactive functionality capable of chemical cross-linking was incorporated directly into the polymer backbone by synthesis and copolymerization of phenyl azide and acyl hydrazine-functional cyclooctene derivatives. Upon assembly, these reactive polymers were cross-linked by photolysis (in the phenyl azide case) or by addition of glutaraldehyde (in the acyl hydrazine case) to form mechanically robust polymer capsules with tunable degradability ( i.e. non-degradable or pH-dependent degradability). This process permits the preparation of both oil-in-water and water-in-oil capsules, thus enabling the encapsulation of hydrophobic or hydrophilic reagents in the capsule core. Furthermore, the assemblies can be sized from tens of microns to the 150 nm - 1 µm size range by either membrane extrusion or ultrasonication techniques. These novel capsules may be well-suited for a number of controlled release applications, where the transport of encapsulated compounds can be regulated by factors such as cross-link density, hydrolytic stability, and environmental triggers such as changes in pH.

Tesis por compendio / [ES] La criopreservación de semen de peces es una técnica que puede aumentar la eficiencia de la reproducción en cautiverio de especies de peces de agua dulce y marinas. A lo largo de las últimas décadas, se han establecido protocolos para criopreservación de semen de diversas especies de peces. Sin embargo, el foco principal de los pescadores ha sido en tener éxito en el congelamiento y descongelamiento de los espermatozoides, no llevando en cuenta el período en que los gametos se quedan expuestos a la solución crioprotectora en un momento previo a la fertilización. Esta exposición de los espermatozoides a las soluciones crioprotectoras después del descongelamiento puede ser perjudicial a la calidad de los gametos, ya que pueden ser tóxicos. La mayoría de los protocolos establecidos utilizan recipientes de plástico ultrarresistentes para almacenar el semen durante el proceso de criopreservación. Estos recipientes normalmente no se reutilizan, generando residuos altamente contaminantes al medio ambiente. Así, el objetivo principal de la tesis fue crear y probar métodos de bajo costo que potencialicen el uso de los espermatozoides descongelados de peces y torne el proceso de criopreservación de semen menos contaminante al medio ambiente. Los experimentos de los capítulos 1 y 2 se llevaron a cabo en Brasil. Utilizamos el jundiá gris Rhamdia quelen, especie considerada modelo experimental para peces nativos de América del Sur. En el capítulo 1, probamos el uso de la dilución de semen descongelado para disminuir la toxicidad de la solución crioprotectora. La técnica se utiliza comúnmente en protocolos de criopreservación de semen de mamíferos, pero nunca antes se había aplicado al semen descongelado de peces suramericanos. Muestras de semen descongelado de R. quelen se diluyeron en un diluyente salino (NaCl al 1,1% - 325 mOsm kg-1; pH 7,6). Después, observamos que los espermatozoides de muestras diluidas mostraron mayores velocidades, rectitud, progresión y frecuencia de batido flagelar que las muestras no diluidas. La dilución del semen descongelado también proporcionó mayores tasas de fertilización y eclosión que el grupo no diluido. De esta manera, la dilución de semen descongelado de R. quelen resultó ser una metodología sencilla, económica y eficiente que debe incluirse en el protocolo de criopreservación del semen de la especie. En los capítulos 2 y 3 desarrollamos y probamos la metodología para el uso de cápsulas de gelatina biodegradables (colágeno) y cápsulas de hipromelosa biodegradables (HPMC) como recipiente alternativo al uso de pajuelas de plástico en la criopreservación de semen de peces. En el segundo capítulo observamos que las cápsulas biodegradables mantuvieron los parámetros cinéticos y la capacidad reproductiva del esperma de R. quelen así como las pajuelas de plástico.Los procedimientos experimentales del capítulo 3 se llevaron a cabo en España. En este capítulo aplicamos la metodología desarrollada en el capítulo 2 para la criopreservación del semen de anguila europea Anguilla anguilla, dorada Sparus aurata y lubina Dicentrarchus labrax. En estas tres especies, las cápsulas biodegradables conservaron los parámetros cinéticos y la integridad de la membrana de los espermatozoides, así como las pajuelas de plástico. Además, observamos que el daño al ADN en muestras de semen de anguila europea y lubina europea criopreservadas en cápsulas y pajuelas no difirió. Sin embargo, las muestras de semen de dorada mostraron mayor daño en el ADN que las criopreservadas en pajuelas. Aunque, el nivel de daño que observamos en las muestras almacenadas en las cápsulas se considera bajo, por lo que pueden no comprometer el desarrollo embrionario. Evaluamos los resultados y concluimos que las cápsulas de gelatina biodegradables y las cápsulas de HPMC biodegradables pueden utilizarse como recipientes alternativos al uso de pajuelas de plástico para la criopreservación de semen de las cuatro especies de peces. / [CA] La criopreservació de l'esperma de peixos és una tècnica que pot augmentar l'eficiència de la reproducció en captivitat d'espècies de peixos d'aigua dolça i marins. Al llarg de les dècades passades, s'han establert protocols per a la criopreservació de l'esperma de diverses espècies de peixos. No obstant això, el focus principal dels pescadors ha estat tenir èxit en la congelació i descongelació dels espermatozoides, sense tenir en compte el temps en què els gamets queden exposats a la solució crioprotectora abans de la fecundació. Aquesta exposició dels espermatozoides a les solucions crioprotectores després de la descongelació pot ser perjudicial per a la qualitat dels gàmetes, ja que poden ser tòxics. La majoria dels protocols establerts utilitzen recipients de plàstic ultrarresistents per emmagatzemar l'esperma durant el procés de criopreservació. Aquests recipients normalment no es reutilitzen, generant residus altament contaminants per al medi ambient. Així, l'objectiu principal de la tesi va ser criar i provar mètodes de baix cost que potenciïn l'ús dels espermatozoides descongelats de peixos i facin que el procés de criopreservació de l'esperma sigui menys contaminant per al medi ambient. Els experiments dels capítols 1 i 2 es van dur a terme a Brasil. Vam utilitzar el jundia gris Rhamdia quelen, una espècie considerada com a model experimental per a peixos natius d'Amèrica del Sud. En el capítol 1, vam provar l'ús de la dilució de l'esperma descongelat per reduir la toxicitat de la solució crioprotectora. Aquesta tècnica s'utilitza comúment en protocols de criopreservació de l'esperma de mamífers, però mai abans s'havia aplicat a l'esperma descongelat de peixos sud-americans. Mostres d'esperma descongelat de R. quelen es van diluir en un diluent salí (NaCl al 1,1% - 325 mOsm kg-1; pH 7,6). Després, vam observar que els espermatozoides de mostres diluïdes mostraven majors velocitats, rectitud, progressió i freqüència de batuda flagel·lar que les mostres no diluïdes. La dilució de l'esperma descongelat també va proporcionar majors taxes de fecundació i eclosió que el grup no diluït. D'aquesta manera, la dilució de l'esperma descongelat de R. quelen va resultar ser una metodologia senzilla, econòmica i eficient que ha d'incloure's en el protocol de criopreservació de l'esperma de l'espècie. En els capítols 2 i 3 vam desenvolupar i provar la metodologia per a l'ús de càpsules de gelatina biodegradables (col·lagen) i càpsules d'hipromelosa biodegradables (HPMC) com a recipient alternatiu a l'ús de canuts de plàstic en la criopreservació de l'esperma de peixos. En el segon capítol vam observar que les càpsules biodegradables mantenien els paràmetres cinètics i la capacitat reproductiva de l'esperma de R. quelen així com els canuts de plàstic. Els procediments experimentals del capítol 3 es van dur a terme a Espanya. En aquest capítol vam aplicar la metodologia desenvolupada al capítol 2 per a la criopreservació de l'esperma d'anguila europea Anguilla anguilla, daurada Sparus aurata i llobarro Dicentrarchus labrax. En aquestes tres espècies, les càpsules biodegradables van conservar els paràmetres cinètics i la integritat de la membrana dels espermatozoides, així com els canuts de plàstic. A més, vam observar que el dany a l'ADN en mostres d'esperma d'anguila europea i llobarro europeu criopreservades en càpsules i canuts no es va diferir. No obstant això, les mostres d'esperma de daurada van mostrar més dany a l'ADN que les criopreservades en canuts. Tot i això, el nivell de dany que vam observar a les mostres emmagatzemades en càpsules es considera baix, pel que poden no comprometre el desenvolupament embrionari. Vam avaluar els resultats i vam concloure que les càpsules de gelatina biodegradables i les càpsules d'HPMC biodegradables es poden utilitzar com a recipients alternatius a l'ús de canuts de plàstic per a la criopreservació de l'esperma de les quatre espècies de peixos. / [EN] Fish sperm cryopreservation is a technique that can increase the reproduction in captive efficiency of freshwater and marine fishes.Over the last few decades, protocols for sperm cryopreservation from many fishes have been established. However, the researchers' main focus was successfully freezing and thawing sperm, neglecting the period in which the gametes remain in contact with the cryoprotective solution until fertilization. Exposure of sperm to cryoprotectant solutions after thawing can be harmful to the quality of the gametes since they can be toxic. The majority of the established protocols use ultra-resistant plastic containers to store sperm during the cryopreservation process. These containers usually are not reused, generating highly polluting waste for the environment. Furthermore, in some countries, the containers usually used are sold by a few industries, which makes acquisition difficult and increases the product's price. Thus, the main objective of the thesis was to create and test low-cost methodologies that enhance the use of fish post-thaw sperm and make the sperm cryopreservation process more environmentally friendly. The experiments in the Chapters 1 and 2 were developed in Brazil. We used the South American silver catfish Rhamdia quelen, a species considered an experimental model for native South American fishes. In Chapter 1, we tested the use of post-thawing dilution to reduce the toxicity of the cryoprotectant solution. This technique is commonly used in mammalian sperm cryopreservation protocols but has never before been applied to post-thaw sperm of South American fishes. South American silver catfish post-thaw sperm samples were diluted in a saline extender (1.1% NaCl - 325 mOsm kg-1; pH 7.6). The post-thaw sperm diluted samples showed higher velocities, straightness, progression, and flagellar beat frequency than the cells of undiluted samples (control). The post-thawing dilution also provided higher fertilization and hatching rates than the control group. Thus, the post-thawing sperm dilution proved to be a simple, cheap, and efficient methodology that should be included in the silver catfish sperm cryopreservation protocol. In Chapters 2 and 3, we developed, tested, and described the methodology for using biodegradable gelatin (collagen) and hypromellose (HPMC) capsules as an alternative container to plastic straws in the fish sperm cryopreservation. In the second chapter, we observed that the biodegradable capsules maintained the kinetic parameters and reproductive capacity of South American silver catfish sperm just as effectively as plastic straws. The experimental procedures in Chapter 3 were carried out in Spain. We apply the methodology developed in Chapter 2 to the cryopreservation of sperm from European eel Anguilla anguilla, gilthead seabream Sparus aurata, and European sea bass Dicentrarchus labrax. In these three species, biodegradable capsules preserved the sperm kinetic parameters and membrane integrity just as effectively plastic straws. We observed that DNA damage in European eel and European sea bass sperm samples cryopreserved in capsules and straws did not differ. On the other hand, gilthead seabream sperm samples showed higher DNA damage than those cryopreserved in straws. However, the damage level observed in samples stored in capsules is considered low, thus, may not compromise embryonic development. We observed the results and concluded that biodegradable gelatin and HPMC capsules could be used as alternative containers to plastic straws for sperm cryopreservation from the tfour aquaculture fishes. / This study was supported by MICINN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (THINKINAZUL/2021/012;THINKINAZUL/2021/024;THINKINAZUL/2021/042) including the contract of FF-G.WAG-L has a Margarita Salas postdoctoral contract (RD 289/2021. UAB) by the Spanish Ministry of Universities. LF has a PhD contract from Generalitat Valenciana (GRISOLIAP/2020/063). TSF (141717/2019-0 and 200285/2021-1) and MPS (200452/2022-3) have fellowships from Brazilian National Council for Scientific and Technological Development (CNPq). / De Souza França, T. (2024). Development and Application of Low-Cost and Environment-Friendly Techniques for Fish Sperm Cryopreservation [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204486 / Compendio

Avec près de 25 000 cas en 2015, le cancer du sein est de loin le cancer le plus fréquent chez les femmes canadiennes. En termes de mortalité, le cancer du sein se place au deuxième rang (13,8%), derrière le cancer du poumon (26,5%). Suite au diagnostic, 70% des femmes avec un cancer de stade avancé et 30% des femmes avec cancer précoce choisiront de subir une mastectomie totale. Plusieurs options de reconstructions mammaires sont ensuite offertes. Plus de 70% des reconstructions se feront par la mise en place d’implants mammaires (plutôt que par reconstruction autologue utilisant les tissus de la patiente). Parmi les complications survenant après cette intervention, la contracture capsulaire en est une des plus fréquente. La contracture capsulaire se définit comme la contraction anormale de la capsule fibreuse se formant autour des implants mammaires, pouvant causer des douleurs en plus d’un mauvais résultat sur le plan esthétique. Les études cliniques ont démontré un risque accru de développer une contracture capsulaire chez les femmes ayant du suivre un protocole de radiothérapie. Si plusieurs études ont cherché à démontré l’influence des radiations sur l’histologie et l’immunologie du tissu capsulaire, aucune étude ne s’était intéressée à leur effet sur l’ultrastructure capsulaire. Notre équipe a récemment démontré que l’ultrastructure capsulaire dépendait de plusieurs facteurs, notamment de la texture prothétique et du délai de reconstruction. L’objectif de ce mémoire était de décrire l’impact de la radiothérapie sur l’ultrastructure capsulaire en comparant des capsules irradiées et non-irradiées en utilisant la microscopie éléctronique à balayage. Nous avons observé, chez les échantillons issus de capsules irradiés, une perte de relief au niveau de l’interface prothèse-capsule par rapport au échantillons non irradiés. Nous avons aussi démontré que cette caractéristique ultrastructurale est associée à la présence de contracture capsulaire. Enfin, ces effets néfastes de la radiothérapie semblent altérer la capacité de la capsule à adhérer à la prothèse mammaire. En conclusion, mon projet de maîtrise aura permis de décrire pour la première fois l’impact de la radiothérapie sur l’ultrastructure des capsules périprothétiques. Bien que nos résultats ne nous permettent pas d’expliquer de façon certaine le lien potentiel avec l’évolution vers un état de contracture capsulaire, ils laissent croire que cela serait associé avec un diminution de la stabilité de la capsule sur son implant, facteur auparavant évoqué comme ayant une rôle protecteur sur la contracture capsulaire. / With around 25,000 cases in 2015, breast cancer is the most common cancer among Canadian women. In term of mortality, breast cancer is second (13.8%), only behind lung cancer (26.5%). As part of the therapeutic approaches, around 70% and 30% of women with advanced and early breast cancer (respectively) will chose to have a total mastectomy. Following a mastectomy, few options of breast reconstruction are offered to the patients. More than 70% of breast reconstructions are implant-based. Among the complications following this surgery, capsular contracture (CC) is one of the most common. CC is defined as the abnormal, and sometimes painful, contraction of the fibrous capsule that normally grows aroung breast implants, resulting in a aesthetic failure and associated with a higher surgical revision rate. Many studies have demonstrated that the incidence of CC was higher among women who had undergone radiotherapy prior to their implant-based breast breast reconstruction procedure. Most of published studies focus on the influence of radiotherapy on capsular tissue at the immune-histological level but no study has described its influence on capsular architecture. As our team recently demonstrated that capsular ultrastructure is influenced by many factors (prosthetic texture, expansion timing). The main purpose was to describe the impact of radiotherapy on periprosthetic capsule ultrastructural features by comparing irradiated and non-irradiated capsules using scanning electron microscopy (SEM). We observed a significant loss of tridimensional texture at the implant-capsule interface in irradiated capsular samples. This feature was also found to be associated with CC. Finally, our results tend to show that radiotherapy impaires the stability of capsules by preventing them to grow withing their surrounding implant texure. In conclusion, the present research project offers the first description of the capsular response following exposiong to radiations. Even though our results do not allow us to conlude to a clear relation between loss of capsular architecture and a higher risk of CC, we think that the impairement of capsular stability might be part of the answer

The present thesis focuses on the fabrication of bio-stimuli responsive micro- and nano-carriers for drug delivery applications. In particular, the objective of this work is to investigate the possibility of using polypeptide drug protamine and glycosaminoglycan drug, chondroitin sulphate as stimuli responsive components in the design of bioresponsive carriers. These biopolymers are biocompatible, biodegradable and clinically used for various applications. Two designs that incorporate these stimuli responsive components have been studied in this thesis. The first design involves hollow micro and nanocapsules that have been fabricated by incorporating the stimuli responsive biopolymers as wall components. Upon exposure to biological triggers, these hollow capsules disintegrate releasing the encapsulated drug. The second design consists of mesoporous silica nanoparticles-biopolymer hybrids. The mesoporous silica nanoparticles act as a gated scaffold that carries the drug molecules. The mesopores of these drug loaded nanoparticles are then blocked with the bioresponsive polymers. Upon exposure to the bio-triggers which consist of enzymes over-expressed in conditions such as cancer and inflammation, these “molecular gates” disintegrate allowing the drug trapped in the mesoporous silica nanoparticles to escape into the surroundings. The thesis has been divided into five chapters: Chapter 1 is an introduction to bio-responsive drug delivery. The broad classification of stimuli used in responsive drug delivery systems are visited. A brief discussion on the various types of bio-stimuli that can be utilized in designing bio-responsive systems is also included in this chapter. Chapter 2 defines the aims and scope of the thesis which is followed by an overview of the various design parameters involved in the fabrication of systems presented in this work. The major stimuli responsive components and the architectures incorporating these elements are discussed in detail here. A literature review of the various carrier designs involved in the study is provided , with special emphasis on stimuli responsive drug delivery. Chapter 3 gives an overview of the various materials and methods involved in this work. A summary of the various characterisation techniques used in the thesis is also included along with the details of the experiments that has been carried out. Chapter 4 provides an overview of the results and discussions of the thesis. The chapter has been divided into six sections: Chapter 4.1 deals with the fabrication of a hollow microcapsule system incorporated with protamine as the stimuli responsive element for bio-responsive drug delivery. The hollow microcapsules that were fabricated by Layer by Layer assembly of protamine and heparin display pH responsive variations in permeability and disintegrate in the presence of the enzyme trypsin that degrades protamine. The biologically triggered enzyme responsive drug release from these microcapsules is also demonstrated using enzymes secreted by colorectal cancer cells. Chapter 4.2 presents nanocapsules fabricated from protamine and heparin. The pH and enzyme responsive drug release of this systems is evaluated in vitro. A wall crosslinking strategy has been tested to control the rate of drug release under physiological pH conditions in the absence of the trigger. The cellular interactions of these nanocapsules loaded with an anticancer drug, doxorubicin was studied using cancer cell lines. Bioavailability studies of doxorubicin encapsulated in these nanocapsules were performed using a BALB/c mice model. Chapter 4.3 discusses the fabrication of a hollow microcapsule system that can disintegrate in response to dual biological stimuli. These carriers have been fabricated by incorporating protamine and chondroitin sulphate as the wall components. Due to the incorporation of two separate stimuli responsive components in the walls, these capsules are expected to be sensitive to the enzymes trypsin or hyaluronidase I. Chapter 4.4 deals with the fabrication of dual enzyme responsive hollow nanocapsule which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements integrated in the hollow nanocapsule walls can undergo degradation in presence of either of the enzymes trypsin or hyaluronidase I leading to the release of encapsulated drug molecules. The drug release from these nanocapsules which were crosslinked and functionalised with folic acid, is evaluated under varying conditions. The cellular uptake and intracellular drug delivery by these nanocapsules were evaluated in cervical cancer cell lines. Chapter 4.5 introduces a mesoporous silica nanoparticle − protamine hybrid system. The system consists of a mesoporous silica nanoparticle support whose mesopores are capped with protamine which effectively blocks the outward diffusion of the drug molecules from the mesopores of the mesoporous silica nanoparticles. Upon exposure to the enzyme trigger, the protamine cap disintegrates opening up the molecular gates and releasing the entrapped drug molecules. The drug release from this system is evaluated in different release conditions in the presence and absence of the enzyme trigger. The ability of these particles to deliver hydrophobic anticancer drugs and induce cell death in colorectal cancer cells has also been demonstrated. Chapter 4.6 discusses the fabrication of another mesoporous silica nanoparticles based bio-responsive drug delivery system consisting of mesoporous silica and chondroitin sulphate hybrid nanoparticles. The ability of the system to modulate drug release in response to hyaluronidase I is demonstrated. By utilizing a cervical cancer cell line, we have demonstrated the cellular uptake and intracellular delivery of hydrophobic drugs encapsulated in these particles. Interestingly, the system showed ability to enhance the anticancer activity of hydrophobic drug curcumin in these cancer cells. Chapter 5 gives a summary of the general conclusions drawn from the thesis work.

O presente trabalho teve como objetivo o estudo da variabilidade genética e dos fatores de virulência de isolados de U. diversum. As cepas foram submetidas a sequenciamento dos genes da urease e 16S rRNA e a testes para verificar os fatores de virulência: cápsula, fosfolipase C, IgAse e adesão e invasão. A análise do sequênciamento parcial do gene 16S rRNA resultou na presença de polimorfismos em 44 posições da seqüência, que diferenciou as amostras em sete grupos. Em relação aos fatores de virulência, os dados mostraram que as cepas estudadas apresentaram uma camada densa ao redor da membrana celular dos microrganismos e atividade de fosfolipase C. No entanto, não foi observado a atividade de IgAse nas cepas. Em relação a atividade de invasão, observou-se que os ureaplasma estudados puderam ser visualizados no interior de células Hep-2 com apenas um minutos de infecção, sendo observados em uma região perinuclear, mas não no interior do núcleo. Além disto, pode verificar que entre 1% a 10% dos ureaplasmas estudos penetraram na célula pelo teste da gentamicina. / The aim of the present study was the study of genetic variability and virulence factors of U. diversum clinical isolates. The strains were submitted to sequencing for 16S rRNA and urease genes. Moreover, the strains were analyzed to the virulence factors: capsule, phospholipase C, IgA protease and adhesion and invasion into Hep-2 cells. The sequencing of parcial 16S rRNA gene showed polymorphic patterns into 44 positions. These polymorphisms clustered the strains in seven groups. For the virulence factors, ureaplasma cells showed a dense-stained external capsule-like structure surrounding the cell membrane. A high level of phospholipase C activity was also detected in 31 studied ureaplasma. However, no strains showed IgA protease activity. For the invasion assay, the isolates and strains used were detected inside the cells after infection of one minute. The invasions of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% to 10% of studied ureaplasmas were inside the infected cells.

O presente trabalho teve como objetivo o estudo da variabilidade genética e dos fatores de virulência de isolados de U. diversum. As cepas foram submetidas a sequenciamento dos genes da urease e 16S rRNA e a testes para verificar os fatores de virulência: cápsula, fosfolipase C, IgAse e adesão e invasão. A análise do sequênciamento parcial do gene 16S rRNA resultou na presença de polimorfismos em 44 posições da seqüência, que diferenciou as amostras em sete grupos. Em relação aos fatores de virulência, os dados mostraram que as cepas estudadas apresentaram uma camada densa ao redor da membrana celular dos microrganismos e atividade de fosfolipase C. No entanto, não foi observado a atividade de IgAse nas cepas. Em relação a atividade de invasão, observou-se que os ureaplasma estudados puderam ser visualizados no interior de células Hep-2 com apenas um minutos de infecção, sendo observados em uma região perinuclear, mas não no interior do núcleo. Além disto, pode verificar que entre 1% a 10% dos ureaplasmas estudos penetraram na célula pelo teste da gentamicina. / The aim of the present study was the study of genetic variability and virulence factors of U. diversum clinical isolates. The strains were submitted to sequencing for 16S rRNA and urease genes. Moreover, the strains were analyzed to the virulence factors: capsule, phospholipase C, IgA protease and adhesion and invasion into Hep-2 cells. The sequencing of parcial 16S rRNA gene showed polymorphic patterns into 44 positions. These polymorphisms clustered the strains in seven groups. For the virulence factors, ureaplasma cells showed a dense-stained external capsule-like structure surrounding the cell membrane. A high level of phospholipase C activity was also detected in 31 studied ureaplasma. However, no strains showed IgA protease activity. For the invasion assay, the isolates and strains used were detected inside the cells after infection of one minute. The invasions of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% to 10% of studied ureaplasmas were inside the infected cells.

Made available in DSpace on 2016-04-28T20:38:37Z (GMT). No. of bitstreams: 1 Andrea de Davide Ratto Morelli.pdf: 720847 bytes, checksum: f67ae12322be10f9bae642d62c44bd21 (MD5) Previous issue date: 2013-03-15 / This study aims to gather information on some types of pathological organizations of the personality, using psychoanalytic knowledge. Several authors underlie it, starting with Sigmund Freud, Melanie Klein, Joan Riviere, Wilfred Bion, Herbert Rosenfeld, Hanna Segal and getting to John Steiner, Donald Meltzer, Frances Tustin, Judith Mitrani and James Grotstein, whose works in these areas are discussed more deeply. Efforts are made to understand and identify points of convergence, divergence and/or intersection among concepts like claustrum, psychic retreats, autistic capsules and adhesive pseudo-object relations. Discussion of the importance of continence and the development of schizo paranoid and depressive positions, permeate all the work and are fundamental to the approach of the clinical material presented. Analyst's psychic continence is questioned in face of difficulties as the claustrum seduction, attraction of adhesive pseudo-object relations, embarrassment of tenderness and struggles for dominate or exclusion of the analyst. Facing the difficulties of handling complex defensive systems, such as pathological organizations, confidence in the existence of unconscious need of psychic truth remains encouraging and cherishing, both to continue the trajectory of psychoanalytic exercise, and to achieve the needs of patients / Este estudo tem por objetivo recolher informações sobre alguns tipos de organizações patológicas da personalidade, utilizando conhecimentos psicanalíticos. Vários autores embasam-no partindo de Sigmund Freud, Melanie Klein, Joan Riviere, Wilfred Bion, Herbert Rosenfeld, Hanna Segal e chegando a John Steiner, Donald Meltzer, Frances Tustin, Judith Mitrani e James Grotstein, cujos trabalhos nessas áreas são discutidos mais profundamente. Esforços são realizados para compreender e identificar pontos de convergência, divergência e/ou intersecção entre conceitos como claustros, refúgios psíquicos, cápsulas autistas e pseudorrelações objetais adesivas. Discussões da importância da continência e da elaboração das posições esquizoparanoides e depressivas perpassam todo o trabalho e são fundamentais para a abordagem do material clínico apresentado. A continência psíquica do analista é questionada em face de dificuldades como a sedução dos claustros e a atratibilidade de pseudorrelações objetais adesivas, o embaraço diante da ternura e lutas por dominar ou excluir o analista. Diante da dificuldade de manejo com sistemas defensivos complexos, como os das organizações patológicas, a confiança na existência da necessidade inconsciente da verdade psíquica permanece estimulante e acalentadora tanto para continuar a trajetória do exercício psicanalítico, quanto para alcançar as necessidades dos pacientes

Targeted delivery aids in minimizing most of the drug-originated systemic toxic effects as well as improving the pharmacokinetic properties of anticancer therapeutics. Tumor targeting using hyaluronic acid (HA) as the targeting ligand has attracted a great deal of interest among a host of strategies developed to target the overexpressed tumor specific receptors. HA is an endogenous molecule that possesses a lot of biological functions in the human body. The role of HA synthases, HA degrading enzymes and the interaction of HA with its primary receptor CD44 in tumor metastasis and angiogenesis is really complex and controversial to date. However, overexpression of CD44receptors on tumor surface has been well studied, which have been utilized to direct tumor targeted drugs. Most of the HA based targeting systems were HA drug conjugates and surface modified colloidal carriers which required covalent modification. The lack of accurate structural characterization of these systems resulted in modification of HA binding sites that could affect the efficient cellular uptake. LbL technique is a simple and facile method to incorporate several materials into polyelectrolyte assemblies for drug delivery applications. HA being a negatively charged polysaccharide can be easily incorporated into such systems without any covalent modification. Although HA based polyelectrolyte multilayer films and microcapsules have been reported in combination with polycations like PAH, PLL and chitosan, their application as targeted drug delivery systems have not yet been explored. Herein, two LbL architectures with HA as the terminal layer have been investigated as targeted drug carriers, which can recognize overexpressed CD44 receptors in metastatic breast cancer cells. In the first part of the thesis, a novel polyelectrolyte nanocapsule system composed of biopolymers HA and protamine sulphate (PR) as the wall components was prepared and characterized. These pH and enzyme responsive nanocapsules were then utilized for efficient loading and release of anticancer drug doxorubicin (dox). Higher drug release was observed in simulated intracellular conditions like acidic pH and presence of hyaluronidase enzyme as compared to physiological pH. In the second part of the thesis, dox incorporated bovine serum albumin (BSA) nanoparticles modified with HA-Poly(l-Lysine) multilayers were developed and characterized. The drug release pattern of the dox loaded BSA nanoparticles was found to depend on the presence of a protease enzyme trypsin than pH variations. Both of these drug delivery systems were then evaluated for their cell targeting efficiency and cytotoxicity in CD44+ positive metastatic breast cancer cell line MDA MB 231. The final layer HA facilitated targeted delivery of these drug carriers via CD44 receptor mediated endocytosis. The enhanced cellular uptake followed by sustained delivery of dox by virtue of slow intracellular enzymatic degradation of the drug carriers resulted in their improved cytotoxicity as compared to free dox. Further in vitro biodistribution and tumor suppression efficiency of both the systems were studied in breast cancer xenograft models using BALB/c nude mice. Enhance accumulation of dox in the tumor tissue and significant tumor reduction were observed when treated with encapsulated dox using the HA based nanocarriers as opposed to free dox.

Submitted by Marco Antonio Oliveira da Silva (maosilva@ipen.br) on 2017-05-02T11:34:44Z No. of bitstreams: 0 / Made available in DSpace on 2017-05-02T11:34:44Z (GMT). No. of bitstreams: 0 / Dentre as diversas aplicações dos radioisótopos, a utilização dos radiotraçadores é considerada uma das mais importantes, no diagnóstico de funcionamento dos equipamentos de processos, em plantas de indústrias químicas e petroquímicas. Os radiotraçadores são utilizados em procedimentos analíticos para obtenção de dados qualitativos e quantitativos de sistemas, em estudos de transferências físicas e físico-químicas. Na produção de radioisótopos gasosos utilizados como traçadores em processos industriais, destacam-se o 41Ar e 79Kr, gases nobres (inertes) que possuem baixa reatividade com os demais elementos químicos. O 41Ar é um emissor gama de alta energia (1,29 MeV) e apresenta elevada porcentagem de transformações com essa energia, o que resulta em quantidades relativamente pequenas necessárias em relação a outras para uma detecção eficaz, mesmo em componentes com grandes espessuras. Atualmente, a produção de radioisótopos gasosos em reatores nucleares de pesquisa é realizada em pequenas quantidades (bateladas), por meio de ampolas de quartzo contendo o gás natural 40Ar ou 78Kr. Nesse sentido, o objetivo desse estudo é desenvolver um sistema de irradiação capaz de produzir em escala contínua, o radioisótopo gasoso 41Ar, dentre outros, com atividade de 7,4x1011 Bq (20 Ci) por ciclo de irradiação, por meio do Reator IEA-R1 de 4,5 MW, fluxo de nêutrons térmicos médio de 4,71 x 1013 ncm-2s-1, para suprir uma demanda existente em empresas de END e inspeções, e pelo próprio Centro de Tecnologia das Radiações, no IPEN/CNEN-SP. O sistema de irradiação (SI) é constituído por uma cápsula de irradiação em alumínio, linhas de transferência, válvulas agulhas, conexões anilhadas, conectores rápidos, manovacuômetro, sistema de vácuo, dewar de liquefação, blindagem em chumbo, cilindros de armazenamento e transporte (CAT), dentre outros. O SI foi aprovado nos testes de estanqueidade e estabilidade (testes de formação de bolhas, pressurização, evacuação e com equipamento leak detector SPECTRON 600 T). Na produção experimental para obtenção de 1,07x1011 Bq (2,9 Ci) de 41Ar, distribuíram-se dosímetros de alanina em diversos componentes e dispositivos do SI. Além disso, determinaram-se as taxas de exposição na parede da blindagem em chumbo, ao concentrar o gás radioativo liquefeito e no CAT, após a transferência do 41Ar, pelo medidor de radiação portátil Teletector ® Probe 6150 AD-t/H. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Submitted by Josimara Dias Brumatti (bcgdigital@ndc.uff.br) on 2017-04-10T13:59:32Z No. of bitstreams: 1 Uma metodologia.pdf: 1412283 bytes, checksum: 9a4fe74670e47650eb81f9b516ebadb0 (MD5) / Approved for entry into archive by Josimara Dias Brumatti (bcgdigital@ndc.uff.br) on 2017-04-10T14:11:57Z (GMT) No. of bitstreams: 1 Uma metodologia.pdf: 1412283 bytes, checksum: 9a4fe74670e47650eb81f9b516ebadb0 (MD5) / Made available in DSpace on 2017-04-10T14:11:57Z (GMT). No. of bitstreams: 1 Uma metodologia.pdf: 1412283 bytes, checksum: 9a4fe74670e47650eb81f9b516ebadb0 (MD5) / A presente tese apresenta uma proposta de ensino de língua portuguesa para alunos do Programa Nacional de Integração da Educação Básica com a Educação Profissional na Modalidade de Educação de Jovens e Adultos (PROEJA), baseada na metodologia Molar de abordagem de textos gramaticais do Prof. Dr. Maurício da Silva. A metodologia foi aplicada ao estudo das regras de emprego do hífen explicitadas no texto das Bases XV e XVI do Acordo Ortográfico da Língua Portuguesa de 1990, tendo em vista a grande importância do assunto em razão da entrada em vigor do Acordo em 2016 e a grande dificuldade de apreensão das referidas regras, bem como das convenções ortográficas em geral, manifestada pelos discentes da referida modalidade de ensino. A proposta criada e descrita nesta tese apoiou-se, também, na utilização da teoria de Gêneros Textuais como forma de mediar a transposição didática dos pressupostos teóricos. Assim sendo, apresenta-se aqui a criação de um material didático em que se corporificou essa transposição para textos dos gêneros bula, propaganda e cápsulas hifênicas. / This thesis presents a proposal on Portuguese language teaching to students of the National Elementary Education Integration Program with Vocational Education in the Youth and Adult Education Mode (PROEJA) based on the Molar methodology approach to grammatical texts by Dr. Mauricio da Silva. The methodology was applied to the study of the rules on the use of the hyphen explained in the Bases XV and XVI of the 1990 Portuguese Language Orthographic Agreement, due to the great importance of the subject, which will come into effect in 2016, and the great difficulties in apprehension of those rules, as well as spelling conventions in general, shown by students of the aforementioned mode of education. The proposal created and described in this thesis was supported by the Genre theory as a way to mediate the didactic transposition of the theoretical assumptions. Therefore, this paper presents the creation of pedagogical material that embodied this transposition to texts of CMI (Consumer Medicine Information) leaflets, advertising and hyphenic capsules.