Dissertations / Theses on the topic 'Gem therapy'

Este trabajo consiste en el desarrollo de un plan de negocio de jabones artesanales con propiedades naturales y de gemoterapia teniendo como prioridad satisfacer las necesidades de nuestro público objetivo. Con el objetivo de elaborar un producto de alta calidad, se contó con la colaboración por medio de entrevistas de expertos que nos permitieron implementar diversas mejoras en la adquisición de los insumos correctos y los procedimientos adecuados. En primer lugar, para la elaboración del proyecto se desarrolló una segmentación que nos permitió determinar de manera efectiva nuestro público objetivo. Posteriormente por medio de los diversos experimentos que se desarrollaron se pudo conocer las características que eran apreciadas por los consumidores de nuestro producto, además se estableció el precio de venta por medio del concierge. En segundo lugar, se evaluaron los diversos factores tanto internos como externos que pueden afectar de manera indirecta o directa a nuestra empresa incluyendo la coyuntura relacionada con el COVID-19, elaborando de esta manera estrategias aplicables para el entorno cambiante actual y futuro. Finalmente, se elaboraron presupuestos en las diversas áreas implicadas en los procesos de la empresa como marketing, responsabilidad social empresarial, operaciones, recursos humanos entre otros tanto a corto como largo plazo, permitiéndonos de esta manera evaluar la viabilidad del proyecto.
This assignment consists in the development of a business plan for handmade soaps with natural properties and gem therapy having as a priority to satisfy the needs of our target public. With the objective of elaborating a high-quality product, we had the collaboration through interviews with experts that allowed us to implement several improvements in the acquisition of the correct inputs and the adequate procedures. First, for the elaboration of the project, a segmentation was developed that allowed us to effectively determine our target audience. Later, through the various experiments that were developed, we were able to know the characteristics that were appreciated by the consumers of our product, in addition to establishing the sales price through the concierge. Secondly, we evaluated the various internal and external factors that may indirectly or directly affect our company including the situation related to the COVID-19, thus developing applicable strategies for the current and future changing environment. Finally, budgets were prepared in the various areas involved in the company's processes such as marketing, corporate social responsibility, operations, human resources among others both in the short and long term, thus allowing us to assess the viability of the project.
Trabajo de investigación

There have been developments in radiation therapy treatment techiques, which lead to an increase in the complexity of these treatments. The aim is to deliver highly conformal three-dimensional (3D) dose distributions, such as stereotactic radiosurgery (SRS). Polymer gel dosimetry offers three-dimensional (3D) dosimetry techniques for dose verification of dose distributions. Nisopropyl- acrylamide (NIP AM) polymer gel was the latest to develop and can be prepared under a normal atmospheric environment and has lower toxicity compared with the highly toxic polymer gels used earlier. NIPAM polymer gel using X-ray computed tomography (CT) was experimentally investigated in terms of its X-ray CT dose response, sensitivity and dose resolution. The effect of radiation beam type, radiation beam energy and radiation beam dose rate on X-ray CT dose response have also been studied. The temporal stability of NIP AM polymer gel has been examined over several days post-irradiation. The change in the polymer gel dosimeter's physical and electron densities as a function of absorbed dose was also investigated. In ,this study two new prototype phantoms were designed and constructed for imaging and irradiation of polymer gel dosimeters to provide simplicity and practicality for clinical dosimetry. The dosimetric and water equivalence properties of four NIP AM based polymer gel dosimeter formulations have been studied by examining their physical properties, interaction probability, radiation transport parameters and performing Monte Carlo modelling of depth doses. NIP AM polymer gel dosimeter irradiated at different doses using 6 Me V photon beam and 400 MU min-1 dose rate were found to have higher CT dose response (up to 37.8% at 10 Gy dose point) than results reported in the literature for NIP AM gel using similar concentration. The CT dose sensitivity of NIPAM polymer gel was found to be 0.405±0.014 H Gi1 , which is 26.2% higher than the reported sensitivity of 0.32l±0.008 H Gy-l with similar NIPAM gel concentration. The maximum change in physical density as a function of absorbed dose for polymer gel dosimeters was found to be up to ~1.0% for an absorbed dose of 20 Gy. 111

In the present thesis we propose the development of hybrid polymer titanium dioxide (TiO2) nanoparticles for use in biomedical applications. TiO2 exhibits high biocompatibility in the dark however, upon illumination in aqueous media with near UV light it produces an array of reactive oxygen species (ROS) which have the capability to induce death in neighbouring cells. The process of inducing cell death using a photosensitive material which produces ROS is known as photodynamic therapy (PDT) and is used to treat a wide range of maladies from psoriasis to cancer.We have demonstrated the ability to produce anatase nanoparticles with high control over their resulting size through a novel water mediated sol-gel synthetic method in benzyl alcohol, using either Ti(OnPr)4, Ti(OnBu)4 or Ti(OiPr)4 as the metal precursor. Through dynamic light scattering (DLS) analysis we have shown that the mechanism of nanoparticle growth appears to proceed through the agglomeration of primary nanoparticles formed instantly upon adding the reagents together. After synthesis the nanoparticles could be easily redispersed in aqueous media at pH2 with any further agglomeration being controlled by the parent alkoxide.After synthesis the nanoparticles were coated with PEG, conjugated to either a catechol or phosphate as ligand, in order to stabilise the nanoparticles at neutral pH. Uncoated nanoparticles exhibited good photoactive capability in the photooxidation of methylene blue. However, on coating with catechols the photoactivity of the nanoparticles was abolished. Coating with phosph(on)ates on the other hand preserved or even enhanced the photoactivity which makes this system promising for in vivo applications.At the same time this thesis also reports preliminary investigations on the use of TiO2 embedded into the walls of model drug loaded poly electrolyte multilayer microspheres for UV triggered delivery applications.

A study into the applicability of laser ablation inductively coupled plasma-mass spectrometry (LA ICP-MS) coupled with native polyacrylamide gel electrophoresis (PAGE) to metal speciation in proteins is described in this thesis. Chapter one of the thesis outlines the various roles played by metal ions in clinical samples. Health risks can arise from anthropogenic metal enrichment and metallodrug therapy is identified as a key source. The use of platinum and gold metallodrugs is discussed in detail. Speciation of metals in clinical samples is examined, concentrating on some of the techniques currently employed and their limitations. Particular attention is paid to the techniques employed in the study. The aim of the study is outlined - to develop an alternative speciation strategy for the study of metals in proteins. The reagents and instrumentation used are described in chapter two along with all experimental procedures. Chapter three describes the method development and determination of the analytical performance of the technique. The technique is used to study platinum speciation of protein samples enriched in vitro, the metal distribution profiles obtained are shown. The technique is next applied to the analysis of in vivo samples obtained from platinum therapy patients and a control source. Chapter four outlines the application of the technique to analysis of gold enriched samples both in vitro and in vivo from chrysotherapy patients. The gold distribution profiles obtained are shown and discussed. Chapter five looks at multi-element distribution and interactions occurring in serum. Elements naturally occurring at trace levels in serum, such as Cu and Fe, are studied in vivo. Other, lower level metals are studied following in vitro enrichment. Chapter six concludes the study. It discusses how the technique was found to be applicable for metal speciation of clinical samples by meeting the criteria laid out at the start of the study. Recommendations for future work are also outlined.

During recent years, significant advances have been made in the field of molecular therapy in urologic oncology, mainly for advanced renal cell carcinoma. In this hitherto largely treatment-refractory disease, several agents have been developed targeting the von Hippel-Lindau metabolic pathway which is involved in carcinogenesis and progression of the majority of renal cell carcinomas. Although cure may not be expected, new drugs, such as the multikinase inhibitors sorafenib and sunitinib and the mammalian target of rapamycine inhibitor temsirolimus, frequently stabilize the disease course and may improve survival. Fewer data are available supporting molecular therapies in prostate, bladder, and testicular cancers. Preliminary data suggest a potential role of high-dose calcitriol and thalidomide in hormone-refractory prostate cancer, whereas targeted therapies in bladder and testicular cancers are still more or less limited to single-case experiences. The great theoretical potential and the multitude of possible targets and drug combinations, however, support further research into this exciting field of medical treatment of urologic malignancies
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

During recent years, significant advances have been made in the field of molecular therapy in urologic oncology, mainly for advanced renal cell carcinoma. In this hitherto largely treatment-refractory disease, several agents have been developed targeting the von Hippel-Lindau metabolic pathway which is involved in carcinogenesis and progression of the majority of renal cell carcinomas. Although cure may not be expected, new drugs, such as the multikinase inhibitors sorafenib and sunitinib and the mammalian target of rapamycine inhibitor temsirolimus, frequently stabilize the disease course and may improve survival. Fewer data are available supporting molecular therapies in prostate, bladder, and testicular cancers. Preliminary data suggest a potential role of high-dose calcitriol and thalidomide in hormone-refractory prostate cancer, whereas targeted therapies in bladder and testicular cancers are still more or less limited to single-case experiences. The great theoretical potential and the multitude of possible targets and drug combinations, however, support further research into this exciting field of medical treatment of urologic malignancies.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

A radioterapia com intensidade modulada de feixe (IMRT) possibilitou a realização de tratamentos de múltiplos alvos simultaneamente em um esquema de tratamento acelerado conhecido como SMART (Simultaneous Modulated Accelerated Radiation Therapy). Estes tratamentos requerem um rigoroso controle de qualidade (QA) que deveria ser realizado, idealmente, em três dimensões. Uma potencial ferramenta para dosimetria tridimensional (3D) é o uso de gel polimérico associado à leitura de doses com Imagens de Ressonância Magnética Nuclear (IRMN). Neste trabalho, a dosimetria 3D com o gel MAGIC-f foi aplicada no CQ de 10 planejamentos radioterápicos usando a técnica SMART. Inicialmente, as distribuições de dose dos planejamentos avaliados foram recalculadas na geometria do simulador baseadas em sua tomogra_a computadorizada. Todos os planejamentos utilizados foram aprovados previamente no QA convencional pontual e planar, o que validou a distribuição de dose do sistema de planejamento (TPS) para comparação com a dosimetria gel. Os simuladores foram irradiados com os planejamentos estabelecidos e as distribuições de dose foram obtidas através da relaxometria em IRMN. A comparação das distribuições de dose calculadas pelo TPS e as medidas no gel foi feita pela análise gama (3%/3mm) e pela comparação entre os histogramas dose-volume (DVHs) calculados e medidos para dois volumes criados nas regiões equivalentes aos volumes alvo de tratamento. Esses resultados evidenciam o potencial do uso da dosimetria gel polimérica no QA 3D de planejamentos de IMRT com a técnica SMART, fornecendo informação completa 3D da dose absorvida em cada volume alvo planejado, desde que realizada seguindo os protocolos pré-estabelecidos.
Intensity Modulated Radiation Therapy (IMRT) treatments in the radiotherapy practice made possible the simultaneously treatment of multiple targets at an accelerated treatment regimen, this scheme is known as Simultaneous Modulated Accelerated Radiation Therapy (SMART). These treatments require a strict quality control (QC) that should be done, ideally, in three dimensions. A potential tool for tridimensional dosimetry (3D) is the use of polymeric gel with Nuclear Magnetic Resonance Images (NMRI). In this paper, the 3D dosimetry with MAGIC-f gel was applied in 10 QC radiotherapy planning using the SMART technique. Initially, the dose distributions of assessed plans were remeasured in simulator geometry based on a CT scan. All plans used were previously approved in punctual and planar conventional CQ, which validated the dose distribution planning system (TPS) for comparison with the gel dosimetry. The simulators were irradiated with established schedules and dose distributions were obtained by relaxometry in NMRI. The comparison of dose distributions calculated by TPS and measured in the gel was made by gamma analysis (3% / 3mm) and by comparing the dose-volume histograms (DVHs) calculated and measured for two volumes created in the equivalent regions to target volumes of treatment. These results highlight the potential use of the polymer gel dosimetry in IMRT planning 3D CQ with SMART technique, providing complete 3D information of the absorbed dose in each target volume planned, if carried out following the pre-established protocols.

A mina de Sal-gema em Loulé localiza-se no eixo principal de circulação da cidade, reconhecido como sendo o espaço mais importante da prática social, económica e cultural da cidade. Este complexo mineiro de sal-gema que assume um papel relevante de entrada e saída da cidade, apresenta-se como um espaço de caracter industrial, inserido num enorme vazio urbano, revelando total descaracterização urbana e paiaagistica. Partindo de uma abordagem prática de projecto, procura-se uma exploração consciente do turismo aliando-o à saúde, conhecidas que são os benefícios do sal para a mesma, nomeadamente ao nível das doenças respiratórias. O projecto pretende criar um enquadramento paisagístico, melhoria de acessibilidades e renovação urbana, com vista a dignificar este complexo industrial, inserindo-o na vida da cidade. ABSTRACT: The Loulé Sait mines are located in the main axis of the city´s circulation, recognized as the most important space of social, economic and cultural pratice of the city. This salt mining complex, that assumes a role of entry or exit in the city, presents it self as an industrial space, inserted in a huge urban emptiness, revealing total urban and landscape deprivation. Starting from a practical approach, the project is looking for an conscious exploration of turism combined with health, known as the benefits of salt for health, especially respiratory diseases. The project aims to create a landscape framework better acessibilities and urban renovation, in order to dignity this industrial complex and inserting in the city life.

Anthracene was attached to light activated, ruthenium-based DNA disruptors to probe their distribution in cancer cells. The objective of this research is to understand the photophysical properties (Chapter 2), photoreactivity toward DNA and proteins (Chapter 3), and localization within cancer cells (Chapter 4) of ruthenium complexes that demonstrate promise as photodynamic therapy (PDT) agents. [(AnthbpyMe)(bpy)Ru(dpp)]2+ (1) and [(AnthbpyMe)2Ru(dpp)]2+ (2) absorb visible light with metal-to-ligand charge transfer (MLCT) transitions at 459 nm (16,000 M-1 cm-1 ) and 461 nm (21,000 M-1 cm-1 ), respectively. These species exhibit 3 MLCT emissions at λem = 661 nm and λem = 663 nm for 1 and 2, respectively, while the anthracene show emissions at 450 – 560 nm. The anthracene unit(s) quench the 3 MLCT to give quantum yields (lifetime) of Φem = 0.0059 [398(1) ns] and Φem = 0.0011 [414(1) ns] for 1 and 2, respectively. Voltammetry shows an irreversible anthracene oxidation at 1.23 – 1.28 V, RuIII/II oxidation at 1.53 – 1.55 V, and quasi-reversible reduction couples attributed to dpp0/-1 at 0.98 V. DNA gel shift assays demonstrate that complexes 1 and 2 modify DNA in the presence and absence of 3 O2 upon light activation to convert supercoiled DNA to a mixture of open circular (OC) DNA and a species that exhibit sa distinctly different migration rate than either OC and linear DNA. Binding constants, Kb, for complexes 1 and 2, toward DNA are 3.50 × 105 (3.50 × 104 ) and 4.50 × 103 (4.50 × 102 ) respectively. SDS-PAGE assays show that the complexes 1 and 2 modify bovine serum albumin (BSA) through an 3 O2-dependent mechanism upon light iii activation. The localization and PDT potency of the anthracene-Ru-dpp complexes are tested against F98 cells, which are rat glioma cells that simulate the infiltrative patterns of growth in cancer. Confocal microscopy demonstrates that complexes 1 and 2 internalize and localize primarily along the cell membrane and associate with dot-like vesicles within the cytoplasm. Complexes 1 and 2 show IC50 values of 107 µM and 85 µM, respectively, after 15 min of drug exposure and 1 h of PDT-treatment (λPDT = 455 nm).
Ph. D.

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Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia)
IPEN/D-MPLO
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

A presença de material de alto número atômico adjacente ao tecido mole aumenta localmente a dose absorvida pelo tecido quando submetido à radiação. Este efeito ocorre devido aos fotoelétrons ejetados do material de alto número atômico. Dosímetros de gel polimérico com partículas de ouro foram utilizados para investigar este efeito. Foram realizados cálculos analíticos para estimar o aumento de dose e simulações com o método de Monte Carlo. A irradiação de amostras de gel polimérico (GP) puro e com 0,005 gAu/gGP utilizando um feixe de raios X produzido por um potencial de 150 kV filtrado com 4 mmAl e 5 mmCu resultou em uma dose absorvida pelas amostras com ouro aproximadamente 20% maior que a dose absorvida pelas amostras de gel polimérico puro. Os cálculos analíticos e a simulação com o método de Monte Carlo resultaram em um aumento de aproximadamente 30% na dose absorvida.
The presence of high-Z material adjacent to soft tissue, when submitted to irradiation, enhances locally the absorbed dose in these soft tissues. Such effect occurs due to the outscattering of photoelectrons from the high-Z material. Polymer gel dosimeters have been used to investigate this effect. Analytic calculations to estimate the dose enhancement and Monte Carlo simulations have been performed. Samples containing polymer gel (PG) with 0.005 gAu/gPG and pure polymer gel have been irradiated using an X-rays beam produced by 150 kV, filtered with 4 mm Al and 5 mm Cu, which resulted in an approximately 20% higher absorbed dose in the samples with gold in comparison to those with pure polymer gel. The analytic calculations and the Monte Carlo simulation resulted in a dose enhancement factor of approximately 30%.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults, affecting thousands of people worldwide every year, with a life expectancy, post diagnosis of 12 months. Surgery, radiotherapy and chemotherapy together, result in an overall mean survival not exceeding 15 months. Targeted therapeutic agents sorafenib, an oral multi kinase inhibitor, and lapatinib, an epidermal growth factor receptor (EGFR) inhibitor, used in combination have been shown to kill GBM cells be through inhibition of major growth mediating signaling pathways that are frequently over expressed in gliomas, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/ protein kinase B (PI3K/AKT). Sorafenib can restore lapatinib induced cytotoxicity by down regulation of myeloid cell leukaemia-1 (Mcl-1) expression. Prior studies have shown Mcl-1 to play an important role in resistance to lapatinib. Furthermore, data indicated that this drug combination is able to trigger activation of autophagic and apoptotic pathways and induce endoplasmic reticulum (ER) stress response in GBM cells, collectively resulting in cell death. In conclusion, data presented here demonstrates that the combination of sorafenib and lapatinib can kill GBM cells in a greater than additive fashion, through induction of autophagy, apoptotic events (extrinsic and intrinsic) and ER stress.

A few computational models currently exist to predict heat production and dissipation in tissue when a tumor containing optically-tunable gold nanoparticles such as nanoshells or nanorods is illuminated with near infrared (NIR) laser. The validity of any computational model still needs to be established by experiments before its wide use for various future clinical applications. One of the possible ways to validate the model is through the heat measurements within a phantom made with tissue-equivalent heat-sensitive gel. Currently, there are a few recipes available for this type of gel and the majority of them use severely toxic ingredients. However, none of them seems to perfectly serve the current purposes. Therefore, the primary goal of this thesis work was to develop and characterize two new types of heat-sensitive gels, using relatively non-toxic substances for the in-phantom validation of computational models. Specifically, two novel agar based phantoms, TG1 and TG2, were developed and characterized. The basic optical response of these phantoms at 808 nm NIR light was determined to test their equivalency to human tissue. Thermal damage to the phantoms was quantified by heating them to specific temperatures and obtaining calibration curves to relate temperature and R2 relaxation rates. The phantoms were scanned with magnetic resonance imaging (MRI) to obtain T2 values. TG1 gel, agar and bovine serum albumin (BSA) mixture, was found not to be optically tissue-equivalent. However, TG1 gel demonstrated unambiguous digital response capable of distinguishing temperature of at least 70 °C compared to the sample receiving no heat. Additionally, TG1 gel produced high degree of linearity in the thermal therapy temperature regime (60 - 80 °C). TG2 gel containing agar mixed with BSA and Intralipid has exhibited tissue equivalency based on laser transmission measurements. TG2 gel exhibited heat damage based on T2 values, only when the temperature reaches 80 °C. This digital response is considered less sensitive in view of the fact that BSA starts to undergo denaturing and cause optical density change at approximately 70 °C. Both gels, however, have shown to be thermally stable at temperatures up to 80 °C with no evidence of gel melting being observed.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

La fibrosis quística (fq) es la enfermedad autosómica recesiva más frecuente en la población caucasoide con una frecuencia de portadores de 1/25. Las manifestaciones clínicas más importantes son las infecciones crónicas recurrentes del pulmón conllevando al deterioro del mismo. En esta tesis nos propusimos corregir dos mutaciones en el gen cftr, responsable de la fq, en la linea celular de epitelio bronquial ib3.1 de genotipo (f508del/w1282x). Para ello, mediante citometría de flujo y microscopía confocal pusimos a punto la incorporación no viral (vectores pei, geneporter y citofectina) de oligonucleótidos modificados (quimeraplastos y oligonucleótidos fosforotioato) y de fragmentos sfhr en estas células. Los quimeraplastos son oligonucléotidos quiméricos de rna y dna y los oligonucleótidos fosforotioato contienen enlaces fosforotioato para evitar su degradación además de enlaces fosfodiéster. Ambos, son capaces de estimular los mecanismos de reparación intrínsecos de las células para corregir a nivel de dna mutaciones puntuales. Los fragmentos sfhr (small fragment homologous replacement) son fragmentos de pcr de longitud variable con la secuencia salvaje del gen que se intercambian con las secuencias genómicas diana mutadas por recombinación homóloga para revertir mutaciones puntuales (cambios de 1 nucleótido, pequeñas inserciones o deleciones). Para estimar el porcentaje de corrección génica adaptamos la técnica diagnóstica de pcr-ola a nuestro modelo para utilizarla como metodología de cuantificación. Además, hemos confirmado que las células ib3.1 tienen los mecanismos de reparación de mutaciones puntuales activos tanto por rt-pcr como por un ensayo in vitro en e.coli con el plásmido pksm4021 que contiene el gen de resistencia a ampicilina y el gen de resistencia a kanamicina inactivado por una mutación puntual. Los resultados que se publicaron en los siguientes artículos nos han permitido extraer las siguientes conclusiones: las células Ib3.1 de epitelio bronquial de fq son competentes en cuanto al sistema de reparación mmr. La incorporación celular de quimeraplastos, oligonucleótidos monocadena y fragmentos sfhr es muy eficiente en las células ib3.1. El lípido catiónico citofectina, el policatión pei y la electroporación, aunque no el lípido catiónico geneporter, son sistemas de transfección eficientes para incorporar oligonucleótidos modificados en el núcleo de las células ib3.1. Los poliplejos de pei y los lipoplejos de citofectina son internalizados por distintos mecanismos aunque en ambos casos los oligonucleótidos modificados son degradados significativamente en las células ib3.1. El análisis genescan de electroferogramas fluorescentes constituye un sistema fiable, fácil, sensible y seguro para evaluar y cuantificar la degradación intracelular y extracelular de oligonucleótidos marcados con fluorescencia en fluidos biológicos. La tecnología de pcr-ola constituye un sistema fiable y preciso de cuantificación de reparación génica aplicable a modelos celulares heterocigotos. Los fragmentos sfhr pueden actuar como cebador artefactual en las reacciones de pcr y generar un artefacto que da lugar a falsos positivos en la detección de conversión génica. Es indispensable diseñar los cebadores de detección de la modificación génica fuera de la región de homología con los fragmentos sfhr. La corrección génica de mutaciones puntuales mediada por quimeraplastos, oligonucleótidos monocadena y fragmentos sfhr es un proceso ineficiente en las células ib3.1. Será necesario estimular los mecanismos endógenos de reparación génica para incrementar la frecuencia de reparación génica hasta niveles terapéuticos en dichas células.

Orientadores: Maria Luiza Moretti, Rogerio de Jeus Pedro
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: OBJETIVOS: Avaliar a incidência de colonização oral por Candida spp. em pacientes com HIV em uso de terapia anti-retroviral, comparando os resultados dos grupos de pacientes colonizados e não colonizados, assim como estudar os aspectos microbiológicos das cepas isoladas. PACIENTES E MÉTODOS: Foi realizado estudo transversal de pacientes assistidos no HC/Unicamp, de agosto de 2003 a abril de 2004, com coleta única por paciente de swab da cavidade oral. CHROMagar Candida® e ID32C® foram utilizados no cultivo, isolamento e identificação de Candida spp. e Candida Check® na determinação dos sorotipos de C. albicans. C. dubliniensis foi identificada por seqüenciamento no aparelho ABI PRISM 3100® GENETIC ANALYZER. O perfil genômico foi estudado por PFGE, usando o sistema CHEF e a sensibilidade aos azólicos, 5-fluocitosina, anfotericina B e nistatina, baseada na microdiluição em caldo (CLSI). Por meio da revisão dos prontuários foi avaliado: gênero, idade, raça, ano de diagnóstico da infecção pelo HIV, tipo de exposição ao HIV, carga viral, contagem de linfócito TCD4+, infecções oportunistas, classificação clínica da infecção pelo HIV, terapia anti-retroviral e terapia antifúngica. RESULTADOS: Foram identificados 140/324 pacientes colonizados e 184/324 não colonizados: gênero masculino (63% e 60%), exposição sexual (81,5% e 80,5%) e idade média de 38,9 anos. A presença de colonização/infecção foi significativamente maior em pacientes com carga viral detectável (p=0,002) e CD4+<200/mm3 (p=0,006). Foi evidenciada incidência de candidíase oral (31,2%), tuberculose (20,9%), herpes zoster (16,3%), pneumonia por Pneumocystis carinii (PCP) (15,7%) e toxoplasmose (11,7%), no total de pacientes estudados. Não foi observada diferença significativa de colonização por Candida entre os pacientes em uso de TARV com ou sem IP. O uso prévio de nistatina foi maior no grupo colonizado (p=0,014). Foram isoladas 115/154 C. albicans sorotipo A, 15/154 C. albicans sorotipo B e 24/154 Candida não albicans. Doze pacientes apresentaram colonização mista. O estudo genômico de C. albicans sorotipo A identificou 15 perfis diferentes, com predomínio do A1 (56,5%), que mostrou similaridade de 100% entre o perfil de C. albicans sorotipo B predominante B1 (86,6%). O perfil genômico de C. glabrata mostrou-se heterogêneo. C. albicans sorotipo A e B mostraram-se sensíveis a todos os antifúngicos avaliados. C. glabrata e C. krusei apresentaram S-DD para os azólicos. CONCLUSÃO: O trabalho contribuiu de forma significativa para traçar o perfil epidemiológico/clínico dos pacientes HIV em uso de TARV e verificou que o uso de IP não influenciou na presença ou ausência de colonização oral por Candida
Abstract: OBJECTIVES: Evaluating de incidence of oral colonization by Candida spp. in patients in use of antiretroviral therapy, comparing the results of the groups of patients colonized and non-colonized, as well as study the microbiological aspects of the isolated strains. PATIENTS AND METHODS: It was made a cross sectional study assisted at HC/UNICAMP, from August, 2003 to April, 2004, with unique collect of the oral cavity by patient using a swab. CHROMagar Candida® and ID32C® were used in growth, isolation and identification of Candida spp. and Candida Check® for determination of C. albicans sorotypes. C. dubliniensis was identified by sequencing in ABI PRISM 3100® GENETIC ANALYZER device. The genomic profile was studied by PFGE, using the system CHEF and azoles, 5-FC, amphotericin B and nistatine sensibility, based broth microdilution (CLSI). It was evaluated through the review of the prontuaries: genre, age, race, year of HIV infection diagnosis, type of exposition, viral load, TCD4+ linfocyte counting, opportunistic infections, antiretroviral therapy and antifungal therapy. RESULTS: It was identified 140/324 colonized patients and 184/324 non-colonized patients: male gender (63% and 60%), sexual exposition (81,5% and 80,5%) and average age of 39,8 years old. The presence of colonization was significantly greater in patients with detectable viral load (p=0,002) e CD4+<200/mm3 (p=0,006). The incidence of oral candidiasis (31,2%), tuberculosis (20,9%), herpes zoster (16,3%), Pneumocystis carinii pneumonia (PCP) (15,7%) and toxoplasmosis (11,7%) was seen among the total of studied patients. It was not observed a significant difference regarding colonization by Candida among the patients in use of ARVT with or without usage of PI. The early usage of nistatine was bigger in the colonized group (p=0,014). It was isolated 115/154 C. albicans sorotype A, 15/154 C. albicans sorotype B and 24/154 non albicans Candida . Twelve patients presented mixed colonization. The genomic study of C. albicans sorotype A, identified 15 different profiles, with dominance of A1 (56,5%), which shown 100 % similarity between C. albicans sorotype B and predominant B1 (86,6%). The genomic profile of C. glabrata showed heterogeneous. C. albicans serotype A and B showed sensible to all evaluated antifugicals. C. glabrata e C. krusei showed S-DD to azoles. CONCLUSION: This work contributed significantly to trace an epidemiological/clinical profile of the HIV patients in usage of ARV therapy and the lack of influence of IP in the presence or absence of colonization of oral Candida
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM. Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model. One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice. Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice. Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor. In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM. Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model. One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice. Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice. Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor. In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.

Neste trabalho as principais características dosímetricas da solução Fricke Xilenol Gel (FXG) foram estabelecidas para futura aplicação clínica na dosimetria de elétrons. As curvas de dose resposta para feixes de nêutrons térmicos para pesquisa em Terapia por Captura de Nêutrons (BNCT) e feixes elétrons de aplicação industrial também foram determinadas. A técnica padrão de leitura utilizada foi espectrofotometria. Para o feixe clínico as reprodutibilidades intra e inter-lotes da solução FXG são melhores que 1,4 % e 5,1 % respectivamente, o comportamento da resposta para o intervalo de dose entre 0,2 e 40 Gy é linear e independente da energia e da taxa de dose para o intervalo estudado. Devido aos efeitos da oxidação natural do FXG o tempo ótimo entre o preparo e a irradiação é de 24h e o comportamento da curva de dose resposta não se altera no período estudado para a variação da absorvância líquida do dosímetro. Para o estudo com o campo de nêutrons as curvas de dose resposta do FXG apresentaram comportamento linear em todo intervalo de dose estudado, e para campos industriais de elétrons o comportamento é exponencial decrescente. De acordo com os resultados obtidos para os feixes de radiação estudados, não houve alteração na posição das bandas características do espectro de absorção do FXG. Como testes adicionais, foi determinada a viabilidade do uso do método de leitura do FXG por imagens fotográficas digitais e aplicação do FXG na dosimetria para braquiterapia intracavitária. O bom desempenho do dosímetro FXG nos testes realizados indica que este pode ser utilizado na avaliação tridimensional da dose em tratamento radioterápicos.
In this work the main dosimetric characteristics of the Fricke Xylenol Gel (FXG) solution were established for further application in the measurement of dose distribution of clinical electron fields. The dose-response curves of the FXG in a neutron field were also evaluated for the research in Boron Neutron Capture Therapy (BNCT) and industrial electron fields. The standard reading technique was the spectrophotometric. For the clinical field, the intra and inter-batch reproducibility are better than 1.4% and 5.1 %, respectively, the response presents a linear behavior for doses ranging from 0.2 to 40 Gy independently of the energy and the dose rate in the studied ranges. Due to the effects of the FXG natural oxidation, the optimum elapsed time between FXG preparation and irradiation was established as 24h period and the behavior of the dose-response curve of the FXG using the variation in the absorbance relative to the non-irradiated dosimeter as a basis during the whole studied period were not altered. The dose-response to the industrial electron beam presented an exponential decreasing behavior and the neutron beam for research in BNCT presented a linear behavior for the complete studied dose range. According to the obtained results for the different types of radiation studied for the FXG, there was no change in the position of the characteristic bands of the absorption spectrum due to the interaction of these radiation types. Additional tests were performed to determine the digital photographic imaging of FXG analyses viability and the application of FXG dosimetry on intracavitary brachytherapy. The good performance of the FXG dosimeter in the tests that were carried out indicates that this dosimeter may be applied to the tri-dimensional dose evaluation in radiotherapic treatments using electrons and neutron beams.

Music Therapy
Ph.D.
The purpose of this study was to identify predictors of responsiveness to the Bonny Method of Guided Imagery and Music (BMGIM), as measured by the Responsiveness to Guided Imagery and Music scale (RGIM). It was hypothesized that when combined, Sense of Coherence (SOC), state trait anxiety (STAI), classical music experience (CME), gender, and/or age would account for a significant amount of variance in responsiveness to BMGIM. Sixty volunteer participants were recruited from the general population. They all attended one research session where they participated in a group Guided Imagery and Music (GIM) experience. Prior to the group GIM experience, participants completed a demographic questionnaire (including a CME measure), the Spielberger State Trait Anxiety Inventory (STAI), and the SOC scale. After the group GIM experience, participants completed the RGIM scale. Once data collection was completed, the reliability and construct validity of the RGIM was examined. Exploratory factor analyses revealed that the RGIM contained five distinct factors: (a) Ability to Relax (AR), (b) Ability to Image to Music (AIM), (c) Responsiveness to Music and Guiding (RMG), (d) Comfort with Self-Disclosure (CSD), and (e) Meaningfulness of the Experience (MOE). These were the dependent (criterion) variables in this study. Before analyzing the data, the researcher ran diagnostics to check for assumptions of regression. Correlational statistical techniques were used to identify significant relationships between variables, and three sets of exploratory multiple linear regressions were used to identify which combinations of variables were the most accurate predictors of RGIM factors. Results of the first multiple regression showed that together, SOC, classical music experience, and age are predictors of "Ability to Image to Music," "Responsiveness to Music and Guiding," and "Meaningfulness of the Experience." Results of the second multiple regression showed that together, state anxiety, classical music experience, and age are predictors of "Ability to Image to Music," "Responsiveness to Music and Guiding," and "Meaningfulness of the Experience." Results of the third multiple regression showed that together, trait anxiety, classical music experience, and age are predictors of "Responsiveness to Music and Guiding." A number of other significant and non-significant results were found and are discussed. Limitations of the study as well as recommendations for future research, clinical practice, and education/training are presented.
Temple University--Theses

El MNGIE és una malaltia mitocondrial d’herència autosòmica recessiva causada per mutacions en el gen TYMP, que codifica l’enzim timidina fosforilasa (TP). La TP catalitza la degradació de timidina (dThd) i desoxiuridina (dUrd), i la seva absència en pacients causa l’acumulació sistèmica d’aquests metabòlits, tòxica per la funció mitocondrial. A dia d’avui les úniques teràpies efectives són el transplantament de cèl·lules mare hematopoètiques i el transplantament de fetge. No obstant, els transplantaments estan limitats per la necessitat d’un donant compatible i tenen una taxa de mortalitat i morbiditat associada especialment elevada en pacients de MNGIE. Per a superar aquests inconvenients, fa temps vam proposar la teràpia gènica mitjançant vectors adenoassociats (rAAV) dirigits al fetge com a alternativa terapèutica, i vam demostrar la seva viabilitat en el model animal de MNGIE, el ratolí doble knockout (dKO) Tymp-/-Upp1-/-. Però aquest model només presenta fenotip bioquímic, de manera que només vam poder demostrar la correcció d’aquest fenotip. Al 2014 es va descriure que augmentant el desequilibri metabòlic mitjançant l’administració oral de dThd i dUrd al model dKO durant tota la seva vida provocava l’aparició d’alguns trets que reproduïen la simptomatologia clínica dels pacients. En aquesta tesi hem estudiat l’ús de la teràpia gènica en aquest model millorat, mitjançant el tractament amb tres rAAVs que expressaven la seqüència codificant de TYMP sota el control de 3 promotors hepàtics a diferents dosis. El tractament amb rAAV va incrementar l’activitat TP en fetge i va disminuir la concentració sistèmica de nucleòsids dels ratolins dKO, que sense tractament eren 30 vegades superiors als valors normals. A nivell fenotípic, en la majoria dels ratolins, el tractament també va prevenir l’increment del volum dels ventricles cerebrals i el deteriorament motor observats en els ratolins no tractats. Quan vam comparar els tres vectors utilitzats, el rAAV amb el promotor de l’alfa-1-antitripsina (AAT) va ser el més eficaç. Aquests resultats confirmen que la teràpia gènica mediada per rAAV dirigida al fetge restaura l’homeòstasi bioquímica i demostren la prevenció de l’aparició del fenotip clínic del model animal millorat de MNGIE. Un altre aspecte important per a la translació de la teràpia a la pràctica clínica és optimitzar el vector per tal de reduir-ne la dosi efectiva. En aquest sentit, hem testat dues aproximacions: la modificació de la seqüència codificant (ADNc) del gen segons la freqüència d’ús de codó per a incrementar la seva expressió, i l’eliminació dels dinucleòtids CpG de l’ADNc del gen per a reduir la immunogenicitat del vector. Vam dissenyar quatre seqüències optimitzades de l’ADNc de TYMP amb 4 algoritmes diferents. Vam generar vectors lentivirals per transduir 4 línies cel·lulars humanes i determinar l’eficiència d’expressió de cada seqüència comparada amb la seqüència salvatge, tenint en compte el grau d’activitat TP, el número de còpies del vector i els nivells relatius d’ARNm. De tots els experiments, només una seqüència optimitzada va millorar el grau d’expressió de TYMP respecte el de la seqüència salvatge en la línia cel·lular hepàtica Huh7. Pel que fa a la reducció de la immunogenicitat del vector, vam eliminar els dinucleòtids CpG de les seqüències dissenyades i vam analitzar el grau d’expressió de TYMP. Només la seqüència salvatge sense dinucleòtids CpG es va acostar a l’expressió de la seqüència natural. Tot i que s’hi observa una reducció d’expressió aproximada del 20%, es compensa per l’avantatge que aporta en termes de reducció de la resposta immunològica de cara a l’ús clínic del vector. En conclusió recomanem aquesta versió sota la regulació del promotor AAT per a ús eventual en la pràctica clínica.
El MNGIE (Mitochondrial NeuroGastroIntestinal Encephalomyopathy) es una enfermedad mitocondrial de herencia autosómica recesiva causada por mutaciones en el gen TYMP, que codifica la enzima timidina fosforilasa (TP). La TP cataliza la degradación de timidina (dThd) y desoxiuridina (dUrd), y su ausencia en pacientes causa la acumulación sistémica de estos metabolitos, tóxica para la función mitocondrial. Hoy en día, las únicas terapias efectives son el trasplante de células madre hematopoyéticas y el trasplante de hígado. No obstante, los trasplantes están limitados por la necesidad de un donante compatible y tienen una tasa de mortalidad y morbididad asociada especialmente elevada en pacientes de MNGIE. Para superar estos inconvenientes, hace tiempo propusimos la terapia génica mediante vectores adenoasociados (rAAV) dirigidos al hígado como alternativa terapéutica, y demostramos su viabilidad en el modelo animal de MNGIE, el ratón doble knockout (dKO) Tymp-/-Upp1-/-. Pero este modelo solo presenta fenotipo bioquímico, de forma que solo pudimos demostrar la corrección de este fenotipo. En 2014 se describió que aumentando el desequilibrio metabólico mediante la administración oral de dThd y dUrd al modelo dKO durante toda su vida provocaba la aparición de algunos rasgos que reproducían la sintomatología clínica de los pacientes. En esta tesis hemos estudiado el uso de la terapia génica en este modelo mejorado, mediante el tratamiento con tres rAAVs que expresan la secuencia codificante de TYMP bajo el control de tres promotores hepáticos a distintas dosis. El tratamiento con rAAV incrementó la actividad TP hepática y disminuyo la concentración sistémica de nucleósidos de los ratones dKO, (sin tratamiento eren 30 veces superiores a los valores normales). A nivel fenotípico, en la mayoría de los ratones, el tratamiento también previno el aumento del volumen de los ventrículos cerebrales y el deterioro motor observados en los ratones no tratados. Cuando comparamos los tres vectores utilitzados, el rAAV con el promotor de la alfa-1-antitripsina (AAT) fue el más eficaz. Estos resultados confirman que la terapia génica por rAAV dirigida al hígado restaura la homeóstasis bioquímica y demuestran la prevención de la aparición del fenotipo clínico del modelo animal mejorado de MNGIE. Otro aspecto importante para la translación de la terapia a la práctica clínica es optimizar el vector para reducir la dosis efectiva. En este sentido, hemos testado dos aproximaciones: la modificación de la secuencia codificante (ADNc) del gen según la frecuencia de uso de codón para aumentar su expresión, y la eliminación de los dinucleótidos CpG del ADNc del gen para reducir la immunogenicidad del vector. Diseñamos cuatro secuencies optimizadas del ADNc de TYMP utilitzando 4 algoritmos diferentes. Generamos vectores lentivirales para transducir 4 líneas celulares humanas y determinar la eficiencia de expresión de cada secuencia comparada con la secuencia natural, teniendo en cuenta el grado de actividad TP, el número de copias del vector y los niveles relativos de ARNm. De todos los experimentos, solo una secuencia optimizada mejoró el grado de expresión de TYMP comparado con el de la secuencia natural, en la línea celular hepática Huh7. En cuanto a la reducción de la immunogenicidad del vector, eliminamos los dinucleótidos CpG de las secuencias diseñadas y analizamos el nivel de expresión de TYMP. Solo la secuencia natural sin dinucleótidos CpG se acercó a la expresión de la secuencia natural. Aunque se observa una reducción de expresión aproximada del 20%, se compensa con la ventaja que aporta en términos de reducción de la respuesta inmunológica de cara al uso clínico del vector. En conclusión, entre las opciones testadas, recomendamos el rAAV que contiene el ADNc natural de TYMP sin dinucleótidos CpG bajo el control del promotor AAT para un uso eventual en la práctica clínica.
MNGIE (Mitochondrial NeuroGastroIntestinal Encephalomyopathy) is an autosomal recessive disease caused by mutations in TYMP, which encodes for the enzyme thymidine phosphorylase (TP). TP catalyses the first step of the catabolism of the nucleosides thymidine (dThd) and deoxyuridine (dUrd). The lack of TP activity causes systemic nucleoside accumulation which is toxic for mitochondrial function. Nowadays, the only available therapies for MNGIE are allogeneic hematopoetic stem cell transplantation or liver transplantation. However, these treatments are limited by the need of a compatible donor and are associated to high mortality and morbidity rates in MNGIE patients. To overcome these limitations, our laboratory proposed adenoassociated virus (rAAV) mediated gene therapy targeted to the liver for MNGIE and demonstrated its feasibility in the Tymp-/- Upp1-/- (dKO) mouse model of the disease. However, dKO mice show biochemical phenotype only, therefore we could only demonstrate the efficacy of our approach at the biochemical level. In 2014 it was reported that increasing the biochemical imbalances of the dKO model by chronic oral administration of dThd and dUrd to dKO mice over their entire life induced some features that recapitulate part of the clinical manifestations observed in patients. In this thesis we have studied the effect of gene therapy in this enhanced model, by treating the animals with three rAAVs expressing the human TYMP coding sequence under the control of three different liver-specific promoters. rAAV treatment increased liver TP activity and limited the systemic nucleoside concentration present in the dKO enhanced model, which was 30-fold higher as compared with non-exposed wild-type mice. AAV-treatment also prevented, in most animals, the enlarged brain ventricles and the motor impairment observed in the exposed dKO mice. When we compared the three promoters tested, the rAAV containing the AAT promoter showed the best efficacy. These results confirm that AAV-mediated gene therapy restores the biochemical homeostasis and demonstrate that this treatment prevents the clinical phenotype developed by the enhanced murine model of MNGIE. Another key point for the clinical translation of gene therapy is the optimization of the therapeutic gene expression to reduce the vector dose. For this reason, we tested two approaches: modification of the coding sequence of TYMP based on the codon use frequency to increase the expression of the gene, and CpG dinucleotide removal from the coding sequence to reduce immunogenicity of the vector. We designed four different codon-optimized sequences of the TYMP coding sequence, using four different algorithms. We cloned each optimized sequence in a lentiviral vector and transduced 4 different human cell lines. We analysed the degree of expression achieved with each sequence, as compared with the non-optimized wild-type TYMP sequence through evaluation of TP activity, vector copy number, and mRNA levels in the cell lines. Among all the sequences studied, only one optimized sequence resulted in higher TP activity when expressed per vector copy number in the hepatic cell line Huh7. To reduce the immunogenicity of the vector we removed the CpG dinucleotides from the wild-type coding TYMP sequence and two codon-optimized TYMP sequences. The wild-type sequence without CpG was the only one showing expression levels similar to those of the wild-type sequence. Even though the CpG free sequence shows a reduced expression of about 20% of that observed in the wild-type sequence, it is compensated by the advantages associated to the absence of CpG sites in terms of reduction of the immunological response when considering the vector for clinical use. In conclusion, among the different options tested, we recommend the rAAV vector containing the wild-type coding TYMP CpG-free sequence under the control of the AAT promoter for its use in the clinical practice.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Objetivos: Avaliar a tolerabilidade e obter dados preliminares referentes aos efeitos endometriais da allopregnanolona, administrada sob a forma de gel, por via vaginal, a pacientes na pós-menopausa em uso de estrogenioterapia oral, ao longo de 2 ciclos de tratamento, assim como obter dados preliminares do efeito da droga de estudo, nos níveis séricos de gonadotrofinas, estradiol e progesterona. Pacientes e métodos: Foram incluídas no presente estudo 13 pacientes na pós-menopausa, divididas em 2 grupos: grupo 1: primeiras 7 pacientes incluídas no estudo (mediana de idade = 52 anos; mediana de tempo de amenorréia = 2 anos ) e grupo 2: 6 últimas pacientes (mediana de idade = 55,5 anos; mediana de tempo de amenorréia = 4,3 anos ). Todas as pacientes foram submetidas a 2 ciclos consecutivos de tratamento, cada qual com 28 dias de duração, utilizando 2 mg ao dia de valerato de estradiol, por via oral, continuamente, associado ao gel de allopregnanolona a 8%, por via vaginal, nos 10 últimos dias de um único ciclo (grupo 1) ou dos 2 ciclos (grupo 2). Todas as pacientes foram avaliadas quanto à caracterização dos efeitos adversos e sangramento genital. Coleta de sangue para dosagens hormonais e biópsias de endométrio foram realizadas no 28o e no 56o dia de tratamento, nas pacientes do grupo 1 e, apenas, no 56o, nas do grupo 2. Resultados: A taxa de aderência ao tratamento foi de 100% nas pacientes de ambos os grupos. A mastalgia foi o efeito adverso mais freqüente (4 casos), seguido pela cefaléia e pela dor abdominal (2 casos cada), todos estes relacionados ao uso do valerato de estradiol. Uma paciente referiu prurido vulvar durante o uso do gel de allopregnanolona. Todos os efeitos adversos foram leves e não interferiram na utilização das medicações prescritas. Não houve diferença significativa (P =1,0) entre as proporções de sangramento genital por deprivação hormonal após um (23,1%) ou dois ciclos de utilização da allopregnanolona (33,3%). Não encontramos diferença significativa (P = 0,27) entre as proporções de endométrio secretor e proliferativo após um (57,1% e 42,9%, respectivamente) ou dois ciclos (16,7% e 66,6%) de uso da allopregnanolona. Contudo há uma aparente redução da percentagem de endométrio secretor após 2 meses de utilização da allopregnanolona, quando comparada a um único ciclo de uso desta droga. Não houve diferença estatisticamente significante entre os níveis séricos de FSH, LH, estradiol ou progesterona após um ciclo com valerato de estradiol exclusivamente, quando comparados a um ou dois ciclos de uso da allopregnanolona. Conclusões: Observamos boa tolerabilidade à administração vaginal do gel de allopregnanolona a mulheres na pós-menopausa, em uso de estrogenioterapia oral com valerato de estradiol, refletida pela baixa incidência de efeitos adversos e pela boa aceitabilidade à terapêutica. Aparentemente, a allopregnanolona não interfere nos níveis séricos de gonadotrofinas, estradiol e progesterona. Estudos com maiores casuística e tempo de seguimento são necessários para se determinar os efeitos endometriais desta nova droga, e, conseqüentemente, da sua possível utilização futura, nos diversos esquemas de terapia de reposição hormonal vigentes.
Objectives: To evaluate the tolerability and to obtain preliminary data regarding the endometrial effects of allopregnanolone, administered in the form of a gel by the vaginal route to postmenopausal women on oral estrogen therapy, along two cycles of treatment, and to obtain preliminary data about the effect of the drug under study on serum gonadotropin, estradiol and progesterone levels. Patients and methods: Thirteen postmenopausal women were divided into 2 groups: group 1: the first 7 patients admitted to the study (median age = 52 years; median time of amenorrhea = 2 years), and group 2: last 6 patients (median age = 55.5 years; median time of amenorrhea = 4.3 years). All patients were submitted to 2 consecutive treatment cycles of 28 days each continuously taking 2 mg estradial valerate a day by the oral route in combination with 8% allopregnanolone gel by the vaginal route during the last 10 days of a single cycle (group 1) or of 2 cycles (group 2). All patients were evaluated in terms of adverse effects and genital bleeding. Blood samples were collected for hormonal measurements and endometrial biopsies were taken on the 28th and 56th days of treatment in group 1 patients and only on the 56th day in group 2 patients. Results: The rate of compliance with treatment was 100% for the patients of both groups. Mastalgia was the most frequent adverse effect (4 cases), followed by headache and by abdominal pain (2 cases each), all of them related to the use of estradiol valerate. One patient reported vaginal pruritus during the use of the allopregnanolone gel by the vaginal route. All adverse effects were mild and none of them interfered with the use of the prescribed medications. There was no significant difference (P = 1.0) between the proportions of genital bleeding due to hormonal deprivation after one (23.1%) or two cycles of allopregnanolone (33.3%). Also, no significant difference (P = 0.27) was found between the proportions of secretory and proliferative endometrium after one (57.1% and 42.9%, respectively) or two cycles (16.7% and 66.6%) of allopregnanolone. However, there was an apparent reduction in the percentage of secretory endometrium after 2 months of allopregnanolone compared to a single cycle of this drug. There was no significant difference between serum FSH, LH, estradiol or progesterone levels after one cycle with estradiol valerate exclusively, compared to one or two cycles of allopregnanolone. Conclusions: We observed good tolerability of vaginal administration of allopregnanolone gel to menopausal women on oral estrogen treatment with estradiol valerate, as shown by the low incidence of adverse effects and by the good acceptability of treatment. Apparently allopregnanolone does not interfere with serum gonadotrophin, estradiol or progesterone levels. Studies on a larger series and with a longer follow-up time are needed to determine the endometrial effects of this new drug and consequently the possibility of its future use in the different schemes of hormonal replacement therapy currently available.

With pre-implantation genetic diagnosis (PGD), genetic testing and selective transfer of embryos is possible. In the future, germ-line gene therapy (GLGT) applied to embryos before implantation, in order to introduce missing genes or replace mutant ones, may be possible. The objective of this dissertation is to analyse moral aspects of these technologies, as described by eighteen British, Italian and Swedish gynaecologists and geneticists. The objective is systematised into three parts: research interviews and qualitative analysis, philosophical analysis, and elaboration of a framework that supports the combination of analytic methods. PGD was described as positive since it enabled some couples at risk for a genetic disease to have a child without the disease. PGD was described as in different senses ‘better’ than methods for prenatal diagnosis and selective termination of pregnancy. It was also described as positive since it provided couples at risk with one more option, even if it did not result in the birth of a healthy child. However, interviewees were concerned about the difficulty of defining and evaluating genetic disease. They were also concerned about patients’ choices, and about exaggerated use or misuse. Whereas PGD gave rise to ambivalence in terms of how to understand, describe and evaluate it, GLGT was often described as unrealistic or undesirable. The results of the qualitative analysis are used in a philosophical analysis of the concepts of choice, autonomous choice, ambivalence, trust and ambivalence in trust relations. A set of distinct characteristics of each concept are elaborated. The results of the philosophical analysis are used in the discussion of the results of the qualitative analysis. The study shows that the technologies imply both ‘new’ ways to perform ‘old’ medical practices and ‘new’ practices. Old moral questions are reformulated. New moral questions are added. Against the background of this, the concept of genetic identity is discussed. Key words: empirical ethics, pre-implantation genetic diagnosis, germ-line gene therapy, qualitative research, philosophical analysis, medical progress, genetic disease, choice, autonomous choice, ambivalence, trust, genetic identity.

O conhecimento do genoma humano e os avanços na biologia molecular levaram a descoberta de mecanismos celulares importantes. A terapia gênica surgiu com a proposta de interferir na fisiologia das células, que apresentam alguma disfunção causadora de uma doença, trazendo a possibilidade de tratamento para incontáveis enfermidades. Na tecnologia de RNA de interferência pequenos fragmentos de RNA de fita dupla (small interference RNA, siRNA) são utilizados para inibir a expressão de genes específicos. O olho tem sido um dos órgãos alvo para o uso desse tipo de tecnologia e, além disso, a córnea é uma membrana atrativa devido ao fácil acesso e a existência de diversas enfermidades que acometem a visão. Para aumentar a biodisponibilidade e viabilizar a administração de siRNA utilizando um polímero catiônico, uma formulação termorreversível com quitosana e outra composta de cristais líquidos funcionalizados com ácido hialurônico foram estudadas como possíveis sistemas de liberação ocular de siRNA, sendo que ambas são inéditas para aplicação tópica e oftálmica de siRNA. O gel termorreversível de poloxamer 407 foi associado a dois diferentes tipos de quitosana (LMW com 92,2% de desacetilação e MMW com 77,0% de desacetilação). Ambas as formulações apresentaram características desejáveis tanto como sistema de liberação de genes, tanto por apresentarem residual positivo e capacidade de complexar o siRNA, quanto como sistema compatível com via tópica ocular, devido ao comportamento reológico pseudoplástico. A quitosana LMW e a adição de NaCl à formulação alteraram a Tsol/gel do poloxamer; somente a associação com a quitosana MMW apresentou Tsol/gel adequada. Os sistemas contendo poloxamer em associação com os dois diferentes tipos de quitosana não promoveram a penetração do siRNA in vitro em córnea bovina nas condições experimentais utilizadas. O sistema líquido cristalino foi desenvolvido com monoleína (MO), polietilenimina (PEI), ácido hialurônico (HA) e fase aquosa. A formação de fases cristalinas foi avaliada por microscopia de luz polarizada e a estrutura das mesofases foi confirmada pela difração de raios X a baixo ângulo. O sistema líquido cristalino, composto de uma mistura de cristais de fase cúbica e hexagonal, foi disperso em nanopartículas de tamanho satisfatório, de aproximadamente 166 nm. Os sistemas apresentaram características desejáveis como sistema de liberação de genes, como potencial zeta adequado, capacidade de complexar sem degradar o siRNA e não foram citotóxicos em fibroblastos L929, além de serem compatíveis com a via ocular por serem isotônicos. A dispersão de cristais de MO/PEI foi capaz de transfectar as células L929, entretanto a incorporação do HA diminuiu a absorção celular, provavelmente devido ao elevado peso molecular do ácido hialurônico empregado. O presente estudo permite delinear o futuro desenvolvimento de formulações tópicas para administração de siRNA na córnea.
The understanding of human genome and the evolvement of molecular biology led to discovery of important cellular mechanisms. Gene therapy has emerged as an approach to interfere with cell physiology, whenever it has a dysfunction that causes a disease, bringing new possibility of treatment to countless illnesses. In RNA interference mechanism, small double-stranded RNAs (small interference RNA, siRNA) inhibit specific gene expression. The eye has been one of the targeted organs for the use of such technology and, moreover, the corneal membrane is an attractive tissue due to the easy access and the existence of several diseases that can impair vision. To enhance bioavailability and facilitate the delivery of siRNA using a cationic polymer, a thermoreversible formulation containing chitosan and a liquid crystalline formulation functionalized hyaluronic acid were studied as potential delivery systems for ocular delivery of siRNA and both this systems are inedited as topical and ocular delivery of siRNA. A poloxamer 407 thermoreversible gel of was associated with two different types of chitosan (LMW with 92.2% deacetylation and MMW with 77.0% deacetylation). Both formulations showed desirable characteristics not only as gene delivery systems due to positive residual charge and capacity for complexing the siRNA, but also as a compatible ocular delivery system, due to pseudoplastic rheological behavior. The LMW chitosan and addition of NaCl to the formulation influenced the Tsol/gel of poloxamer gel; only the association with MMW chitosan showed desirable Tsol/gel. The systems containing poloxamer in combination with two different types of chitosan did not promote in vitro penetration of siRNA using bovine cornea under the used experimental conditions. The liquid crystalline system was developed with monoolein (MO), polyethylenimine (PEI), hyaluronic acid (HA) and aqueous phase. The formation of crystalline phases was observed by polarized light microscopy and the mesophases structures were confirmed by small angle X-ray diffraction. The liquid crystalline system composed of a mixture of hexagonal and cubic phases was dispersed into nanoparticles of suitable size, of approximately 166 nm. The formulations showed desirable characteristics as gene delivery systems, such as suitable zeta potential, ability to complex without degrading the siRNA and were not cytotoxic to fibroblasts L929, moreover, were compatible with the ocular administration due to isotonicity. The dispersion of crystals of MO/PEI was able to transfect L929 cells, however the incorporation of the HA decreased cellular uptake, probably due to high molecular weight hyaluronic acid employed. This study provides an outline for the future development of topical formulations to deliver siRNA to the cornea.

碩士
國立陽明大學
醫學工程研究所
93
Aim: Homogeneous head phantom was designed for the verification of three-dimensional(3D) absorbed dose distribution in intensity-modulated radiotherapy(IMRT). Based on a computed tomography scan of the phantom, an intensity-modulated radiotherapy plan for a nasopharynx carcinoma was calculated using an inverse planning system (CadPlan, Varian Medical Systems, U, S,A). Materials and Methods: The plan consisted seven beams delivered in a step and shoot technique out of 163 sub-fields. Immediately before and after irradiation 3D magnetic resonance (MR) imaging of the phantom were performed using a Spin-echo and 3D-Fast spin pulse sequence for induced image intensity change in the gel’s magnetic resonance images. Results and Discussion: Measurement and planning show good agreement in regions of standard Fricke gel with an average deviation below 5%. In these areas also expend sensitivity of the dose response and multi-shape were observed as compared to standard Fricke gel. This MRI-based Fricke dosimeter gel has proven to be a useful and valuable pre-clinical tool for evaluating complex treatment planning delivery systems and procedures.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, South Africa Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, South Africa Johannesburg 2017.
Psoriasis vulgaris is a chronic, hyper-proliferative skin condition which affects the patient’s quality of life. The treatment strategy involves long term use of drugs that maintain the condition, however; playing a pivotal negative role in patient compliance. A constructive development in the design of treatment addressing the disease should focus on the challenges faced by current designs. Hence, cellular internalization and trans-barrier transport of nanoparticles can be manipulated on the basis of the physicochemical and mechanical characteristics of nanoparticles to enhance the treatment options of the condition by reducing dosing and increasing the healing due to intracellular drug delivery. Dictating these characteristics allows for the control of the rate and extent of cellular uptake, as well as delivering the drug-loaded nanosystem intra-cellularly which is imperative for drugs that require a specific cellular level to exert their effects, as is with psoriasis. Additionally, physicochemical characteristics of the nanoparticles should be optimal for the nanosystem to bypass the natural restricting phenomena of the body and act therapeutically at the targeted site. Neo-geometric copper nanoparticles (CuNPs) in the biomedical application ascertained skin permeation and retention of the CuNPs as a drug delivery system. The approach to the use of the nanocrystal exploited the shape properties as a function of enhanced cellular uptake and the copper in the inflamed psoriatic environment acted as a cytotoxic agent against hyper-proliferating keratinocytes. A Self-Oscillating Polymeric Network (SOPN) served as a vehicle for the topical delivery of the geometric CuNPs in addition to its oscillating phenomenon to promote the permeation of the active nanoparticles across the rate limiting barrier of the skin, the stratum corneum. This twofold system adequately targets the key limitations in addressing psoriasis. A statistical experimental design comprising a full factorial model for the optimization of the geometric CuNPs and Box-Behnken design applied on the SOPN served as a refining factor to achieve stable, homogenous, geometric nanoparticles using a one-pot method for the systematic optimization of the geometric CuNPs. The optimization of the SOPN involved amplitude and duration of the oscillations, permeation kinetics and cytotoxicity. After optimization of the nano-shapes and oscillations of the SOPN, extensive ex vivo cellular internalization studies were conducted to elucidate the effect of geometric CuNPs on uptake rates; in addition to the vital toxicity assays to further understand the cellular effect of geometric CuNPs as a drug delivery system. Complementing the geometry analysis; volume, surface area, orientation to the cell membrane and colloidal stability were also addressed. The SOPN was also investigated ex vivo for its biocompatibility to determine the LD50 and permeation kinetics. The in vivo study probed the nanosystem embedded in the innovative SOPN to stimulate the permeation of the CuNPs across the stratum corneum of the induced psoriasiform-plaque in a BALB/c mouse model. The results confirmed an optimized CuNPs-loaded SOPN topical system with promising plaque thickness reduction when compared with a commercial gold standard in the treatment of the skin condition. This novel system can be safely used with less frequent, lower dosing and no odour, therefore promoting patient compliance.
MT2017

碩士
中臺科技大學
醫學工程暨材料研究所
98
Malignant neoplasms (cancer) is the top one of 10 leading causes of death for people in Taiwan. radiotherapy treatment is important for cancer. Polymer gel dosimeters with the characteristics of three-dimensional dose distribution is extremely suitable in radiation therapy dose verification. It`s used to measure plan. In this study, laser optical computed tomography (OCT) was used in dose distribution of the NIPAM polymer gel. The design of experiment (DOE) was used to find the optimal composition of NIPAM gel. The availability of the best formula to increase the stability and sensitivity of NIPAM Gel, and understanding of their properties. Different dosage interval have to change of polymer gel formulations. The polymer gel after irradiation by the radiation produced by polymerization, a result of curbed because of the role of oxygen, it must be produced in anaerobic conditions or by adding antioxidants. Residues of oxygen in the gel will result in an inhibition in the low-dose region, the large deviations encountered in dose-response. Therefore, when receiving the low dose, need to an increase antioxidant (THPC) . In a good save an maintain the stability and regular gel effectiveness. After fabrication had to stored in low temperature to avoid high temperature caused by other chemical reactions. Also should be avoid micro-gel in the light of radiation polymerization. Avoid causing large deviations encountered in dose-response by measurements.

The complex dose distributions delivered by modern, conformal radiation therapy techniques present a considerable challenge in dose verification. Traditional measurement tools are difficult and laborious to use, since complete verification requires that the doses be determined in three dimensions (3D). The difficulty is further complicated by a required target accuracy of ± 5% for the dose delivery. Gel dosimetry is an attractive option for realizing a tissue-equivalent, 3D dose verification tool with high resolution readout capabilities. However, much important work remains to be completed prior to its acceptance in the clinic. The careful development of easily accessible, fast optical readout tools such as cone beam optical computed tomography (CT) in combination with stable and reliable low-toxicity gel dosimeters is one key step in this process. In this thesis, the performance capabilities and limitations of the two main classes of cone beam optical CT-based absorbing and scattering gel dosimetry are characterized, and their measurement improved through careful matching of dosimeter and scanner performance. These systems are then applied to the evaluation of clinically relevant complex dose distributions. Three-dimensional quality assurance assessments of complex treatment plan dose distributions are shown to be feasible using an optically absorbing Fricke-xylenol-orange-gelatin-based gel dosimeter. Better than 95% voxel agreement is achieved between the plan and the delivery, using 3% dose difference and 3 mm spatial distance-to-agreement gamma function comparison criteria. Small field dose delivery evaluations are demonstrated to be viable using an optically scattering N-isopropylacrylamide (NIPAM)-based polymer gel, with the same comparison criteria. Full treatment process quality assurance is also possible using a NIPAM dosimeter in-phantom, but is limited in its accuracy due to the inherent difficulty of managing the effects of stray light pertubation in the optical attenuation-to-dose calibration.
Thesis (Ph.D, Physics, Engineering Physics and Astronomy) -- Queen's University, 2010-09-02 15:01:48.501

The study evaluated the survival of 364 glioblastoma multiforme (GBM) patients who received different modalities of treatment in two Canadian tertiary care centres. Retrospective and prospective databases were utilized to do a retrospective population based cohort study. The thesis question was among treated GBM patients in Edmonton and Halifax; does the survival rate differ with introduction of concomitant temozolomide and radiation therapy (RT) versus non concomitant treatment? Our results indicate that concomitant temozolomide with radiation therapy and surgery was associated with longer survival in comparison to radiation therapy with surgery. We also found that age; surgical resection and shorter time to radiation therapy are important factors for longer survival.
Clinical epidemiology

X-ray computed tomography (CT) polymer gel dosimetry (PGD) is an attractive tool for three-dimensional (3D) radiation therapy (RT) treatment verification due to the availability of CT scanners in RT clinics. Nevertheless, wide-spread use of the technique has been hindered by low signal-to-noise CT images largely resulting from gel formulations with low radiation sensitivity. However, a new gel recipe with enhanced dose sensitivity was recently introduced that shows great promise for use with CT readout. This dissertation describes development of an CT PGD system for 3D verification of RT treatments using the new gel formulation. The work is divided into three studies: gel characterization, commissioning of a multislice CT scanner and investigation of a dose rate dependence observed during gel characterization. The first component of this work examines the dosimetric properties of the new gel formulation. The response of the gel is found to be stable between 15 - 36 hours post-irradiation and excellent batch reproducibility is seen for doses between 0 - 28 Gy. A dose rate dependence is found for gels irradiated between 100 - 600 MU/min, indicating machine dose rate must be consistent for calibration and test irradiations to avoid dosimetric error. An example clinical application is also presented using an IMRT treatment verification that demonstrates the potential of the system for use in modern RT. The second component of this work focuses on commissioning a multislice CT scanner for CT PGD. A new slice-by-slice background subtraction technique is introduced to account for the anode heel effect. Additional investigations show recommendations for optimizing image quality in CT PGD using a single slice machine also apply to multislice scanners. In addition, the consistency of CT numbers across the multislice detector array is found to be excellent for all slice thicknesses. Further work is performed to assess the tube load characteristics of the scanner and develop a scanning protocol for imaging large gel volumes. Finally, images acquired throughout the volume of an unirradiated active gel show variations in CT data across each image on the order of 7 HU. However, these variations are not expected to greatly influence gel measurements as they are consistent throughout the gel volume. The third component of this work examines the dose rate dependence found during gel characterization. Studies using gel vials and 1 L cylinders indicate the response of the gel does not depend on changes in mean dose rate on the order of seconds to minutes. However, the machine dose rate remains, indicating variations in dose rate on the order of milliseconds influence the response of the gel. An attempt is made to mitigate the effect by increasing the concentration of antioxidant in the gel system but results in reduced overall response. Further work is performed to determine if self-crosslinking of one of the gel components contributes to the observed machine dose rate dependence. In summary, this dissertation has significantly advanced the field of gel dosimetry by providing a prototype CT PGD system with enhanced dose resolution for complex RT treatment verification.
Graduate
0992
0495
0756
holly.johnston@utsouthwestern.edu

碩士
中臺科技大學
醫學影像暨放射科學系暨研究所
102
Purpose:Except the NIPAM polymer gel, dose verification in radiotherapy can only be performed in two-dimension using other modern measurement tools. Although the NIPAM polymer gel analyzed by magnetic resonance imaging (MRI) is the only one tool that can measure radiation dose and perform in three-dimension, this technique is still limited in clinical department of radiation oncology without MRI scanner. Recently, computed tomography (CT) is developed to analyze the NIPAM polymer gel. That can improve the 3D radiation dose verification development in general department of radiation oncology. In this study, we used the CT scanner in our department to create the gel dose curve that can perform the relationship of the radiation dose and the density of polymer gel. Materials and Methods:We made intensity modulated radiotherapy (IMRT) plans in home-designed phantom. The dose verifications of these treatment plans will be performed by both MatriXX and the NIPAM polymer gel, and the dose differences with the treatment planning system was estemated. This study present the 3D measurement of dose distribution delivered using IMRT technique and NIPAM polymer gel with X-ray CT as the dose reading tool. The changes of dose response curve between one-field exposure vs multiple-field exposure and X-ray CT vs CBCT were also analyzed in this study. Results:The images acquired with X-ray CT are less noisy and with fewer artifact than those acquired with CBCT. The passing rate of Gamma Index (3%/3mm or 5%/5mm or 7%/7mm) in high dose region (80% prescribed dose) and medium dose region (30~80% prescribed dose) are all larger than 90%. The sensitivity of the dose response curve in one-field exposure is 0.503CTN/Gy, and would be improved to 0.667CTN/Gy in multiple-field exposure. Conclusion:The CT scanner is more convenient, which can be used to analyzed the NIPAN polymer gel and increase the potential of the NIPAN polymer gel used in dose verification in radiotherapy.

碩士
國立成功大學
醫學工程研究所碩博士班
97
How to improve wound healing such as preventing hypertrophic scarring and keloids formation is an important clinical issue. The wound healing process would end in ranging from nearly scarless healing to an extent of abnormal scar formation. There are almost 100 million people annually developing a scar worldwide with 31 percent of the scar as a remain of a trauma. Many physical and chemical treatments for wound healing are intensively studied in order to find the key factors that influence the mechanisms of wound healing and to improve healing. Factors such as closing tension, mechanical stimulation, injection, and nutrition etc. had been studied on wound healing. Wound healing contains a sequence of complicated, concurrent biological responses including inflammation, remodeling, and maturation. In investigating wound healing, the tensile strength of the healing skin ought to be one of the critical indicators of skin recovery because skin must be able to sustain stress. However, most studies on wound healing in the past were mostly relied on gross and pathologic observations. Studies in the past focused on mechanical evaluations are rare seen. Thus, in our previous study, we had investigated the influence of different suture methods on skin wound healing. The present study based on our previous animal model, continued to investigate other parameters on skin wound healing in the mechanical respect. First, we investigated the substance for prevention and managements of abnormal scars formation—Dermatix . We applied Dermatix on the incision wound of our animal model and observed its effects on wound healing at healing time 1, 2, 4 and 6 weeks. We introduced the recovery index to normalize the variations in mechanical properties of each rat skin at different growing conditions. The recovery index (%) was defined as (tensile strength of the wounded skin)/(tensile strength of unwounded skin) on the same rat dorsal skin. The results were that wounds treated with Dermatix did not have significant differences in recovery index from untreated wounds statistically (p=0.093). However, at all the healing time points, there are at least 23% differences between the recovery index of Dermatix treated wounds and untreated wounds. Coupling with pathological observations helped clarify the effects of Dermatix on would healing. Second part of this thesis focused on the influence of far infrared radiation (FIR) on skin wound healing. Recently, FIR had been used to treat partially ischemia and chronic ulcer wound. Thus, the study investigated the influence of FIR on the incision wound of our animal model. Wounds secured with the same suture methods as previous study did were exposed to FIR 5 times per week and observed at healing time 1, 2, 4 and 6 weeks. Results showed that there are no significant improvements comparing the wounds exposed to FIR with the wounds without FIR at healing time 6 weeks.

Photodynamic therapy (PDT) is a cancer treatment method in which a photosensitizer, light of a particular wavelength, and also oxygen are used to destroy cancerous cells. Cancer cells absorb the photosensitizing agent which is injected into the body, and it is triggered to cause cell destruction upon absorption of light. This occurs because of the excitation of the photosensitizer produces reactive oxygen species that induce a cascade of cellular and molecular events in the body. Photosensitizing agents that can photo-cleave DNA at long wavelengths are highly demanded in PDT, because the long wavelengths of light can penetrate through tissue deeply compared to visible light. While most of the photosensitizers are activated at wavelengths less than 690 nm, penetration of light continues to increase at increasing wavelengths. In this thesis, photosensitizers that can be activated to oxidize DNA with long wavelengths of light will be discussed. Using quinoline cyanine dyes, here we report the first example of DNA photocleavage at a wavelength of light above 800 nm.

CUNHA, Pedro Miguel Pacheco de Jesus da - On the development of a novel targeted miRNA-based therapy towards glioblastoma. Coimbra : [s.n.], 2015. Dissertação de Mestrado em Biologia Celular e Molecular.
Glioblastoma (GBM) is a fast growing, highly vascularized and chemoresistant cancer, being the most common and malignant type of brain tumor. Despite recent advances in the identification of genomic/molecular alterations that fuel the tumor and the aggressive standard post-surgery treatment regimen of radiotherapy and concomitant or adjuvant chemotherapy with temozolomide (TMZ), the average patient survival post-diagnosis remains very low (14.6- months). In this work, we aim at finding new therapeutic targets for GBM treatment and eradication. Abnormal microRNA (miRNA) expression has been clearly associated with cancer development and progression, including GBM pathogenesis and resistance to therapy. Therefore, we hypothesized that a gene therapy based on the modulation of miRNAs aberrantly expressed in GBM cells might promote cancer cell killing and tumor eradication, either per se or in combination with chemotherapy. We have found that the transient increase in the levels of miR-128 and miR-302a, downregulated in two human GBM cell lines (U87 and DBTRG), including isolated CD133+ cells, promoted anti-proliferative effects, which were strengthened upon cell treatement with TMZ, sunitinib and axitinib. On the other hand, we have demonstrated that miR-302a arrested cell cycle in S phase, which was further potentiated by chemotherapeutic drug combination. Next, we conducted in vivo studies using an orthotopic GBM mouse model to test the anti-tumoral potential of the combined therapy involving miR- 21 modulation and sunitinib treatment, which previous studies in our laboratory had demonstrated to constitute a promising approach for GBM treatment. We demonstrated that stable nucleic acid lipid particles targeted to GBM cells, through covalent coupling of chlorotoxin (CTX-SNALPs) were able to specifically deliver nucleic acids to intracranial tumors in mice upon intravenous injection, without causing toxicity. Moreover, silencing of miR-21 (overexpressed in GBM) via CTX-SNALPs containing anti-miR-21 oligonucleotides rendered GBM cells highly susceptible to the anti-angiogenic drug sunitinib. Altogether, our results clearly show that miRNA modulation working alongside chemotherapy constitutes a promising strategy towards GBM eradication. Importantly, the CTX-SNALP formulations show to be an appropriate vehicle for a clinical application towards GBM.
O glioblastoma (GBM) é o tipo de cancro cerebral mais comum e mais maligno, caracterizandose por elevada taxa de proliferação, abundante vascularização e alta resistência à quimioterapia. Apesar dos recentes avanços científicos na identificação das alterações genómicas e moleculares responsáveis pela progressão deste tumor e do agressivo tratamento pós-cirúrgico, que tipicamente alia à radioterapia a administração do fármaco temozolomide (TMZ), a verdade é que a sobrevivência dos pacientes após diagnóstico desta patologia permanece muito baixa (em média 14,6 meses). O objetivo do presente trabalho reside em encontrar novos alvos para o tratamento de GBM, com vista à sua erradicação. A expressão anómala de microRNAs (miRNAs) tem sido claramente associada ao desenvolvimento e progressão de tumores, incluindo GBM, e à aquisição de resistência aos tratamentos convencionais. Desta forma, o trabalho realizado no âmbito da presente tese emergiu da hipótese de que uma terapia génica, baseada na manipulação dos níveis de expressão de miRNAs em GBM, per se ou coadjuvada com quimioterapia, seria capaz de conduzir à morte das células cancerígenas e, assim, à erradicação do tumor. Consistentemente, demonstrou-se neste trabalho que um aumento transitório dos níveis de miR-128 e miR-302a, que se encontravam sub-expressos em duas linhas celulares humanas de GBM (U87 e DBTRG), e inclusivamente nas células CD133+ isoladas a partir dessas linhas, promovia efeitos antiproliferativos que se tornavam mais acentuados após tratamento das células com TMZ, sunitinib e axitinib. Por outro lado, verificou-se que miR-302a induzia a paragem do ciclo celular na fase S, efeito que era potenciado pela combinação com fármacos antitumorais. Posteriormente, foram conduzidas experiências in vivo, com recurso a um modelo animal ortotópico de GBM, de forma a testar o potencial antitumoral de uma estratégia combinada, envolvendo regulação dos níveis de expressão de miR-21 e administração de sunitinib, que estudos prévios in vitro, levados a cabo no nosso laboratório, haviam demonstrado constituir uma abordagem promissora para o tratamento de GBM. Partículas lipídicas estáveis de ácidos nucleicos, endereçadas a células de GBM através da conjugação de clorotoxina (CTX-SNALPs), mostraram a capacidade de entregar os ácidos nucleicos a tumores intracranianos em murganhos, após administração intravenosa, sem causarem toxicidade. Para além disso, o silenciamento de miR-21 (sobreexpresso em GBM) através de CTX-SNALPs contendo oligonucleótidos anti-miR-21 tornou as células de GBM mais susceptíveis ao fármaco antiangiogénico sunitinib. No seu conjunto, os nossos resultados mostraram claramente que a manipulação dos níveis de miRNAs em paralelo com quimioterapia constitui uma estratégia promissora visando a erradicação de GBM. Adicionalmente, CTX-SNALPs revelaram-se formulações adequadas ao transporte de fármacos, com potencial aplicação clínica para o tratamento de GBM.

Cyanine dyes that absorb light in the near infrared to far red region of the electromagnetic spectrum are desirable as photosensitizers for photodynamic cancer therapy. Light of wavelengths in this range is able to deeply penetrate tissue allowing for practical in vivo use of these dyes. A series of three structurally similar pentamethine cyanine dyes that absorb light ~800 nm to ~500 nm was tested to determine the effects of structural influences on the yields of supercoiled DNA photo-converted to nicked or linear forms. Possible mechanisms and optimal parameters for near- quantitative DNA photocleavage with a symmetrical quinoline pentamethine cyanine dye are discussed.

Glioblastoma (GBM) is the most common high-grade brain tumor diagnosed in patients who are more than 50 years of age. The standard of care treatment is surgery, followed by radiotherapy and chemotherapy. The median life expectancy of patients is only between 12 to 15 months after receiving current treatment regimes. Hence, identification of new therapeutic compounds and gene targets are highly warranted. This thesis describes four interlinked studies to attain this goal. In study 1, we explored drug combination effects in a material of 41 patient-derived GBM cell (GC) cultures. Synergies between three compounds, pterostilbene, gefitinib, and sertraline, resulted in effective killing of GC and can be predicted by biomarkers. In study 2, we performed a large-scale screening of FDA approved compounds (n=1544) in a larger panel of GCs (n=106). By combining the large-scale drug response data with GCs genomics data, we built a novel computational model to predict the sensitivity of each compound for a given GC. A notable finding was that GCs respond very differently to proteasome inhibitors in both in-vitro and in-vivo. In study 3, we explored new gene targets by RNAi (n=1112) in a panel of GC cells. We found that loss of transcription factor ZBTB16/PLZF inhibits GC cell viability, proliferation, migration, and invasion. These effects were due to downregulation of c-MYC and Cyclin B1 after the treatment. In study 4, we tested the genomic stability of three GCs upon multiple passaging. Using molecular and mathematical analyses, we showed that the GCs undergo both systematic adaptations and sequential clonal takeovers. Such changes tend to affect a broad spectrum of pathways. Therefore, a systematic analysis of cell culture stability will be essential to make use of primary cells for translational oncology. Taken together, these studies deepen our knowledge of the weak points of GBM and provide several targets and biomarkers for further investigation. The work in this thesis can potentially facilitate the development of targeted therapies and result in more accurate tools for patient diagnostics and stratification.