O tema deste trabalho é a modelização computacional das propriedades magnéticas de sistemas nanoparticulados a temperatura finita. Estes materiais, que são de grande interesse acadêmico e aplicado, possuem uma sensibilidade atípica às flutuações térmicas, um fenômeno conhecido como superparamagnetismo. Por essa e outras peculiaridades, eles apresentam um comportamento extremamente rico e complexo que se estende por uma gama ampla de situações experimentais, indo desde eras geológicas em aplicações na área de geomagnetismo, a fenômenos ultra-rápidos em dispositivos eletrônicos e tratamentos clínicos. O modelo empregado, conhecido como teoria de Néel-Brown, introduz na equação dinâmica magnética um termo estocástico para lidar com as flutuações térmicas. Sua validade é bastante geral, podendo ser aplicado para simular uma quantidade enorme de experimentos. Implementamos uma biblioteca numérica extremamente eficiente, que permite tratar sobre um mesmo escopo estas diferentes situações. Neste trabalho, focamos no problema de histerese dinâmica que vêm recebendo considerável atenção nos últimos anos motivado, principalmente, pela aplicação de nanopartículas magnéticas em tratamentos de tumores por uma técnica conhecida como magneto-hipertermia. / This thesis concerns the use of computer models to study the magnetic properties of nanoparticles at a finite temperature. These materials, which are of great academic and applied interest, are known to have an enhanced sensitivity to thermal fluctuations -- a phenomenon known as superparamagnetism. Such a peculiar nature is responsible for a large number of interesting physical phenomena, which are known to extend over a wide range of experimental situations. These include, among others, geomagnetism, ultra-fast devices and oncological treatments. The model employed, known as the Néel-Brown theory, introduces in the dynamical equation an stochastic term representing the thermal fluctuations. It\'s range of validity is quite broad, thus being applicable to all of the aforementioned situations. We implemented a highly efficient numerical library, whose scope extends over a large range of experiments. In this thesis we focused on the problem of dynamic hysteresis, which has receive considerable attention in recent years. This was motivated, among other things, by the potential use of nanoparticles in magneto-hyperthermia treatments.

The human brain functions within a narrow range of temperatures and variations outside of this range incur cellular damage and death and, ultimately, death of the organism. Other organisms, like the poikilotherm Drosophila melanogaster, have adapted mechanisms to maintain brain function over wide ranges in temperature and, if exposed to high temperatures where brain function is no longer supported, these animals enter a protective coma to promote survival of the organism once the acute temperature stress is alleviated. This research characterized the role of different neuronal cell types, including glia, in the protection of brain function during acute hyperthermia, specifically looking at two protective pathways: the heat shock protein (HSP) pathway and the cGMP-dependent protein kinase G (PKG) pathway. Whole animal behavioral assays were used in combination with tissue-specific genetic manipulation of protective pathways to determine the specific cell types sufficient to confer protection of neuronal function during acute hyperthermia. Using the neuromuscular junction (NMJ) preparation, calcium imaging techniques were combined with pharmacological and genetic manipulations to test the hypothesis that alterations in ion channel conductance via endogenous mechanisms regulating the cellular response to high temperature stress alter neuronal function. Expression of foraging RNAi to inhibit PKG expression in neurons or glia demonstrated protection of function during acute hyperthermia measured behaviorally through the extension of locomotor function. This extension of function with the tissue-specific inhibition of PKG was also confirmed at the cellular level using the genetically encoded calcium indicator (GECI), GCaMP3, to image calcium dynamics at the NMJ, where preparations expressing foraging RNAi could continue to elicit changes in calcium dynamics in response to stimulation. Over the course of this study, the mechanism underlying a novel glial calcium wave in the peripheral nervous system was characterized in order to elucidate glia’s role in the protection of neuronal function during acute hyperthermia. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-05-24T14:27:28Z No. of bitstreams: 1 FABIANO_PERES_MENEZES_TES.pdf: 24793474 bytes, checksum: 8b7fdf3efa2bb4839dd339d485cee522 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-06-11T14:26:49Z (GMT) No. of bitstreams: 1 FABIANO_PERES_MENEZES_TES.pdf: 24793474 bytes, checksum: 8b7fdf3efa2bb4839dd339d485cee522 (MD5) / Made available in DSpace on 2018-06-11T14:31:14Z (GMT). No. of bitstreams: 1 FABIANO_PERES_MENEZES_TES.pdf: 24793474 bytes, checksum: 8b7fdf3efa2bb4839dd339d485cee522 (MD5) Previous issue date: 2018-03-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Epilepsy is the most serious neurological condition in the world. It is characterized by recurrent seizures from synchronous neuronal discharges. Disturbances in neuronal signaling in the early stages of development may lead to increased susceptibility to seizures in adulthood, as well as seizures in the early stages of development may lead to alterations in neurotransmission systems. Adenosinergic signaling is known to act as an endogenous anticonvulsant through its neuromodulatory function. Disturbances in adenosinergic signaling in early stages of development lead to changes in the susceptibility to seizures conditionally at the stage of development in which the disturbance occurs, and time of exposure to the disturbing agent. In the four chapters of this thesis, it was discussed about factors that influence the susceptibility to pentylenetetrazole (PTZ)-induced seizure under different aspects using zebrafish. In the first chapter, it was analyzed the influence of temperature on zebrafish sensitivity to PTZ as well as the ability of the MK-801 antagonist to reverse the effects of hyperthermia on susceptibility to PTZ-induced seizures. In addition, it was verifyed possible differences in the susceptibility to seizures according to gender or weight. In the second chapter, it was used transient molecular blockade through the morpholine technique to block the translation of the transcripts corresponding to the adenosinergic A1 and A2A receptors at the beginning of embryogenesis. The animals that underwent transient blockade were evaluated for survival rate and morphology, at 7 days post-fertilization (dpf) and locomotor activity and susceptibility to seizures caused by PTZ at 7 dpf and in adulthood. In the third chapter, it was used the morpholine technique to block the translation of the transcripts corresponding to the enzyme ecto-5'-nucleotidase and concentrative nucleoside transporters type 2 (CNT2) at the beginning of embryogenesis. The animals that underwent transient blockade were evaluated for survival rate and morphology at 7 days post-fertilization (dpf) and locomotor activity and susceptibility to seizures caused by PTZ at 7 dpf and in adulthood. In the fourth chapter, it was performed microinjection of the 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), antagonist of A1 receptor; ZM241385, A2A antagonist; caffeine, non-selective adenosine receptor antagonist; dipyridamole, equilibrative nucleoside transporter blocker (ENT) and Adenosine 5 '- (?, ?-methylene) diphosphate (AMPCP), ecto-5'-nucleotidase enzyme inhibitor, in zebrafish eggs (1 hour post -fertilization). The animals exposed to these drugs were evaluated for survival rate, morphology and locomotor activity at 7 dpf and susceptibility to seizures caused by PTZ at 7 dpf and in adulthood. These results indicated that hyperthermia increases the susceptibility of zebrafish to PTZ-induced seizures and that this effect is prevented by the administration of MK-801. In addition, there was no difference in susceptibility to PTZ dependent on gender or body mass. These results indicated that disturbances in adenosinergic signaling through blockade via morpholine or in the higher doses of the drugs mentioned above, caused a decrease in the survival rate and high rates of morphological changes. None of the approaches caused alterations in the locomotor activity in the initial phase of development, whereas in the adult phase, there were occasional changes. At 7dpf, none of the targets blocked by morpholine caused alterations in the susceptibility to seizures caused by PTZ, whereas among the targets blocked by drugs there was alteration mainly in animals microinjected with DPCPX, Caffeine and Dipyridamole. However, in the adult phase all the targets blocked by morpholine triggered in greater susceptibility to seizures, while those blocked by drugs showed changes in specific doses and seizure stage. These results corroborate a series of studies that report the importance of adenosinergic signaling in the early stages of development as well as the deleterious effects of both exogenous and endogenous perturbations in this signaling pathway. / A epilepsia ? a condi??o neurol?gica grave de maior incid?ncia no mundo. ? caracterizada por crises convulsivas recorrentes, provenientes de descargas neuronais sincr?nicas. Dist?rbios na sinaliza??o neuronal na fase inicial do desenvolvimento podem acarretar em aumento na suscetibilidade a crises convulsivas na fase adulta, assim como crises convulsivas na fase inicial do desenvolvimento podem acarretar em altera??es nos sistemas de neurotransmiss?o. A sinaliza??o adenosin?rgica reconhecidamente ? capaz de agir como um anticonvulsivante end?geno, atrav?s de sua fun??o neuromoduladora. Perturba??es na sinaliza??o adenosin?rgica em fases inicias do desenvolvimento acarretam em altera??es na suscetibilidade a crises convulsivas de forma condicional ao est?gio de desenvolvimento em que a perturba??o ocorre e tempo de exposi??o ao agente perturbador. Nos quatro cap?tulos integrantes dessa tese foram abordados, sob diferentes aspectos, fatores que influenciam a susceptibilidade a crise convulsiva provocada pela exposi??o ao pentilenotetrazol (PTZ) utilizando peixe-zebra. No primeiro cap?tulo, foi analisada a influ?ncia da temperatura na sensibilidade do peixe-zebra ao PTZ, bem como a capacidade do antagonista MK-801 de reverter os efeitos provocados pela hipertermia na suscetibilidade a crises convulsivas induzidas por PTZ. Al?m de serem verificadas as poss?veis diferen?as na suscetibilidade a crises convulsivas em fun??o do g?nero ou peso. No segundo cap?tulo, foi descrito o uso do bloqueio molecular transit?rio atrav?s da t?cnica de morfolinos para bloquear a tradu??o dos transcritos correspondentes aos receptores adenosin?rgicos A1 e A2A no inicio da embriog?nese. Os animais que sofreram o bloqueio transit?rio foram avaliados quanto a taxa de sobreviv?ncia e morfologia at? os 7 dias p?s-fertiliza??o (dpf) e atividade locomotora e suscetibilidade a crises convulsivas provocadas por PTZ aos 7 dpf e na fase adulta. No terceiro cap?tulo, foi descrito o uso da t?cnica de morfolinos para bloquear a tradu??o dos transcritos correspondentes a enzima ecto-5?-nucleotidase (e5?nt) e transportadores concentrativos de nucleos?deo tipo 2 (CNT2) no inicio da embriog?nese. Os animais que sofreram o bloqueio transit?rio foram avaliados quanto a taxa de sobreviv?ncia e morfologia aos 7 dpf e atividade locomotora e suscetibilidade a crises convulsivas provocadas por PTZ aos 7dpf e na fase adulta. No quarto cap?tulo, foi abordado o efeito da microinje??o de 8-Ciclopentil-1,3-dipropilxantina (DPCPX), antagonista do receptor A1; ZM241385 antagonista do receptor A2A; cafe?na, antagonista n?o-seletivo dos receptores de adenosina; dipiridamol, bloqueador do transportador equilibrativo de nucleos?deo (ENT) e Adenosina 5?-(?,?-metileno)difosfato (AMPCP), inibidor da enzima ecto-5?-nucleotidase, nos ovos do peixe-zebra (1 hora p?s-fertiliza??o). Os animais expostos a estes f?rmacos foram avaliados quanto a taxa de sobreviv?ncia, morfologia, atividade locomotora aos 7 dpf e suscetibilidade a crises convulsivas provocadas por PTZ aos 7dpf e na fase adulta. Nossos resultados apontam que a hipertermia aumenta a suscetibilidade do peixe-zebra a crises convulsivas provocadas por PTZ e que esse efeito ? prevenido pela administra??o de MK-801. Al?m disso, n?o houve diferen?a na suscetibilidade do PTZ dependente de g?nero ou massa corporal. Nossos resultados indicam que perturba??es na sinaliza??o adenosin?rgica atrav?s de bloqueio via morfolinos ou nas doses mais altas dos f?rmacos acima citados, provocaram diminui??o na taxa de sobreviv?ncia e altas taxas de altera??es morfol?gicas. Nenhuma das abordagens provocou altera??es na atividade locomotora na fase inicial do desenvolvimento, enquanto que na fase adulta foram verificadas altera??es pontuais. Aos 7dpf nenhum dos alvos bloqueados por morfolinos provocou altera??o na suscetibilidade a crises convulsivas provocadas por PTZ, enquanto que entre os alvos bloqueados por f?rmacos houve altera??o principalmente em animais microinjetados com DPCPX, Cafe?na e Dipiridamol. J? na fase adulta todos os alvos bloqueados por morfolinos desencadearam em maior suscetibilidade a crises convulsivas enquanto os bloqueados por f?rmacos exibiram altera??es em doses e est?gio de convuls?o espec?ficos. Esses resultados corroboram com uma s?rie de estudos que reportam a import?ncia da sinaliza??o adenosin?rgica na fase inicial do desenvolvimento, bem como os efeitos delet?rios provenientes de perturba??es tanto ex?genas quanto end?genas nessa via de sinaliza??o.

Neste trabalho, pós magnéticos nanoestruturados de PrxFe94-xB6 (x = 6, 8, 10 e 12) foram preparados a partir da combinação do processo de hidrogenação, desproporção, dessorção e recombinação (HDDR) e moagem de alta energia entre uma liga em estado bruto de fusão (Pr14Fe80B6) e Fe-α em pó. As nanopartículas produzidas apresentaram propriedades magnéticas e microestruturais comparáveis aos estudos realizados em hipertermia. O tempo ideal para obtenção de nanopartículas magnéticas é de 5 horas (a 900 rpm). Foi constatado que quanto maior o tempo de moagem, maior o percentual de carbono nas partículas (0,05 - 3,43 % C). O carbono é proveniente do ácido oléico adicionado como surfactante na etapa de moagem. Os nanocompósitos obtidos exibiram forças coercivas entre 80 Oe (6,5 kAm-1) e 170 Oe (13,5 kAm-1), e momentos magnéticos variando entre 81 - 129 Am2kg-1. A partir da difração de raios X foram identificadas apenas duas fases em todas as amostras: Fe-α e a fase magnética Pr2Fe14B. Nanopartículas isoladas com diâmetro aproximado de 20nm foram analisadas. Todas as composições estudadas apresentaram aquecimento proveniente da exposição a um campo magnético alternado (f = 222 kHz e Hmax ~3,7 kAm-1) comparáveis aos reportados na literatura. As variações de temperaturas (ΔT) dos pós foram: 51 K referente à composição de Pr6Fe88B6, 41 K para Pr8Fe86B6, 38 K no composto com 10% at. Pr (Pr10Fe84B6) e 34 K em Pr12Fe82B6. As taxas de absorção específicas estimadas foram de 201 Wkg-1 para a composição Pr6Fe88B6, 158 Wkg-1 para a composição Pr8Fe86B6 e 114 Wkg-1 para as composições Pr10Fe84B6 e Pr12Fe82B6. / In this work, PrxFe94-xB6 (x = 6, 8, 10 and 12) nanostructured powders were prepared by a combination of hydrogenation, disproportionation, desorption and recombination (HDDR) process with high energy milling applied to the mixture of an as-cast alloy (Pr14Fe80B6) and α-Fe. The produced nanoparticles showed magnetic properties comparable to those reported in hyperthermia studies. The optimal time to obtain the magnetic nanoparticles is 5 hours (at 900 rpm). It was verified that longer milling times cause an increase in carbon percentage on the particles. The carbon is derived from oleic acid added as a surfactant in the milling step. The nanocomposites exhibit coercive force ranging from 80 Oe (6.5 kAm-1) to 170 Oe (13.5 kAm-1) and magnetic moments in the range of 81 129 Am2kg-1. From the x-ray diffraction analyses, only two phases were found in all samples: α-Fe and the magnetic phase Pr2Fe14B. Individual nanoparticles with diameter of about 20 nm were verified. The samples studied presented heating when exposed to an alternating magnetic field (f = 222 kHz e Hmax ~3.7 kAm-1) comparable to reported in literature. Temperature variations (ΔT) of the powders were: 51 K for Pr6Fe88B6, 41 K for Pr8Fe86B6, 38 K for Pr10Fe84B6 and T = 34 K for Pr12Fe82B6. The specific absorption rates (SARs) of the powders were 201 Wkg-1 for Pr6Fe88B6 composition, 158 Wkg-1 on the composition Pr8Fe86B6, and 114 Wkg-1 for Pr10Fe84B6 and Pr12Fe82B6 compositions.

O uso da elevação da temperatura local como recurso adjuvante no combate ao câncer tem sido explorado intensamente nas últimas décadas. A hipertermia, como é chamada essa elevação de temperatura local, apresenta seu maior potencial clínico quando combinada com a quimioterapia e/ou radioterapia, sendo capaz de promover benefícios terap êuticos significativos. Apesar dos resultados positivos, a hipertermia, até o momento, não se estabeleceu como terapia padrão, devido a limitações no controle da deposição de energia e no monitoramento da distribuição de temperatura em tempo real. Neste trabalho, discutem-se características fundamentais da eficiência da hipertermia no tratamento de tumores cerebrais e de bexiga. O projeto foi todo ele desenvolvido em colaboração com o grupo de hipertermia do Department of Radiation Oncology da Duke University. Com relação à hipertermia aplicada ao cérebro, primeiramente apresenta-se o procedimento de desenvolvimento e teste de eficiência de um mini-aplicador de micro-onda dedicado ao aquecimento do cérebro de camundongos. Após estes, avaliou-se a capacidade de disponibilização termo-estimulada da doxorrubicina a modelos tumorais de glioblastoma. O método utilizado para monitoramento da liberação e distribuição da doxorrubicina foi a microscopia confocal de fluorescência intravital. O estudo do impacto da hipertermia sob a distribuição da formulação de doxorrubicina encapsulada em lipossomos termosensíveis demonstrou que a elevação moderada de temperatura promove alterações significativas na permeabilidade da barreira hematoencefálica, além de promover aumento do acúmulo total de droga e aumento no grau de penetração. Para a hipertermia aplicada à bexiga, apresenta-se um estudo de viabilidade de aquecimento para uma metodologia alternativa ao dispositivo de uso clínico padrão (_Synergo_), denominada magneto-hipertermia. Os ensaios com a magneto-hipertermia apontam que o uso de nanopartículas magnéticas sob influência de um campo magnético alternado (40 kHz) é capaz de elevar a temperatura do lúmen da bexiga a 42_C de forma localizada, não promovendo efeitos significativos de aquecimento a tecidos do entorno. / The use of local heating to achieve adjuvant response in cancer treatment has been widely explored in the last decades. The thermal therapy has a well-known clinical bene_t when combined to chemotherapy and/or radiotherapy. Despite all positive results, the use of thermal therapy has not yet been established as standard treatment, mainly due to the limitation on the control of energy deposition and real-time temperature mapping. This thesis discuss some of the fundamentals of the application of hyperthermia to treat bladder and brain tumors. All experiments were performed in collaboration with the Department of Radiation Oncology of Duke University. As regards brain experiments, we developed and built a microwave antenna dedicated to locally heating the mouse brain. After that, we assessed the ability of thermo release and thermo delivery of doxorubicin to glioblastoma tumor models. Intravital confocal fluorescence microscopy was used to monitor the drug release and distribution into brain tissue as a whole. Our findings indicated that a mild elevation in brain temperature (42_C) modulates the permeability of the blood-brain barrier and promotes an increase on both total drug accumulation and drug penetration. Concerning bladder hyperthermia, we investigated the feasibility of magnetic-hyperthermia as an alternative heating source to the standard clinical device (_Synergo_). The magnetic-hyperthermia results indicate that the amount of heat dissipation by the magnetic nanoparticles, under the influence of alternating magnetic field (40KHz), was able to raise the temperature in the bladder lumen to 42_C and did not promote any significant heating effects on surrounding tissues.

O principal interesse neste estudo foi a proposição de uma nova classe de material que permite a ação combinada da Terapia Fotodinâmica (TFD) e da Hipertermia (HPT), projetadas para trabalhar sinergicamente, que possa levar a uma considerável regressão de tumores neoplásicos após mínimas doses de dissipação de calor e/ou fotossensitização luminosa. Esta nova classe de material se baseia em um lipossoma de longo tempo de circulação associado ao fármaco fotossensibilizador (FS) zinco ftalocianina (ZnPC), na presença de fluido magnético (FM) constituído por nanopartículas de ferrita de cobalto (CoFe2O4) recobertas com ácido cítrico. As propriedades fotofísicas (em meio orgânico e em meio lipossomal) e os estudos fotobiológicos em células da linhagem B-16 foram desenvolvidos para avaliar as propriedades da ZnPC na ausência e na presença de FM. As propriedades fotofísicas da ZnPC em meio orgânico e lipossomal, na ausência e na presença de FM, foram realizadas empregando-se técnicas de espectroscopia no estado estacionário e resolvido no tempo. Foi possível determinar importantes parâmetros que elucidaram o potencial fotodinâmico da partícula mista ZnPC/FM com um apropriado sistema de liberação, confirmando sua viabilidade para aplicação em estudos in vitro e in vivo. A interação de fluidos magnéticos biocompatíveis (FMBs) com a macromolécula biológica, a soro albumina bovina (BSA), foi estudada por meio da determinação da constante de ligação (Kb) e do número de sítios de ligação (n),. Este formalismo aplicado para nanopartículas magnéticas usadas em aplicações biológicas foi realizado pela primeira vez neste rabalho. As toxicidades da ZnPC, do FM e da partícula mista ZnPC/FM, em meio homogêneo e lipossomal, na ausência e na presença de luz e/ou campo magnético, foram estudadas. Na última parte deste trabalho iniciou-se o desenvolvimento de um modelo tumoral subcutâneo na região dorso-lateral de camundongos C57BL6J. Este é considerado o primeiro passo para a transferência dos resultados fotobiológicos no desenvolvimento de um tratamento clínico proposto para a terapia de humanos. Os nossos resultados demonstraram que o complexo ZnPC/FM em meio lipossomal apresenta propriedades fotofísicas e fotobiológicas úteis, ativados pela luz e campo magnético, como uma geração de fármacos atuando sinergicamente pela TFD e pela HPT. / The main goal in this study was to introduce a new material class that allows the combined action of Photodunamic therapy (PDT) and Hyperthermia therapy (HPT), designed to work in a synergetic ways, leading to an expected enhancement of the tumor damage after minimum drug doses and based on heat dissipation and /or light photosensitization. This new material class is a ?stealth? liposome associated with the photosensitizer drug zinc phthalocyanine (ZnPC), in the presence of a magnetic fluid (MF) based on nanoparticles of cobalt-ferrite (CoFe2O4) surface-coated with citric acid. Photophysical properties (in organic and liposomal medium) and photobiological studies in B-16 tumor cell lines were developed to evaluate the properties of the ZnPC in the absence and in the presence of MF. The photophysical properties of the ZnPC in organic and liposomal medium, were realize using spectroscopy techniques in the steady state and by time resolved studies. It was possible to quantify important parameters that elucidated and confirm the photodynamic potential of the ZnPC/MF complex as an appropriate drug delivery system, confirming its viability for application in vitro and in vivo studies. The interaction of the biocompatible magnetic fluids (FMBs) with the biological macromolecule, serum albumin proteins (BSA),was investigated through the determination of the binding constant (Kb) as well as the binding stoichiometry of the complex (n). This is the first time that this formalism was applicable to magnetic nanoparticles used in biological application. The toxicities of the ZnPC, the MF and also for the combined particle (the ZnPC/MF complex), in homogeneous and liposomal medium, in the absence and in the light presence and/or magnetic field were studied, and all the parameters that will allow the used of this synergic compound have been defined. In the last part of this work it was also started the establishment of one animal model by development of subcutaneous tumoral skin cancer in the back-lateral area of C57BL6J mice. This are the first step in the transfer of the photobiological results to the development of a clinical trial proposal for human therapy Our results demonstrated that the ZnPC/MF complex in liposome medium showed useful photophysical and photobiological properties, acting by light activation and AC magnetic field as a new generation of synergic drugs for TFD and cellular HPT.

by Lam Kai Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 156-165). / Abstract also in Chinese. / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Tumor Necrosis Factor-α in Cancer Treatment --- p.1 / Chapter 1.1.1 --- Historical Background --- p.1 / Chapter 1.1.2 --- Mechanisms of Action --- p.2 / Chapter 1.1.2.1 --- Production of Reactive oxidative Species / Chapter 1.1.2.2 --- Increase of Intracellular Free Calcium Concentration / Chapter 1.1.2.3 --- Activation of Ca2+/Mg2+-dependent Endonuclease / Chapter 1.1.2.4 --- Decrease of glucose uptake and Protein Synthesis / Chapter 1.1.2.5 --- Formation of Ion-permeable Channel / Chapter 1.1.2.6 --- Activation of Phospholipase / Chapter 1.1.2.7 --- Increase of S-phase Cells / Chapter 1.1.2.8 --- Immunomodulatory Effects / Chapter 1.1.3 --- Resistance of Cells to TNF-α --- p.7 / Chapter 1.1.4 --- Clinical Studies --- p.11 / Chapter 1.1.5 --- Side Effects --- p.12 / Chapter 1.2 --- Hyperthermia and Cancer Treatment --- p.14 / Chapter 1.2.1 --- Hyperthermic Agents --- p.15 / Chapter 1.2.2 --- Intrinsic Heat Sensitivity --- p.15 / Chapter 1.2.3 --- Mechanisms of Action --- p.17 / Chapter 1.2.3.1 --- Depolarization of Membrane Potential / Chapter 1.2.3.2 --- "Reduction of glucose transport and DNA, mRNA and Protein Synthesis" / Chapter 1.2.3.3 --- Decrease of Intracellular pH / Chapter 1.2.3.4 --- Calcium Imbalance / Chapter 1.2.3.5 --- Effect on Nucleolar Protein / Chapter 1.2.3.6 --- Apoptosis / Chapter 1.2.3.7 --- Induction of Autologous Tumor Killing / Chapter 1.2.3.8 --- "Blood Flow, Tumor Oxygenation and Vascular Damage" / Chapter 1.2.4 --- Clinical Studies --- p.20 / Chapter 1.3 --- Combined Treatment --- p.21 / Chapter 1.3.1 --- Combined Treatment with TNF-α and Fixed-temperature Hyperthermia --- p.22 / Chapter 1.3.2 --- Combined Treatment with TNF + Step-down Hyperthermia --- p.22 / Chapter 1.3.3 --- In Vivo Study --- p.23 / Chapter 1.3.4 --- Sequence of Treatment --- p.24 / Chapter 1.3.5 --- Proposed Mechanism of Synergism --- p.24 / Chapter 1.4 --- Objective of Study --- p.26 / Chapter 1.4.1 --- Sequence of Treatments --- p.26 / Chapter 1.4.2 --- Comparison of Treatments' Effectiveness --- p.27 / Chapter 1.4.3 --- Effect on Normal Cell --- p.27 / Chapter 1.4.4 --- Effect on Distribution of Cells in Cell Cycle Phases --- p.28 / Chapter 1.4.5 --- In Vivo Study --- p.28 / Chapter Chapter Two: --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.30 / Chapter 2.1.1 --- For Cell Culture --- p.30 / Chapter 2.1.2 --- In vitro Treatments --- p.31 / Chapter 2.1.3 --- DNA Electrophoresis --- p.31 / Chapter 2.1.4 --- Flow Cytometry --- p.32 / Chapter 2.2. --- Reagent Preparation --- p.33 / Chapter 2.2.1 --- Culture Media --- p.33 / Chapter 2.2.2 --- Human Recombinant Tumor Necrosis Factor alpha (rhTNF-α) --- p.33 / Chapter 2.2.3 --- Phosphate Buffered Saline (PBS) --- p.33 / Chapter 2.2.4 --- Lysis Buffer --- p.34 / Chapter 2.2.5 --- TE Buffer --- p.34 / Chapter 2.2.6 --- Proteinase K and Ribonuclease A (RNase A) --- p.34 / Chapter 2.2.7 --- 100 Base-Pair DNA Marker --- p.34 / Chapter 2.2.8 --- Propidium Iodide (PI) --- p.35 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Cell Culture --- p.35 / Chapter 2.3.1.1 --- Ehrlich Ascitic Tumor (EAT) and Human Leukemia (HL-60) / Chapter 2.3.1.2 --- Human Coronary Artery Endothelial Cells (HCAEC) / Chapter 2.3.2 --- In vitro Experiments --- p.36 / Chapter 2.3.3 --- Tumor Necrosis Factor Treatment --- p.37 / Chapter 2.3.4 --- Hyperthermia Treatments --- p.37 / Chapter 2.3.5 --- Cell Counting --- p.38 / Chapter 2.3.5.1 --- Trypan Blue Exclusion Assay / Chapter 2.3.5.2 --- Neutral Red Assay / Chapter 2.3.6 --- Determination of Additive or Synergistic Effect --- p.39 / Chapter 2.3.7 --- DNA Electrophoresis --- p.40 / Chapter 2.3.8 --- Flow Cytometry --- p.42 / Chapter 2.3.7.1 --- Preparation of Samples / Chapter 2.3.7.2 --- Flow Cytometry Acquisition / Chapter 2.3.7.3 --- Analysis / Chapter 2.3.9 --- In vivo Experiments --- p.44 / Chapter 2.3.8.1 --- Animal Strain / Chapter 2.3.8.2 --- Cell Line / Chapter 2.3.8.3 --- Tumor Necrosis Factor Treatment / Chapter 2.3.8.4 --- Hyperthermia Treatments / Chapter 2.3.8.5 --- Test of Body Temperature / Chapter 2.3.8.6 --- Cell Harvesting / Chapter Chapter Three: --- Result --- p.50 / Chapter 3.1 --- Optimal Sequence of Treatments --- p.50 / Chapter 3.1.1 --- Optimal Sequence of Treatments on Murine Ehrlich Ascitic Tumor (EAT) cells --- p.50 / Chapter 3.1.1.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.1.1.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.1.1.3 --- TNF + Step-down Hyperthermia3 / Chapter 3.1.2 --- Optimal Sequence of Treatments on Human Leukemia cells HL-60 --- p.60 / Chapter 3.1.2.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.1.2.2 --- TNF + Step-Down Hyperthermia2 / Chapter 3.1.2.3 --- TNF + Step-Down Hyperthermia3 / Chapter 3.2 --- Comparison of Effectiveness of Treatments --- p.72 / Chapter 3.2.1 --- Effectiveness of Various treatments on EAT cells --- p.72 / Chapter 3.2.2 --- Synergistic Effect between rhTNF-α and Hyperthermia on EAT cells --- p.74 / Chapter 3.2.3 --- Decrease of Relative Growth and Viability of EAT with Time --- p.79 / Chapter 3.2.3.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.2.3.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.2.3.3 --- TNF + Step-down Hyperthermia3 / Chapter 3.2.4 --- Comparison of Effectiveness of Various Treatments on HL-60 cells --- p.82 / Chapter 3.2.5 --- Synergistic Effect between rhTNF-α and Hyperthermia on HL-60 cells --- p.87 / Chapter 3.2.6 --- Change of Relative Growth and Viability of HL-60 with Time --- p.90 / Chapter 3.2.6.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.2.6.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.2.6.3 --- TNF + Step-down hyperthermia3 / Chapter 3.3 --- Cell Death Pathway --- p.96 / Chapter 3.3.1 --- Experiments on Ehrlich Ascitic Tumor (EAT) Cells --- p.96 / Chapter 3.3.2 --- Experiments on Human Leukemia (HL-60) Cells --- p.100 / Chapter 3.4 --- Experiment on Normal Cell --- p.104 / Chapter 3.5 --- Effect of TNF + Fixed-temperature Hyperthermia on the Cell Cycle Progression --- p.107 / Chapter 3.5.1 --- Different Times of TNF Administration and Distribution of EAT cells in Cell cycle --- p.107 / Chapter 3.5.2 --- Different Times of TNF Administration and Distribution of HL-60 cells in Cell Cycle --- p.114 / Chapter 3.5.3 --- Shift of Cells Cycle after TNF Treatment --- p.120 / Chapter 3.5.3.1 --- Response of Ehrlich Ascitic Tumor Cells / Chapter 3.5.3.2 --- Response of Human leukemia Cells / Chapter 3.6 --- Effectiveness of Treatments in vivo: --- p.129 / Chapter 3.6.1 --- Dose-dependent Response --- p.129 / Chapter 3.6.2 --- Change of Body Temperature During Hyperthermia --- p.131 / Chapter 3.6.3 --- Comparison of Effectiveness of Various Treatments in vivo --- p.133 / Chapter 3.6.4 --- Synergistic Effect Between rhTNF-α and Hyperthermia in vivo --- p.135 / Chapter Chapter Four: --- Discussion --- p.138 / Chapter 4.1 --- Optimal Sequence of Treatments --- p.139 / Chapter 4.2 --- Comparison of Various Treatments --- p.143 / Chapter 4.3 --- Distribution of Cells in Cell Cycle Phases --- p.149 / Chapter 4.4 --- In vivo Study --- p.153 / Chapter Chapter Five: --- References --- p.156

Physical activity of moderate intensity and duration leads to healthy biological adaptations in humans. However, very intense and prolonged exercise may induce disruption in redox balance, potentially increasing oxidative stress. In addition, exposure to environmental heat stress and associated hyperthermia further increases oxidative stress and may induce the expression of heat shock proteins. However, antioxidant supplementation is believed to minimise the effect of oxidative stress and may therefore help reduce or limit the heat shock response to exercise heat stress. The first study (Chapter 4) examined whether exertional heat illness (EHI) casualties among military recruits may exhibit greater disturbances in redox balance following exercise compared to non-EHI controls. Nine (n=9) recruits were identified as having suspected EHI during the Loaded March (LM) on day 1, with a peak mean (SD) body core temperature of 40.1 (0.5) °C. Fifteen (n=15) recruits were identified as having suspected EHI during the Log Race (LR) on day 2, with a peak mean (SD) body core temperature of 39.7 (0.5) °C. A further twenty-one (n=21) recruits, which successfully finished both LM and LR events, were treated as controls (CON). Interestingly, the plasma antioxidant concentration was significantly elevated from pre to post-exercise (p < 0.001) for EHI and CON groups, during both LM and LR events, with no changes on lipid peroxide protein carbonyl concentrations. These data suggest there is no increase in lipid peroxide or protein carbonyl level damage in response to intense hyperthermic military exercise, regardless of acute heat illness. It is possible that military training augments the body's defence capabilities, thus reducing oxidative stress and damage induced by free radical production. To date there is a scarcity of data examining the effects of acute intake of antioxidant supplements on oxidative stress and heat shock response during continuous exercise in a hot environment. Hence, the aims of the second study (Chapter 5) were to examine the effects of acute ingestion of Quercetin (Q), Quercetin + vitamin C (QC) or placebo (P) 14 hours before, 2 hours before and every 20 minutes during trials on oxidative stress and heat shock response. In this randomised, crossover study 10 recreationally active males (age 21±2 y, V̇ O2max 54.9±8.4 ml.kg.min-1) completed three running trials at 70% V̇ O2max for 60 minutes in the heat (33.0±0.3°C; 28.5±1.8% relative humidity). Exercise heat stress significantly elevated plasma quercetin (p=0.02), antioxidant power (FRAP) (p < 0.001),plasma heat shock protein 70 (HSP70) (p=0.009) and plasma heat shock protein 90α(HSP90α) (p < 0.001) over time, but no differences were detected between trials. Also, no changes were observed in protein carbonyl concentration. Acute intake of quercetin significantly increased the level of plasma quercetin however, this did not affect the plasma antioxidant capacity or heat shock response to exercise heat stress. The increases in plasma HSP70 and HSP90α concentrations might act as supplementary antioxidants, reducing the oxidative damage reflected in the absence of changes in protein carbonyl. Exercise heat stress is effective in inducing both intracellular HSP70 (muscle and peripheral blood mononuclear cell (PBMC)) and extracellular HSP70 (plasma) concentrations. Thus, the third study (Chapter 6) tested the hypothesis that this acute quercetin supplementation would induce similar trends in plasma HSP70 and intracellular HSP70 concentrations 2 days following exercise heat stress. In this randomised, crossover study, 9 recreationally active males (age 22±2y, V̇ O2max 50.3±3.3ml.kg.min-1) completed three running trials at 70% V̇ O2max for 60 minutes in the heat (32.9±0.3°C; 28.3±1.2% relative humidity). This study demonstrated that there is no positive relationship between both intracellular of HSP70 (muscle and PBMC) and plasma HSP70 (eHSP70) 2 days following exercise heat stress. These data suggest that the release of eHSP70 could originate from others tissue or cells. Additionally, the absence of differences between trials in the expression of muscle HSP70, PBMC HSP70 and plasma HSP70 might indicate it is implausible that quercetin might inhibit the expression of HSP70 in plasma, muscle and PBMC 2 days following the exercise heat stress stimulus. Overall, the results from this thesis emphasise that the hyperthermia experienced in response to exercise and environmental heat stress could potentially influence the human redox response and heat shock response. Besides, there is reasonable evidence that acute quercetin co-ingestion with vitamin C has the potential to improve the bioavailability and bioactive effects of quercetin, however, the effects of quercetin supplementation in reducing oxidative stress in response to exercise heat stress remains to be elucidated. In addition, the anti-oxidative ability of acute ingestion of quercetin to suppress the intracellular and extracellular heat shock response remains uncertain and worthy for further investigation.

by Li Jian Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 157-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

O Câncer nas últimas décadas tem se tornado um evidente problema de saúde pública mundial. Os números de novos casos que surgem a cada ano e as altas taxas de mortalidade levam os pesquisadores a procurar formas de conter o avanço dessa doença. A principal forma de tratamento e que possui a maior incidência de cura é o uso de quimioterápicos, que são substâncias químicas utilizadas isoladas ou em combinação, com o objetivo de tratar as neoplasias malignas. Entretanto eles atuam sem especificidade, não destruindo seletivamente e exclusivamente as células tumorais o que causa graves efeitos colaterais aos pacientes. Com o intuito de aumentar a seletividade do tratamento, diminuir a toxicidade e aumentar o poder de cura o presente trabalho utiliza duas abordagens para o combate do câncer, a nanotecnologia (uso de sistemas de liberação de fármacos) e a hipertermia. Foram preparadas, caracterizadas e avaliadas quanto à atividade antitumoral in vitro nanocápsulas contendo o agente quimioterápico selol (composto semi-sintético provindo do óleo de girassol e que possui selênio na sua estrutura) e fluído magnético iônico (composto por nanopartículas magnéticas de maghemita, ?-Fe2O3. Ao total foram preparadas quatro diferentes formulações pelo método de nanoprecipitação descrito por Fessi com algumas modificações. As nanocápsulas apresentaram um tamanho de partícula máximo de 230,5 nm (± 4,5), com índice de polidispersividade < 0,267 (± 0,05) e potencial zeta que variou de -54,4 mV (± 3,4) a -28,6 mV (± 4,3). Foram realizadas análises da morfologia das nanocápsulas através de microscopia eletrônica de transmissão que confirmaram o tamanho nanométrico do sistema preparado. Todas as formulações demonstraram ser estáveis durante o tempo 3 meses quando armazenadas a temperatura de 4oC. Nos estudos celulares foram utilizadas as linhagens B16- F10 (melanoma murino) e OSCC (carcinoma epidermóide de boca humano), sendo que as mesmas mostraram diferentes comportamentos quando incubadas com as formulações em diferentes concentrações. Na linhagem B16-F10 foi observado um maior efeito de morte causado pelo selol (a viabilidade celular chegou a 52,5 % ± 8,4), já quando o campo magnético foi utilizado não foi possível observar um aumento da morte celular. No estudos com a linhagem OSCC, a mesma demonstrou resistência quando foi tratada com selol e na ausência de campo magnético, já quando o campo magnético foi utilizado a viabilidade celular chegou a 33,3% (± 0,3), indicando um forte efeito hipertêmico nesta linhagem. Mais estudos devem ser realizados para entendermos o efeito do sistema preparado perante diferentes linhagens celulares, no entanto podemos confirmar o sucesso no preparo do mesmo e a capacidade de causar morte de diferentes células neoplásicas, o que indica uma importante arma para atuar futuramente no combate do câncer. / In the latest decades, cancer has become a clear public health problem worldwide. The neoplastic diseases increase each year and high mortality rates lead researchers to develop new approaches able to contain the progress of this disease. The main treatment type which has the highest incidence of cure is based on chemotherapeutic agents used alone or in combination. However, they act without specificity and selectively destroying both tumor and normal cells causing serious side effects to patients. In order to enhance the selectivity of the treatment decreasing toxicity and increase the healing power, the present study employs two approaches to treat the cancer, nanotechnology (the use of drug delivery systems) and hyperthermia (magnetic fluid). Nanocapsules containing the chemotherapeutic agent selol (semi-synthetic compound coming from sunflower oil and that has selenium in its structure) and maghemite magnetic nanoparticles (?-Fe2O3) were prepared, characterized, and evaluated in respect with their in vitro antitumor activity. Four different formulations were prepared by the nanoprecipitation method described by Fessi et al. with some modifications. The nanocapsules presented a particle size up to 230.5 nm (±4.5) with polydispersity index of 0.267 (±0.05), and zeta potential ranged from - 54.4 mV (±3.4) to - 28.6 mV (±4.3). The transmission electron microscopy analysis of nanocapsules confirmed the nanometric size system prepared. All formulations proved to be stable during 3 months as stored at 4°C. The cell lines studied were B16-F10 (murine melanoma) and OSCC (oral squamous cell carcinoma). These cell lines showed different behavior after incubation at different formulation concentrations. For cytotoxicty study on B16-F10 cells, it was observed a strong effect caused by Selol (cell viability reached 52.5% ±8.4). On the other hand, there was no cytotoxic effect on B16-F10 cells (p > 0.05) under magnetic field application. OSCC cell line showed a resistance to treatment with selol and in the absence of AC magnetic field. However, after magnetic field activation the cell viability reached 33.3% (±0.3) indicating a strong hyperthermic effect on OSCC cells. Therefore, it has been confirmed nanocapsules containing selol and magnetic fluid are able to destroy B16-F10 or OSCC neoplastic cells indicating an important weapon for future work in the treatment against cancer.