The elucidation of the molecular mechanism of the cell response to moderate heating is of importance for understanding the events that occur in the cell upon use of heating for therapeutic purpose or during illnesses that are associated with fever. The aim of this work was to investigate and to compare the effects of mild (fever) and severe hyperthermia on functional properties of oxidative phosphorylation system in normal tissue mitochondria. Modular kinetic analysis for the first time was applied to evaluate effects of hyperthermia on oxidative phosphorylation in rat heart and liver mitochondria. We demonstrated that changes in mitochondrial functions induced by mild hyperthermia (42 ºC) are reversible but more severe hyperthermia (45 ºC) causes partially irreversible uncoupling and inhibition of mitochondrial respiration in state 3, hyperthermia remarkably (3.6-2.1 fold) activates ROS generation in heart mitochondria and that maximal increase in rate of H2O2 production and lipid peroxidation is observed in the fever temperature range. We show that the response of liver mitochondria and hepatocytes to hyperthermia is to certain extent dependent on gender and temperature. Specific differences of male rat liver and heart mitochondrial components phase transitions have been revealed by DSC analysis. / Ląstelių atsako į nuosaikią hipertermiją molekulinio mechanizmo išaiškinimas yra labai svarbus norint suprasti procesus, kurie vyksta ląstelėse jas kaitinant gydymo tikslais ar organizmui karščiuojant. Šio darbo tikslas buvo nustatyti ir palyginti švelnios (karščiavimo) ir šiurkščios hipertermijos poveikį oksidacinės fosforilinimo sistemos funkcijoms normalių audinių mitochondrijose. Pirmą kartą panaudojome modulių kinetinę analizę hipertermijos poveikiui širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai tirti. Mes nustatėme, kad švelnios hipertermijos (42 ºC) poveikis širdies mitochondrijų funkcijoms yra grįžtamas, bet šiurkštesnė hipertermija (45 ºC) sukelia dalinai negrįžtamą kvėpavimo ir fosforilinimo atskyrimą bei mitochondrijų kvėpavimo greičio trečioje metabolinėje būsenoje slopinimą. Hipertermija didino ROS gamybos greitį ir lipidų peroksidaciją, kurie buvo didžiausi karščiavimo temperatūroje. Nustatėme, kad kepenų mitochondrijų ir hepatocitų atsakas į hipertermiją priklauso nuo žiurkės lyties ir temperatūros. Atlikome palyginamąjį širdies ir kepenų mitochondrijų sandų fazinių virsmų analizę diferencine skenuojamaja kalorimetrija ir nustatėme būdingus skirtumus.

Ląstelių atsako į nuosaikią hipertermiją molekulinio mechanizmo išaiškinimas yra labai svarbus norint suprasti procesus, kurie vyksta ląstelėse jas kaitinant gydymo tikslais ar organizmui karščiuojant. Šio darbo tikslas buvo nustatyti ir palyginti švelnios (karščiavimo) ir šiurkščios hipertermijos poveikį oksidacinės fosforilinimo sistemos funkcijoms normalių audinių mitochondrijose. Pirmą kartą panaudojome modulių kinetinę analizę hipertermijos poveikiui širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai tirti. Mes nustatėme, kad švelnios hipertermijos (42 ºC) poveikis širdies mitochondrijų funkcijoms yra grįžtamas, bet šiurkštesnė hipertermija (45 ºC) sukelia dalinai negrįžtamą kvėpavimo ir fosforilinimo atskyrimą bei mitochondrijų kvėpavimo greičio trečioje metabolinėje būsenoje slopinimą. Hipertermija didino ROS gamybos greitį ir lipidų peroksidaciją, kurie buvo didžiausi karščiavimo temperatūroje. Nustatėme, kad kepenų mitochondrijų ir hepatocitų atsakas į hipertermiją priklauso nuo žiurkės lyties ir temperatūros. Atlikome palyginamąjį širdies ir kepenų mitochondrijų sandų fazinių virsmų analizę diferencine skenuojamaja kalorimetrija ir nustatėme būdingus skirtumus. / The elucidation of the molecular mechanism of the cell response to moderate heating is of importance for understanding the events that occur in the cell upon use of heating for therapeutic purpose or during illnesses that are associated with fever. The aim of this work was to investigate and to compare the effects of mild (fever) and severe hyperthermia on functional properties of oxidative phosphorylation system in normal tissue mitochondria. Modular kinetic analysis for the first time was applied to evaluate effects of hyperthermia on oxidative phosphorylation in rat heart and liver mitochondria. We demonstrated that changes in mitochondrial functions induced by mild hyperthermia (42 ºC) are reversible but more severe hyperthermia (45 ºC) causes partially irreversible uncoupling and inhibition of mitochondrial respiration in state 3, hyperthermia remarkably (3.6-2.1 fold) activates ROS generation in heart mitochondria and that maximal increase in rate of H2O2 production and lipid peroxidation is observed in the fever temperature range. We show that the response of liver mitochondria and hepatocytes to hyperthermia is to certain extent dependent on gender and temperature. Specific differences of male rat liver and heart mitochondrial components phase transitions have been revealed by DSC analysis.

Šilumos taikymas įvairioms ligoms gydyti jau buvo naudojamas senovės Egipto, Graikijos, Romos civilizacijose daugiau kaip prieš 2000 m. pr. Šiuo metu hipertermija plačiai taikoma visame pasaulyje gydant vėžį, nes tai pigus ir patogus metodas turintis mažą šalutinį poveikį. Mokslininkai nustatė, kad vėžinės ląstelės greičiau žūsta esant aukštesnei už fiziologinę (41-45°C) temperatūrai, todėl hipertermija, derinama su kitais vėžio gydymo metodais (radioterapija, chemoterapija, imunoterapija ir chirurgija), tampa efektyvesniu metodu. Kadangi yra mažai žinoma apie hipertermijos poveikio mechanizmą sveiko audinio ląstelėms karščiavimo, hiperterminio vėžio gydymo ar gydymo termoabliacija metu, todėl yra svarbu nustatyti hipertermijos paveiktų ląstelių gyvybingumą bei hipertermijos poveikį adenino ir piridino nukleotidų koncentracijoms. Šio darbo tikslas buvo įvertinti hipertermijos, būdingos nutolusioms nuo termozondo audinio sritims poveikį, adenino ir piridino nukleotidų koncentracijoms žiurkės kepenų ląstelėse bei audinyje. Buvo naudojamas jonų porų efektyviosios skysčių chromatografijos metodas, leidžiantis vienoje chromatografinėje analizėje išskirstyti labai skirtingo hidrofobiškumo junginius. Taip pat buvo vertintas gyvų ir negyvų ląstelių skaičius gautoje hepatocitų suspensijoje panaudojant tripano mėlio metodą bei NAD(P)H fluorescencijos pokyčiai kepenų audinyje termoabliacijos metu. Gauti rezultatai parodė, kad išskirti hepatocitai pasižymėjo dideliu gyvybingumu (8... [toliau žr. visą tekstą] / The application of heat in the treatment of disease was first recorded in the ancient civilizations of Egypt, Greece, and Rome from as early as 2000 BC. Nowadays hiperthermia is widely using in cancer diseases in all the world. It was determined by many scientists that cancer cells are more sensitive for supraphysiological temperature (41-45°C) killing compared to normal cells. There are numerous evidences that hyperthermia can increase the effectiveness of other cancer therapies: radiotherapy, chemotherapy, immunotherapy and surgery. There is little known about the mechanisms of hyperthermia effects on healthy tissue, which are important in fever, in hyperthermic treatment of neighboring tumour and in thermoablation. Therefore it is very important to determinate the vability of cells during different hyperthermic treatment and hyperthermic effects of adenine ir pyridine nucleotides concentrations.The aim of study was to value the effect of hyperthermia,which is typical remote from thermoprobe tissue areas, on the concentration of adenine and pyridine nucleotides in hepatocytes and liver tissue. It was used ion-pair high-performance liquid chromatography method, which allows to disperse different combinations of hydrophobicity. Also were evaluated live and dead cells quantity in the suspension through tripan blue method and NAD(P)H fluorescence changes in liver tissue during the ablation. The results showed that isolated hepatocytes exhibited with high viability (80%)... [to full text]

The present study was performed to determine if pigs varying in their susceptibility to stress, adapted to a casein-based diet, experience an improvement in biochemical parameters related to stress and behaviour compared to pigs adapted to the traditional western canadian cereal-based swine diet. Experiment 1 involved separating fifty-eight, 8-week old pigs according to genotype with respect to the halothane gene. Within each genotype pigs were divided into 2 groups and assigned to either a control diet or to a diet where most of the protein source was substituted for casein. All animals were adapted to diet for 6 weeks and experienced a weekly blood sampling stressor. Day 1, 14 and 35 of the plasma samples were analyzed for glucose, cortisol, ACTH, insulin, pyridoxal 5$ sp prime$-phosphate (PLP), amino acid concentrations and dopamine-$ beta$-hydroxylase (DBH) activity; metabolic indices known to be responsive to stress. Experiment 2 involved separating fifty-seven, 14-week old pigs in the same manner, then adapting the pigs to their respective diets for a period of 4 weeks. Afterwards, pigs were transferred from their pen to a novel pen-maze situation where they had their behaviour monitored for a period of one hour. Results of these experiments have revealed that (1) the stress susceptible and carrier pigs experienced reduced day 35 plasma glucose, PLP concentrations and DBH activity compared to normal pigs; (2) dietary adaptation to the casein diet resulted in greater day 14 and 35 PLP levels and day 35 essential amino acid lysine, threonine, methionine, tryptophan and arginine concentrations compared to control-adapted pigs; (3) the carrier pigs investigated their surroundings more frequently than the stress susceptible pigs, and the normal pigs engaged in the through-maze behaviour more often than the stress susceptible pigs; and (4) adaptation to the casein diet, compared to the control diet, resulted in fewer displacement-type behaviours such as drinking

<p>Traditional small molecule chemotherapeutics show limited effectiveness in the clinic as their poor pharmacokinetics lead to rapid clearance from circulation and their exposure to off-target tissues results in dose-limiting toxicity. The objective of this dissertation is to exploit a class of recombinant chimeric polypeptides (CPs) to actively target drugs to tumors as conjugation to macromolecular carriers has demonstrated improved efficacy by increasing plasma retention time, reducing uptake by healthy tissues, and enhancing tumor accumulation by exploiting the leaky vasculature and impaired lymphatic drainage characteristic of solid tumors. CPs consist of two principal components: (1) a thermally responsive elastin-like polypeptide (ELP) that displays a soluble-to-aggregate phase transition above a characteristic transition temperature (Tt); and (2) a cysteine-rich peptide fused to one end of the ELP to which small molecule therapeutics can be covalently attached (the conjugation domain). This work describes the development of CP drug-loaded nanoparticles that can be targeted to solid tumors by the external application of mild regional hyperthermia (39-43°C). </p><p>Highly repetitive ELP polymers were assembled by Plasmid Reconstruction Recursive Directional Ligation (PRe-RDL), in which two halves of a parent plasmid, each containing a copy of an oligomer, were ligated together to dimerize the oligomer and reconstitute the functional plasmid. Chimeric polypeptides were constructed by fusing the ELP sequence to a (CGG)8 conjugation domain, expressed in Escherichia coli, and loaded with small molecule hydrophobes through site specific attachment to the conjugation domain. Drug attachment induced the assembly of nanoparticles that retained the thermal responsiveness of the parent ELP in that they experienced a phase transition from soluble nanoparticles to an aggregated phase above their Tt. Importantly, the Tt of these nanoparticles was near-independent of the CP concentration and the structure of the conjugated molecule as long as it displayed an octanol-water distribution coefficient (LogD) > 1.5. </p><p>A series of CP nanoparticles with varying ratios of alanine and valine in the guest residue position was used to develop a quantitative model that described the CP transition temperature in terms of three variables - sequence, chain length, and concentration - and the model was used to identify CPs of varying molecular weights that displayed transition temperatures between 39°C and 43°C. A murine dorsal skin fold window chamber model using a human tumor xenograft was used to validate that only the thermoresponsive CP nanoparticles (and not the controls) exhibited a micelle-to-aggregate phase transition between 39-43°C in vivo. Furthermore, quantitative analysis of the biodistribution profile demonstrated that accumulation of these thermoresponsive CP nanoparticles was significantly enhanced by applying heat in a cyclical manner. It is hoped that this work will provide a helpful resource for the use of thermoresponsive CP nanoparticles in a variety of biomedical applications.</p> / Dissertation

Malignant hyperthermia is a potentially fatal pharmacological disorder and is triggered by volatile anaesthetics in predisposed individuals. Mutations in the RYR1 gene, encoding the skeletal muscle calcium receptor channel have been linked to MH susceptibility. Over 200 point mutations have been have been found to date in the RYR1 gene linked to MHS worldwide. EBV-immortalization is regularly used worldwide as an effective procedure for inducing long-term growth of human B lymphocytes. In the current study, it was observed that immortalized lymphocytes from MHS patients heterozygous for the missense mutation H4833Y when initially cultured expressed both wild type and mutant allele but after a few weeks of culture they seemed to lose the mutant allele. High resolution melting assays and hybridization probe assays showed the loss of heterozygosity and this was confirmed using DNA sequencing. Genotyping and haplotype analysis using three intragenic RFLPs and two (CA)n repeat microsatellite markers tightly linked to the RYR1 gene showed a definite change in the haplotype, suggesting more widespread changes in the genome upon short-term culture of EBV-immortalized B-lymphocytes

Malignant hyperthermia (MH) is a rare pharmacogenetic disorder in humans induced by volatile anaesthetics and depolarising muscle relaxants. An MH reaction shows abnormal calcium homeostasis in skeletal muscle leading to a hypermetabolic state and increased muscle contracture. A mutation within the skeletal muscle calcium release channel ryanodine receptor gene (RYR1) is associated with MH and is thought to cause functional defects in the RYR1 channel leading to abnormal calcium release to the sarcoplasm and consequent MH reactions. Mutations within RYR1 are also associated with a rare congenital myopathy, central core disease (CCD). CCD is characterised by muscle weakness and is thought to be caused by insufficient calcium release from the RYR1 channel during excitation-contraction (EC) coupling. To investigate functional effects of RYR1 mutations, the entire coding region of human RYR1 was assembled and cloned into an expression vector. Mutant clones containing RYR1 mutations linked to MH or CCD were also constructed. Wild-type (WT) and mutant RYR1 clones were used for transient transfection of HEK-293 cells. Western blotting was performed after harvesting and expressed WT and mutant RYR1 proteins were successfully detected. Immunofluorescence showed co-localisation of RYR1 proteins and the endoplasmic reticulum in HEK-293 cells. [3H]ryanodine binding assays showed that RYR1 mutants linked to MH were more sensitive to the agonist 4-chloro-m-cresol (4-CmC) and less sensitive to the antagonist Mg2+ compared with WT. Two C-terminal RYR1 mutants T4826I and H4833Y were very significantly hypersensitive to 4-CmC and they may also result in a leaky channel. This hypersensitivity of mutants linked to MH may result in abnormal calcium release through the RYR1 channel induced by triggering agents leading to MH reactions. RYR1 mutants linked to CCD showed no response to 4-CmC showing their hyposensitive characteristics to agonists. This study showed that the human RYR1 proteins could be expressed in HEK-293 cells. Moreover, using the recombinant human RYR1 clone, a single mutation within RYR1 resulted in a functional defect in expressed RYR1 proteins and functions of mutant RYR1 proteins varied from hypersensitive to hyposensitive depending on the mutation and whether it was linked to MH or CCD.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Magnetic hyperthermia (MH) is an anti-cancer treatment which exploits the heat produced by tumour-targeted magnetic nanoparticles (MNPs) subjected to an alternating magnetic field (AMF). A problem limiting the clinical use of MH, however, is the inability to adequately localise the MNPs at the tumour site. A cellular approach using mesenchymal stem cells (MSCs) as carriers has been proposed as these cells are believed to home to sites of tissue injury and tumour growth, however problems with MNPs uptake and toxicity retard progress and need to be overcome. The aim of this project was to find an alternative approach in MH treatment, creating engineered human MSCs able to biosynthesise MNPs. To achieve this goal, MSCs were transfected with either, or both, M. magneticum AMB-1 mms6 and mmsF genes. M. magneticum AMB-1 is a genus of magnetotactic bacteria, containing magnetosomes, which are lipidic organelles containing single crystals of magnetite. M. magneticum-AMB1 mms6 and mmsF genes are important for final crystal morphology and are known to play a role in crystal synthesis and growth respectively. The originality of this study was in using mms6 and mmsF genes, which were codon-optimized for mammalian expression, alone or in combination, for transfection of human MSCs, which have known tumour homing capacity. The transfected MNPs-bearing MSCs, able to migrate into the tumour tissue, were subjected to AMF in MH experiments in an attempt to induce cancer cell death. mms6 and mmsF gene expression, following transfection, was investigated in the human osteosarcoma cell line MG63 by reverse transcription polymerase chain reaction (RT-PCR). The cellular ultrastructure of transfected MG63 cells was investigated by transmission electron microscopy (TEM), revealing the presence of nanoparticles. The magnetism of transfected MG63 cells was proved by superconducting quantum interference device (SQUID) and supported by in vitro MH experiments. Then, human MSCs were transfected with mms6 and mmsF genes, alone or in combination. The effect of transfection experiments and MNPs synthesis on MSCs markers of stemness, cell proliferation and differentiation ability were investigated. The MTB genes expression in human MSCs was assessed by RT-PCR and cell magnetism was confirmed by SQUID, in vitro MH experiments and by magnetic force microscopy (MFM). Then, in vitro studies of MH were undertaken to establish whether mms6 transfected MSCs expressing MNPs supported a MH effect when exposed to an AMF. Cells were initially exposed to an AMF of 565.3 kHz frequency in monolayers and in 3D arrangements and cell death/viability was assessed. Subsequently, the effect of the same AMF on 3D models of mixed populations of mms6-expressing MSCs and cancer cells was assessed. The results indicate that viability of MNPs-expressing MSCs and adjacent cancer cells is reduced following AMF exposure. In vivo studies of MH were undertaken following intracardiac injection of mms6-expressing MSCs in tumour-bearing mice (epidermoid carcinoma). The expression of mms6-expressing MSCs inside mice organs was confirmed by RT-PCR, fluorescence microscopy and immunohistochemistry. The effect of the application of an AMF of 565.3 kHz on mice tumours was studied with different techniques (tumour size and volume measurement, multiphoton microscopy, haematoxylin and eosin staining, and activated Caspase 3 expression), to understand if MNPs created inside mms6- expressing MSCs, following AMF exposure, could lead to cancer cell death. Results indicate that mice tolerate the treatment well, however no appreciable tumour reduction or necrosis was evident. Overall the results suggest that mms6 transfection alone confers the highest magnetisation to MSCs compared to mmsF alone or mms6+mmsF co-transfected, and that mms6 expression in human MSCs does not have an adverse effect on important cell functions. mms6-expressing MSCs, when exposed to an AMF, show reduced viability and enhanced cell cytotoxicity in vitro. When co-cultured with cancer cells in 3D models in vitro, mms6-expressing MSCs are able to reduce viability of adjacent cancer cells confirming the potential applicability of mms6- expressing MSCs for MH treatment. In vivo proof of concept experiments show that mms6-expressing MSCs can locate to the tumour tissue, and mms6-expressing intracardiac injected MSCs mice exposed to AMF tolerate the treatment well. However, the number of mms6-expressing MSCs able to localize to the tumour tissue in this experiment was too low to give an appreciable tumour reduction, so more experiments are needed to enhance the experimental protocol. A number of improvements are required to progress this novel technique towards clinical application. Gene transfection and MNPs production need to be optimised, the best frequency for MH needs to be established and MSCs delivery to the tumour has to be significantly increased to allow concentration of MNPs. The study has helped to increase our knowledge on the creation of magnetic human MSCs to potentially use these cells in MH cancer treatment.

Orientadora: Profa. Dra. Maria Camila Almeida / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2017. / Eventos estressantes que ocorrem ao longo da vida são um importante foco nos estudos de doenças psiquiátricas. Para este estudo, utilizou-se a privação materna no início da vida que favorece o aparecimento de comportamentos análogos à depressão, além de alterações no sistema neuroendócrino. A depressão é caracterizada por humor deprimido e pacientes deprimidos apresentam disfunção de mecanismos de refrigeração termorreguladora. O objetivo deste estudo foi avaliar o perfil termorregulatório de ratos Wistar machos adultos submetidos a um modelo de separação materna (SM). Os animais foram submetidos à SM do dia pós-natal 2 ao 14, durante 3 horas por dia. Após 3 meses do nascimento, os animais foram submetidos a testes comportamentais, a um procedimento cirúrgico para colocação de datalogger e, posteriormente, a testes termorregulatórios. Os testes comportamentais demonstraram que os animais SM apresentaram comportamentos relacionados à ansiedade (menor tempo gasto no centro do campo aberto e nos braços abertos do labirinto em cruz elevado) e à depressão (menor motivação observada no maior tempo de imobilidade no teste de natação forçada). Os animais SM apresentaram maior temperatura corporal no período escuro quando comparados aos controles. Além disso, os animais SM apresentaram maior resposta hipertérmica quando expostos ao ambiente de calor e, quando possível selecionar sua temperatura ambiente de preferência, permaneceram por períodos mais longos a temperaturas ambientes mais baixas quando comparados aos controles. Os resultados sugerem que em uma condição de SM, há uma mudança no perfil termorregulatório em ratos. A compreensão dos mecanismos relacionados a essas mudanças é importante para conhecer melhor os sintomas e o tratamento das patologias relacionadas ao estresse. / Stressful lifelong events are an important focus on psychiatric disease studies. For this study, early maternal deprivation was used to induced depression-like behaviors. Depression is characterized by depressed mood and depressed patients have dysfunction of thermoregulatory cooling mechanisms. The objective of this study was to evaluate the thermoregulatory profile of adult male Wistar rats submitted to a maternal separation model (MS). The animals were submitted to MS from postnatal day 2 to 14, for 3 hours per day. After 3 months of birth, the animals were submitted to behavioral tests, a surgical procedure to place a datalogger and, later, to thermoregulatory tests. The behavioral tests showed that SM animals exhibited anxiety-like behaviors (less time spent in the center of open field and in the open arms of the elevated plus maze) and depression (less motivation in the forced swimming test). SM animals presented higher body temperature in the dark period when compared to controls. In addition, SM animals presented a higher hyperthermic response when exposed to the heat environment and, when allowed to select their preferred ambient temperature, they remained for longer periods at lower ambient temperatures when compared to controls. The results suggest that in a condition of MS, there is a change in the thermoregulatory profile in rats. Understanding the mechanisms related to these changes is important to a better comprehension of the symptoms and treatment of stress-related pathologies.