There is a large amount of interest and research currently going into studying the effects of low dose radiation on humans, and bridging the gap with the data from the effects of high dose radiation. Much work is to be done to understand low dose exposures such as from medical treatments and those who work with or around radiation. Two popular and widely known examples of low-dose phenomena are the radiation induced bystander effects and the radioadaptive response (RAR). This research involves the study of the impact of a low dose of radiation that is administered several hours after a high – even fatal – dose is given, which contrasts the traditional RAR where a low priming dose is given before a high dose and can lead to increased cell survival. Many different parameters were checked to see if cell survival can be enhanced or diminished depending on the stage of the cell cycle, cell growth conditions, and cell profiling differences in protein function (namely the TP53 gene). Additionally, the post-conditioning response was contrasted to see if it was possible to see any effects from the newly emerging area of bystander signalling, UV BioPhotons, would be present in cell lines that either did or did not exhibit a post-conditioning effect. It was shown that post-conditioning has a protective effect on survival of the cells in certain dose ranges and certain cell lines. The post-conditioning effect also appears to be stronger in magnitude than the classic RAR. No relationship between gamma-induced biophoton signalling and post-conditioning was observed, nor is it certain whether an acute gamma-field can induce significant UV biophoton damage. This thesis is aimed to explore the various parameters by which post-conditioning effects occur on various Human cancers. / Thesis / Master of Science (MSc) / This research looks at the effects of radiation on cells. More specifically, how do low doses of radiation affect cells after they have already been treated with higher doses of radiation. Moreover, can cells communicate through non-physical methods, such as through invisible light? The research focuses primarily on cancer cells and their responses to varying doses of radiation treatments.

Η μοριακή σηματοδότηση κατά την αρχή της επαναιμάτωσης η οποία οδηγεί σε προστασία του μυοκαρδίου περιλαμβάνει το μονοπάτι διάσωσης NO/cGMP/PKG/KATP, το μονοπάτι διάσωσης των κινασών RISK(PI3K/Akt, ERK 1/2, GSK3β) και το μονοπάτι JAK/STAT έχοντας ως τελικό στόχο την αναστολή της διάνοιξης των mPTP, το οποίο θεωρείται το τελικό σημείο της επαγόμενης καρδιοπροστασίας. Η παραγωγή του H2S εμπλέκεται στους μηχανισμούς της ισχαιμικής προετοιμασίας και της μετισχαιμικής προστασίας. Διάφορες μελέτες σε απομονωμένα μυοκάρδια (Langendorff isolated perfused hearts) υποστηρίζουν ότι η επαγωγή της καρδιοπροστασίας από την εξωγενή χορήγηση H2S επιτυγχάνεται μέσω της ενεργοποίησης των KATP διαύλων. Ωστόσο, δεν υπάρχουν ολοκληρωμένες μελέτες οι οποίες να καταδεικνύουν τους μοριακούς μηχανισμούς οι οποίοι εμπλέκονται στην καρδιοπροστατευτική δράση του συγκεκριμένου αέριου διαβιβαστή σε in vivo πειραματικά μοντέλα. Ο σκοπός της παρούσας μελέτης είναι ο έλεγχος της υπόθεσης ότι η θεραπευτική χορήγηση ενός ανόργανου δότη H2S (NaHS) στο τέλος της ισχαιμίας και κατά την επαναιμάτωση μειώνει την έκταση του εμφράγματος του μυοκαρδίου σε αναισθητοποιημένους κόνικλους. Επιπλέον μελετήθηκαν οι υποκείμενοι μοριακοί μηχανισμοί. Αναισθητοποιημένοι αρσενικοί κόνικλοι Νέας Ζηλανδίας διαχωρίσθηκαν σε 7 ομάδες και υπεβλήθησαν σε 30 λεπτά παρατεταμένης/συνεχούς ισχαιμίας του μυοκαρδίου ακολουθούμενης από 3 ώρες επαναιμάτωσης με τις ακόλουθες παρεμβάσεις: 1) Ομάδα ελέγχου (control) : χωρίς περαιτέρω παρεμβάσεις, 2) Ομάδα NaHS: χορήγηση ενός δότη Η2S (ΝaΗS) με IV bolus έγχυση και δόση 100μg/kg στο 20 λεπτό της ισχαιμίας ακολουθούμενη από έγχυση σταθερού ρυθμού με δόση 1mg . kg-1 . h-1 για τα επόμενα 120 λεπτά , 3) Ομάδα ΝaHS και DT-2: χορήγηση ΝaHS όμοια με την ομάδα 2 και DT-2 με IV bolus έγχυση και δόση 0,25 mg. kg-1 10 λεπτά πριν την παρατεταμένη ισχαιμία, 4) Ομάδα TAT και ΝaHS: χορήγηση NaHS όμοια με την ομάδα 2 και ΤΑΤ με IV bolus έγχυση και δόση 0.143 mg. kg-1 10 λεπτά πριν την παρατεταμένη ισχαιμία (Η δόση επελέγει ισομοριακά ως προς το DT-2), 5) Ομάδα NaHS+5-HD: χορήγηση NaHS όμοια με την ομάδα 2 και 5-HD με IV bolus έγχυση σε δόση 5mg/kg 40 λεπτά πριν την παρατεταμένη ισχαιμία., 6) Ομάδα NaHS+L Name: χορήγηση NaHS όμοια με την ομάδα 2 και L Name με IV bolus έγχυση σε δόση 10mg/kg στο 19 λεπτό της παρατεταμένης ισχαιμίας, 7) Ομάδα NaHS+Wortmannin: χορήγηση NaHS όμοια με την ομάδα 2 και Wortmannin με IV bolus έγχυση σε δόση 60μg/kg στο 19 λεπτό της παρατεταμένης ισχαιμίας. Μετά το τέλος των πειραμάτων εκτιμήθηκε η εμφραγματική(Ι) και η περιοχή σε κίνδυνο (R). Σε δεύτερη σειρά πειραμάτων ελέγθηκε η ενεργοποίηση των Akt, ERK 1/2 ,eNOS, GSK3β, STAT3, VASP και της PLB σε δείγμα ισχαιμικού ιστού των ομάδων ελέγχου, NaHS και NaHS + DT-2. Για επιβεβαίωση της μη φωσφορυλίωσης ορισμένων από τις προαναφερθείσες πρωτεΐνες χρησιμοποιήθηκε ως ομάδα αναφοράς, μία πρόσθετη ομάδα PostC η οποία υπεβλήθη σε 30 λεπτά παρατεταμένη ισχαιμίας ακολουθούμενης από 10 λεπτά επαναιμάτωσης στην έναρξη της οποίας εφαρμόσθηκαν 8 κύκλοι των 30 δευτερολέπτων ισχαιμίας/επαναιμάτωσης. Ο δότης H2S, NaHS, μείωσε την έκταση του εμφράγματος σε σύγκριση με την ομάδα ελέγχου(12.3±3.3% vs 46.4±1.8%,p<0.05), ενώ η προσθήκη του DT-2 ανέστειλε την καρδιοπροστατευτική δράση του NaHS(39.8±3.4%,p=NS vs Control). Η χορήγηση του πεπτιδίου ελέγχου ΤΑΤ δεν τροποποίησε τη δράση του NaHS(23.0±3.4%,p=NS vs H2S group). Ο αναστολέας διάνοιξης των mitoKATP (5-HD) και της ενεργότητας της eNOS (L-NAME) δεν μείωσε την ανασταλτική δράση του NaHS στην έκταση του εμφράγματος(14.1±2.0% και 14.7±2.2% αντίστοιχα, p=NS). Ωστόσο, η χορήγηση του αναστολέα των PI3K/Akt (wortmannin) ανέστρεψε την καρδιοπροστατευτική δράση του NaHS(41.8±1.4% vs 12.3±3.3%, p<0.05). Η φωσφορυλίωση των VASP,και PLB ήταν σημαντικά υψηλότερη στην ομάδα NaHS σε σχέση με τις ομάδες ελέγχου και NaHS+DT-2,οι ERΚ 1/2 φωσφορυλιώθηκαν στις ομάδες NaHS και PostC σε σχέση με τις ομάδες ελέγχου και NaHS+DT-2, οι Akt και STAT3 ήταν εξίσου ενεργοποιημένες στις ομάδες NaHS, NaHS+DT-2 και PostC σε σχέση με την ομάδα ελέγχου, ενώ δεν παρατηρήθηκε φωσφορυλίωση των eNOS και GSK3β στις ομάδες ελέγχου, ΝaHS και NaHS+DT-2 σε σχέση με την ομάδα PostC. Η εξωγενής χορήγηση H2S στο τέλος της παρατεταμένης ισχαιμίας και κατά την επαναιμάτωση επάγει φαρμακολογική μετισχαιμική προστασία σε αναισθητοποιημένους κόνικλους μέσω των μονοπατιών Akt/PKG/PLB και PKG/ ERK 1/2 ανεξάρτητα από την eNOS, την GSK3β, το JAK/STAT μονοπάτι και την διάνοιξη των mitoKATP. / The signal transduction pathways which are recruited during early reperfusion include the nitric oxide/cGMP/PKG/KATP pathway, the reperfusion injury salvage kinase pathway (PI3K /Akt, ERK 1/2, GSK3α), and the JAK/STAT pathway targeting the inhibition of mPTP opening which is considered the end-point for inducing cardioprotection. The production of H2S plays a role in myocardial pre-and post-conditioning responses. Several studies in isolated hearts support cardioprotection from exogenous H2S due to KATP channels activation. However, there is a lack of evidence for the molecular mechanism underlying the protection of H2S in in vivo experimental models of ischemia/reperfusion injury. The aim was to elucidate the hypothesis that therapeutic administration of the H2S donor NaHS before and during reperfusion reduces the infarct size in anesthetized rabbits. Additional the molecular mechanisms underlying the induced cardioprotection from exogenous administrated H2S were studied. Anesthetized male rabbits were divided into 7 groups and were subjected to 30 min regional ischemia of the heart and 3 hours reperfusion with the following additional interventions: 1) Control group no further intervention, 2) NaHS group was treated with the H2S donor sodium hydrosulphide (NaHS) at a dose of 100 ιg.Kg-1 bolus on the 20th min of ischemia followed by infusion of 1mg.Kg-1. h-1 for the next 120 min, 3) NaHS +DT-2 group, treated with NaHS and the PKG inhibitor DT-2 that was given at a dose of 0.25 mg.kg-1 bolus 10 min before sustained ischemia, 4) NaHS +TAT group, treated with NaHS and the control peptide TAT that was given at a dose of 0.143 mg.kg-1 bolus 10 min before sustained ischemia, 5) NaHS+5-hydroxydecanoic acid (5-HD) treated with NaHS and mitoKATP channels inhibitors 5-HD iv bolus 40 minutes before occlusion at a dose of 5 mg.kg-1, 6) NaHS+ L-NAME treated with NaHS and the synthase of NO inhibitor L NAME IV bolus on the 19th min of ischemia at a dose of 10 mg.kg-1 and 7) NaHS+ Wortmannin treated with NaHS and the PI3/Akt inhibitor Wortmannin on the 19th min of ischemia at a dose of 60ιg.kg-1. After the end of the experiments the infarct size (I) and the area at risk (R) were estimated. In a second series of experiments, determination of activation of Akt, ERK 1/2 ,eNOS, GSK3α, STAT3, VASP and phopsholamban (PLB) was investigated in tissue samples from ischemic area of myocardium from Control, NaHS and NaHS + DT-2 groups. As positive control of no phosphorylation observed, PostC group was used. In PostC group animals were subjected in 30 minutes sustained ischemia followed by 10 minutes of reperfusion, were 8 cycles of 30 seconds of ischemia/ reperfusion were applied immediately after the onset of reperfusion. H2S donor NaHS reduced the infarct size compared to Control (12.3 ± 3.3% vs 46.4 ± 1.8%, p<0.05), whereas the addition of the PKG inhibitor DT2 abrogated the infarct size limiting effect (39.8 ± 3.4%, p=NS vs Control). Treatment with the control peptide TAT did not alter the effect of NaHS (23.0 ± 3.4%, p=NS vs H2S group). Administration of mitoKATP inhibitor (5-HD) and eNOS inhibitor (L-NAME) did not alter the infract limiting effects of NaHS (14.1±2.0% and 14.7±2.2% respectively, p=NS). However, administration of the PI3K/Akt inhibitor wortmannin reversed this cardioprotection (41.8±1.4% vs 12.3±3.3%, p<0.05). Phosphorylation of VASP, ERK ½ and PLB was significantly higher in NaHS treated group versus control and NaHS+DT-2 groups, in PostC group ERK ½ were phosphorylated respectively to NaHS treated group, Akt and STAT3 were phosphorylated in NaHS, NaHS+DT-2 and PostC groups vs Control group, whereas no phosphorylation of eNOS and GSK3α was observed in NaHS, NaHS+DT-2 and control groups compared to PostC group. Exogenous administration of H2S at the end of ischemia and during reperfusion induces pharmacological postconditioning in anesthetized rabbits due to Akt/PKG/PLB and PKG/ ERK 1/2 activation independently of eNOS, GSK3α, JAK/STAT and mitoKATP activation.

L'infarctus du myocarde est la première cause de mortalité dans le monde. La reperfusion précoce est le traitement central de la prise en charge thérapeutique. Mais bien que salvatrice, la reperfusion s'accompagne elle-même de lésion dite de reperfusion. La mitochondrie et l'ouverture du pore de transition de perméabilité mitochondriale (mPTP) sont au centre de ces lésions de reperfusion. Le dysfonctionnement mitochondrial et la nécrose entraine par ailleurs une intense réponse inflammatoire locale et systémique. Le post-conditionnement ischémique et pharmacologique (par la cyclosporine A, CsA) constitue une voie de recherche importante afin de limiter les lésions de reperfusion. Mes travaux de thèse se sont attachés à étudier le rôle de la Sirtuine 3 dans la cardioprotection ainsi que d'étudier la réponse inflammatoire locale et systémique induite par l'ischémie/reperfusion (I/R) myocardique.Nous avons précisé le mécanisme d'action du post-conditionnement, qui semble médié par l'activation de la sirtuine 3 et la désacétylation de la cyclophiline D qui contribue à inhiber l'ouverture du mPTP. Nous avons ensuite montré que l'I/R myocardique induisait une intense réponse inflammatoire chez l'homme avec un rôle particulier d'IL-17A, IL-6, IL-8 et IL-10. Cependant, cette réponse inflammatoire n'était pas modifiée par l'utilisation de CsA. Enfin, nous avons pu montrer que le facteur induit par l'hypoxie (HIF-1a), qui est surexprimé lors de l'I/R est un important activateur la réponse inflammatoire, notamment sur l'inflammasome, et que le Nicotinamide Mononucléotide possède un rôle anti-inflammatoire en empêchant la stabilisation de HIF-1a. Tous ces éléments sont autant de cibles thérapeutiques potentielles à développer avec de nouvelles études / Myocardial infarction (MI) is the first cause of death in the world. Reperfusion is the key treatment of MI. However, reperfusion can cause reperfusion injuries. Mitochondria and mitochondrial permeablility transition pore are the target of reperfusion injuries. Mitochondrial dysfonction and necrosis lead to an intense local and systemic inflammation. Ischemic post-conditioning (PC) and pharmacologic PC (with cyclosporine A, CsA) are used to limit reperfusion injuries. During my thesis, I worked on cardioprotective effet of sirtuin 3 and I studied inflammation induced by myocardial ischemia/reperfusion (I/R). I have shown that ischemic PC involve sirtuin 3 and deacetylation of cyclophilin D. I demonstrated that myocardial I/R induce an intense inflammatory response in Human with a key role of IL-17A, IL-6, IL-8 and IL-10. However, this inflammatory response is not modulated by the administration of CsA. A least, we studied the role of HIF-1a that is over expressed during I/R. We showed that HIF-1a activate inflammasome and the secretion of IL-1beta and IL-18. Furthermore, Nicotinamied Mononucleotide has anti-inflammatory effets with an action of HIF-1a. Taken together, these data contribute to develop new target for cardioprotection

Introdução: O pré-condicionamento isquêmico tem a capacidade de minimizar as lesões decorrentes do processo de isquemia e reperfusão. O pós-condicionamento isquêmico, descrito posteriormente, apresenta resultados semelhantes na proteção contra as lesões por isquemia e reperfusão em miocárdio, cérebro, rins, músculo esquelético e medula espinhal. Objetivo: Avaliar o efeito do pré e do pós-condicionamentos isquêmicos bem como sua associação sobre a lesão tecidual em músculo esquelético de ratos submetidos ao processo de isquemia e reperfusão. Método: Foram utilizados 50 ratos Wistar, distribuídos em cinco grupos de 10 animais: grupo Controle, em que se realizou isquemia parcial por clampeamento aórtico infra-renal (240 min) e reperfusão (60 min); Grupo Sham, fez-se o mesmo procedimento cirúrgico, porem sem o clampeamento da aorta abdominal; grupo Pós-condicionamento, onde precedendo o início da reperfusão, foi realizado o pós-condicionamento isquêmico (três ciclos de um minuto de reperfusão intercalados por três ciclos de um minuto de isquemia); grupo Pré-condicionamento, precedendo ao período isquêmico procedeu-se ao pré-condicionamento isquêmico(três ciclos de 5 min de isquemia intercalados por três ciclos de 5 min de reperfusão); grupo Pré e Pos-condicionamento, neste grupo associaram-se os dois métodos. Avaliaram-se os resultados pela dosagem de enzimas tissulares (aspartato aminotransferase-AST, creatinofosfoquinase-CK e desidrogenase lática-DHL), malondialdeído (MDA) e glicogênio tissular com tratamento estatístico. Resultado: Houve elevação significativa dos níveis de CK em todos os grupos em relação ao grupo sham. A AST elevou-se no grupo pré+pós-condicionamento em relação ao grupo controle. O comportamento da LDH foi semelhante entre os grupos. O marcador de lesão de membrana celular representado pelo MDA mostrou-se elevado em todos os grupos, exceto no grupo pós-condicionamento em relação ao grupo sham. Não ocorreu diferença entre os grupos isquêmicos e reperfundidos. Quanto à reserva energética ocorreu queda significativa do glicogênio no grupo controle em relação aos grupos sham, pré-condicionamento e pré+pós-condicionamento, exceto no grupo pós. Conclusão: Concluiu-se que o pré-condicionamento bem como a associação do pré com o pós-condicionamento isquêmico foram capazes de minimizar a lesão tecidual em musculatura estriada de ratos submetidos à isquemia e reperfusão, porém a associação dos métodos não trouxe vantagens adicionais sobre os mesmos aplicados isoladamente / Background: Ischemic preconditioning has the ability to reduce injuries resulting from ischemia and reperfusion. Ischemic post-conditioning, described later, shows similar protective results against injury by ischemia and reperfusion in myocardium, brain, kidney, skeletal muscle, and spinal cord. Objective: To evaluate the effect of ischemic pre- and post-conditioning and their combination on protection against skeletal muscle tissue injury in rats subjected to ischemia and reperfusion. Methods: We used 50 Wistar rats divided into five groups of 10 animals each: Control group, subjected to partial ischemia by infrarenal aortic clamping (240 min) and reperfusion (60 min); Sham group, subjected to the same surgical procedure but without clamping of the abdominal aorta; Post-conditioning group, in which ischemic post-conditioning was performed before the onset of reperfusion (three cycles of one min of reperfusion interspersed with three cycles of one min ischemia); pre-conditioning group, subjected to ischemic pre-conditioning prior to the ischemic period (three cycles of 5 min ischemia interspersed with three cycles of 5 min reperfusion); pre-and post-conditioning group, in which the two methods were used in combination. Results were evaluated by measuring levels of tissue enzymes (aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH), malondialdehyde (MDA) and tissue glycogen, with statistical analysis. Results: There was a significant elevation of CK levels in all groups compared to the sham group. AST increased in the pre- + post-conditioning group compared to the control group. The behavior of LDH was similar for all groups. The marker of cell membrane damage represented by MDA was high in all groups except the post-conditioning one compared to the sham group. No significant difference was observed between the ischemic and reperfused groups. As to the energy reserve (glycogen), a significant decrease occurred in the control group compared to the sham, pre-conditioning and pre- + post-conditioning groups, but not in the post-conditioning group. Conclusion: We conclude that pre-conditioning and combined pre - and post-conditioning were able to minimize tissue injury in the skeletal muscle of rats subjected to the ischemia and reperfusion process, but the combined methods did not bring about additional advantages compared to each method applied aloneT

Introdução: O pré-condicionamento isquêmico tem a capacidade de minimizar as lesões decorrentes do processo de isquemia e reperfusão. O pós-condicionamento isquêmico, descrito posteriormente, apresenta resultados semelhantes na proteção contra as lesões por isquemia e reperfusão em miocárdio, cérebro, rins, músculo esquelético e medula espinhal. Objetivo: Avaliar o efeito do pré e do pós-condicionamentos isquêmicos bem como sua associação sobre a lesão tecidual em músculo esquelético de ratos submetidos ao processo de isquemia e reperfusão. Método: Foram utilizados 50 ratos Wistar, distribuídos em cinco grupos de 10 animais: grupo Controle, em que se realizou isquemia parcial por clampeamento aórtico infra-renal (240 min) e reperfusão (60 min); Grupo Sham, fez-se o mesmo procedimento cirúrgico, porem sem o clampeamento da aorta abdominal; grupo Pós-condicionamento, onde precedendo o início da reperfusão, foi realizado o pós-condicionamento isquêmico (três ciclos de um minuto de reperfusão intercalados por três ciclos de um minuto de isquemia); grupo Pré-condicionamento, precedendo ao período isquêmico procedeu-se ao pré-condicionamento isquêmico(três ciclos de 5 min de isquemia intercalados por três ciclos de 5 min de reperfusão); grupo Pré e Pos-condicionamento, neste grupo associaram-se os dois métodos. Avaliaram-se os resultados pela dosagem de enzimas tissulares (aspartato aminotransferase-AST, creatinofosfoquinase-CK e desidrogenase lática-DHL), malondialdeído (MDA) e glicogênio tissular com tratamento estatístico. Resultado: Houve elevação significativa dos níveis de CK em todos os grupos em relação ao grupo sham. A AST elevou-se no grupo pré+pós-condicionamento em relação ao grupo controle. O comportamento da LDH foi semelhante entre os grupos. O marcador de lesão de membrana celular representado pelo MDA mostrou-se elevado em todos os grupos, exceto no grupo pós-condicionamento em relação ao grupo sham. Não ocorreu diferença entre os grupos isquêmicos e reperfundidos. Quanto à reserva energética ocorreu queda significativa do glicogênio no grupo controle em relação aos grupos sham, pré-condicionamento e pré+pós-condicionamento, exceto no grupo pós. Conclusão: Concluiu-se que o pré-condicionamento bem como a associação do pré com o pós-condicionamento isquêmico foram capazes de minimizar a lesão tecidual em musculatura estriada de ratos submetidos à isquemia e reperfusão, porém a associação dos métodos não trouxe vantagens adicionais sobre os mesmos aplicados isoladamente / Background: Ischemic preconditioning has the ability to reduce injuries resulting from ischemia and reperfusion. Ischemic post-conditioning, described later, shows similar protective results against injury by ischemia and reperfusion in myocardium, brain, kidney, skeletal muscle, and spinal cord. Objective: To evaluate the effect of ischemic pre- and post-conditioning and their combination on protection against skeletal muscle tissue injury in rats subjected to ischemia and reperfusion. Methods: We used 50 Wistar rats divided into five groups of 10 animals each: Control group, subjected to partial ischemia by infrarenal aortic clamping (240 min) and reperfusion (60 min); Sham group, subjected to the same surgical procedure but without clamping of the abdominal aorta; Post-conditioning group, in which ischemic post-conditioning was performed before the onset of reperfusion (three cycles of one min of reperfusion interspersed with three cycles of one min ischemia); pre-conditioning group, subjected to ischemic pre-conditioning prior to the ischemic period (three cycles of 5 min ischemia interspersed with three cycles of 5 min reperfusion); pre-and post-conditioning group, in which the two methods were used in combination. Results were evaluated by measuring levels of tissue enzymes (aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH), malondialdehyde (MDA) and tissue glycogen, with statistical analysis. Results: There was a significant elevation of CK levels in all groups compared to the sham group. AST increased in the pre- + post-conditioning group compared to the control group. The behavior of LDH was similar for all groups. The marker of cell membrane damage represented by MDA was high in all groups except the post-conditioning one compared to the sham group. No significant difference was observed between the ischemic and reperfused groups. As to the energy reserve (glycogen), a significant decrease occurred in the control group compared to the sham, pre-conditioning and pre- + post-conditioning groups, but not in the post-conditioning group. Conclusion: We conclude that pre-conditioning and combined pre - and post-conditioning were able to minimize tissue injury in the skeletal muscle of rats subjected to the ischemia and reperfusion process, but the combined methods did not bring about additional advantages compared to each method applied aloneT