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The central thesis of this project is that damage caused by reactive nitrogen species, e.g. 3-nitrotyrosine (Tyr-NO2), constitutes a marker of disease progression/severity. A new sensitive electrochemiluminescence ELISA was optimised and validated for Tyr-NO2 measurement, giving a lower limit of quantification of 0.04 nM BSA-NO2, intra- and inter-assay CVs of 6.5% and 11.3%, an average recovery of 106 ± 3% and average linearity 0.998 ± 0.001. Nitrative stress, carbonyl stress and C-reactive protein (CRP) concentrations were measured before and after major elective surgery. CRP measurements confirmed the induction of an inflammatory response. Median serum Tyr-NO2 levels increased post-surgery to a median (inter-quartile range) value of 0.97 (0 – 1.7) fmol nitrated BSA (BSA-NO2) equivalents/mg protein compared with a pre-surgery level of 0.59 (0 – 1.3) fmol BSA-NO2 equivalents/mg protein (p<0.05). Oxidative damage was confirmed by serum protein carbonyl levels (p<0.05). In a second pre-/post- surgery study, patients who developed sepsis postoperatively had significantly higher serum Tyr-NO2 levels one day prior to diagnosis (median (IQR) 4.5 (1.65 – 8.21) fmol BSA-NO2 equivalents/mg protein) compared to patients without sepsis (1.2 (0.74 – 5.97) fmol BSA-NO2 equivalents/mg protein; p<0.05). Tyr-NO2 levels have not previously been measured before clinical diagnosis. However, Tyr-NO2 did not improve upon CRP as a diagnostic marker (area under the curve: Tyr-NO2 0.69 versus CRP 0.88). Nitrate (NO3¯) supplementation in healthy smokers was also studied. Plasma Tyr-NO2 levels were unaltered by supplementation or smoking status. Salivary nitration was unaffected by smoking and decreased with NO3¯ supplementation: the median (IQR) pre-supplementation was 0.67 (0.31-1.14) and post-supplementation was 0.43 (0.12-0.61) pmol BSA-NO2 equivalents/mg protein. Ozone-based chemiluminescence was utilised for nitrite (NO2¯) and NO3¯ measurement as indicators of ˙NO production. Plasma and salivary NO2¯ and NO3¯ concentrations increased significantly with NO3¯ supplementation (p<0.05). In contrast to published studies, brain frontal lobe Tyr-NO2 levels were not higher in dementia: the median (IQR) levels in dementia were 0.29 (0.19-0.57) and in non-dementia controls were 0.3 (0.22-0.55) pmol BSA-NO2 equivalents/mg protein. However, the median brain tissue NO2¯ concentration was significantly higher in the Alzheimer’s disease group (p<0.05). Western blotting revealed that nitration was predominantly in a few select proteins, with TOF-MS/MS analysis suggesting haemoglobin is one of these proteins. Measurement of nitrative stress using ozone-based chemiluminescence and an electrochemiluminescence-based-ELISA overcomes earlier methodological flaws, such as low sensitivity. Detection of total Tyr-NO2 in different inflammatory states indicates that its measurement could have potential as a marker of disease, but measurement of nitration in specific proteins may be more informative than total Tyr-NO2.

Les substituts sanguins à base d’hémoglobine (HBOC) en cours de développement et d’évaluation, sont susceptibles, selon leur formulation et le protocole d’évaluation envisagé, d’induire des modifications du comportement des cellules endothéliales (CE). Nous avons développé des protocoles visant à évaluer le rôle de trois paramètres : les contraintes de cisaillement, l’hypoxie et la présence d’HBOC au contact des CE. Plusieurs facteurs, comme le stress oxydatif, la vasomotricité et l’inflammation ont été étudiés par qPCR, RPE, cytométrie en flux et dosage des métabolites. Dans un premier temps, nous avons observé l’action simultanée des contraintes de cisaillement et des HBOC (Hb-Dex-BTC, Oxyglobin®, aa-Hb) sur le comportement des CE. Nos résultats montrent combien le cisaillement influence la réponse des CE en présence d’HBOC. Pour se rapprocher de la situation clinique d’un usage potentiel d’HBOC, nous avons élaboré un protocole d’hypoxie/réoxygénation des CE par ces Hb. Au travers des deux protocoles d’hypoxie développés (brève et chronique), nous avons montré des mécanismes différents de réponse cellulaire : après 4 h, la réoxygénation avec les substituts induit une diminution de l’inflammation engendrée par l’hypoxie. Alors qu’après 24 h, nous constatons son induction quel que soit le substitut utilisé. D’une façon générale, la présence de dextran dans l’Hb-Dex-BTC rend celle-ci plus furtive vis-à-vis des mécanismes de détection de la présence d’Hb par les CE. Dans une dernière partie, nous avons effectué des tests de cytotoxicité sur des Hb de nouvelle génération (PEG-Hb et Hb octamérique produite par génie génétique) par mesure de la viabilité cellulaire et du taux d’apoptose-nécrose, Ces résultats encourageants confirment l’intérêt de poursuivre les évaluations. / Hemoglobin based oxygen carriers (HBOC), under development and evaluation, are potentially able to induce modifications of endothelial cells (EC) behaviour. We developed protocols to evaluate the role of three different parameters, like shear stress, hypoxia and HBOC presence on EC. Several factors, like oxidative stress, vasomotion and inflammation were studied using qPCR, EPR, flow cytometry and metabolites quantification. At first, we observed simultaneously the action of shear stress and HBOC (Dex-BTC Hb, Oxyglobin®, aa-Hb) on EC behaviour. Our results show how shear stress influences EC response face to HBOC. Then, we elaborated a hypoxia/reoxygenation protocol with HBOC. Using two different protocols of hypoxia (brief and chronic) we demonstrated that different mechanisms drive cell response. After 4 h of hypoxia, reoxygénation with blood substitutes induces a diminution of the inflammation engender by hypoxia. However, after 24 h of hypoxia, we observed an over inflammation whatever was the blood substitute used. In conclusion, among the three HBOC tested, we highlighted that the presence of dextran in Dex-BTC-Hb solutions makes Hb furtive for EC Hb detection mechanisms. Finally, we developed cytotoxicity tests on new Hb generation (PEG-Hb, octameric Hb) based on cellular viability and apoptosis-necrosis. These tests were encouraging for both Hb.

THESIS ABSTRACT This thesis work was undertaken to investigate the effects of internal (i.e. morphology, physiological capacity, stress and inflammatory cytokines) and external (i.e. heat exposure) factors on military physical performance in members of the Canadian Armed Forces (CAF). By gaining insight into how these factors affect military physical performance, training and intervention strategies could be better tailored for optimizing performance. Both morphology and physiological capacity have previously been recognized to affect performance on certain military physical performance tasks. However, the effect of such factors on the newly implemented Common Military Task Fitness Evaluation (CMTFE), the current physical employment standard for the CAF, has not been determined. Stress and inflammatory cytokines have also been shown to affect physical performance in the general population, but there is no data available on levels of stress and inflammatory cytokines in the CAF, or their potential effect on military physical performance. Recently, the CAF have also implemented a loaded march, followed by an assessment of military physical performance (FORCEcombat), as a part of the physical performance testing for all members of the Canadian Army. However, there is a lack of research currently available on how factors like heat exposure can affect thermoregulatory and cardiovascular response, as well as performance on a loaded march, and the following FORCEcombat test. In order to provide key information about the requirements and delivery of the military physical performance tests currently used in the CAF, my thesis focused on four main parts to better understand the importance of some of the internal and external factors known to affect physical performance. Firstly, my thesis assessed CAF members’ morphological and physiological characteristics that may affect overall performance on the CMTFE. In Chapter 2, results showed that both characteristics of morphology and physiological capacity separated the top and bottom performers. Even though a difference in morphology was observed between top and bottom performers, performance on the CMTFE was mainly dependent on aerobic capacity and measures of strength, rather than morphology. Aerobic capacity explained ~36% of variability in performance among women, and ~32% variability in performance among men. Core strength also had a significant effect on performance in both groups, however, men relied more on upper body strength than did women. Apart from showing that physiological capacity, rather than morphology was the main component affecting performance outcome on the CMTFE, it was also concluded that, unlike physical performance tests used by the U.S. Armed Forces (i.e. push-ups, sit-ups, mile run), no body mass bias exists against larger individuals performing the CMTFE. Chapter 3 and 4 focused on describing levels of stress and inflammatory cytokines among CAF members, and their effect on military physical performance. Stress exposure is known to induce an increase in the production of stress and inflammatory cytokines, and an increase in levels of inflammatory cytokines have been shown to be associated with a decrease in physical performance in general population. Members of the armed forces are susceptible to high stress exposure, but no current data exist on basal levels of stress and inflammatory cytokines in a military population. We therefore performed a descriptive analysis of levels of stress and inflammatory cytokines among CAF members. The results from this analysis showed a generally low detection rate of most of the inflammatory cytokines measured in our military population. However, we did observe a higher detection rate for IFN-γ, TNF-α, IL-2, IL-5, IL-8, IL-17a, IL-23 and IL-31 with increasing age. Adiponectin levels were higher in women compared to men (5.81 (3.52-13.19) µg/ml vs 16.71 (7.68-25.32) µg/ml), whereas IL-18 levels were higher in men compared to women (89.25 (84.03-94.48) vs 75.91 (69.70-82.13) pg/ml). Increasing age was associated with higher basal levels of C-Reactive Protein (CRP), Adiponectin, IL-18 and IL-2, and we also found a significant positive correlation between body fat percentage (BF%) and CRP levels. Following the outcomes of the descriptive study, the associations between levels of stress and inflammatory cytokines and military physical performance were elucidated. Using multiple linear regression, controlling for covariates such as age, sex and BF%, a significant negative association was observed between CRP levels and Total Performance on the CMTFE (p=0.01), picking and digging performance (p=0.04), aerobic capacity (p=0.05) and plank time (p<0.01) among CAF members. Finally, in Chapter 5, the effect of heat exposure on the capacity to perform a task oriented military test was quantified. Many CAF members are subject to a significant heat exposure on a daily basis, and studies from the general population have shown that heat exposure can have a detrimental effect on physical performance. As mentioned previously, The Canadian Army have recently implemented a test of loaded march performance, as a part of their occupational physical performance testing. The loaded march will be performed while wearing military personal protective equipment (PPE) and a daypack (~35 kg), and all members of the Canadian Army will be required to perform this test. Due to this need for mass testing, a large part of the loaded march performance assessments will be required to take place during the summer months. Temperature and relative humidity (RH) can reach high levels on several of the Canadian Army bases located around Canada, during the summer months. However, there is currently limited research available assessing the thermoregulatory and cardiovascular responses to performing a loaded march in the heat while wearing military PPE. Consequently, a study was designed to determine the thermoregulatory and cardiovascular responses to a loaded march (60 min, 5.17 km*h-1, ~35 kg external load) at normal temperature (21°C, 50% RH) and in the heat (30°C, 50% RH). This study also aimed to quantify the effect of heat exposure and previous experience on loaded march and military physical performance (FORCEcombat). Ten participants experienced with loaded march (military reservists), and ten participants inexperienced with loaded march (civilians) were recruited for this study. The results showed that whereas nine out of ten participants in the experienced group completed the loaded march in the heat, only five of the ten participants in the inexperienced group were able to do the same. Performing the loaded march in the heat while wearing military PPE led to a state of uncompensable heat stress, for both the experienced and the inexperienced group. Both groups showed a continuous increase in core temperature and heat storage (0.025°C/min and 0.02°C/min mean increase in core temp, 8.7 kj/min and 6.7 kj/min mean increase in heat storage, for the inexperienced and experienced group respectively) throughout the heat trial. Apart from the difference in completion rate on the loaded march, experienced participants also had a lower heart rate (134.2±11.9 vs 143.1±8.9 bpm, p≤0.05), perceived exertion (10.2±1.4 vs 13.0±0.9, p≤0.05), thermal comfort (1.9±0.5 vs 2.4±0.4, p≤0.05), and FORCEcombat completion time (662±133 vs 530±49 sec, p≤0.05) compared to the inexperienced participants. The overall results from this thesis show that physiological capacity, inflammatory cytokines, heat exposure and previous experience, all have an effect on military physical performance. It was found that physiological capacity rather than morphology, was the superior predictor of performance on the CMTFE: inflammatory cytokines are present in CAF members and CRP levels increased with increasing age, CRP levels were negatively associated with military physical performance, performing a loaded march in the heat while wearing military PPE exposed both experienced and inexperienced participants to a state of uncompensable heat stress and decreased performance on the FORCEcombat test, and that previous experience has a positive effect on loaded march completion rate and FORCEcombat performance.