Cytoglobin (Cygb) and neuroglobin (Ngb) are among the newest members of vertebrate globin family characterized by a classical 3-over-3 α-helical fold and a heme prosthetic group capable of reversibly binding small ligands such as O2, CO and NO. The physiological functions of Cygb and Ngb remain to be determined; however, current data suggest that both proteins have a significant role in cytoprotection in hypoxic and genotoxic conditions. Cytoglobin and Ngb are distinct from their better-known counterparts, hemoglobin (Hb) and myoglobin (Mb), in several structural features. First, in the absence of an external ligand, the sixth coordination site of the heme iron in Cygb and Ngb is occupied by a distal histidine residue, leading to a complex ligand rebinding mechanism dependent on the rate of distal His dissociation from the heme iron. Although hexacoordination was observed before in plant and bacterial hemoglobins, the physiological role of this feature remains unknown. Second, both Ngb and Cygb are capable of forming an intraprotein disulfide bond, which has been shown to regulate ligand binding affinity, leading to a hypothesis that intracellular function of these proteins is redox-dependent. Lastly, Cygb contains 20 amino acid long extensions on both N- and C- termini, a unique feature among vertebrate globins with unknown physiological function. The work presented in the dissertation reveals that hexacoordinate heme reactivity is distinct from that of pentacoordinate heme and is strongly influenced by the distal histidine residue and the disulfide bond. In the case of human Cygb, experimental and computational approaches demonstrated that the disulfide bond regulates the flexibility of the N terminus and the accessibility of the 1,8-ANS binding site. Furthermore, molecular dynamics of the hexa- and pentacoordinate human Ngb were probed computationally to elucidate structural requirements that govern signal transmission between CD loop and the distal pocket. Lastly, Ngb and Cygb were reconstituted with a fluorescent analog of the native heme group to produce hexacoordinate variants with favorable photophysical properties that can be used to characterize protein-protein interactions.

Le stress oxydatif joue un rôle majeur dans la mort des cellules neuronales dans diverses conditions neuropathologiques. Cependant, les astrocytes réactifs, en produisant des facteurs neuroprotecteurs et antioxydants, sont capables de protéger les neurones contre le stress oxydatif. De ce fait, la protection des cellules gliales contre les facteurs nocifs, s’avère indispensable pour prévenir les dommages des cellules nerveuses. Les globines du cerveau, en particulier, la neuroglobine (Ngb) et l’hémoglobine (Hb), exprimées dans les cellules nerveuses, jouent un rôle important dans le métabolisme de l’oxygène. Récemment, il a été démontré, que ces protéines exercent des effets neuroprotecteurs dans les modèles expérimentaux de maladies neurodégénératives. Cependant, aucun effet glioprotecteur n’a été rapporté. Les objectifs de ce travail de thèse sont, de mettre en évidence les effets protecteurs de l’Hb et la Ngb dans les astrocytes en culture en présence d’un stress oxydant, et d’élucider les mécanismes intracellulaires mis en jeu. Nous avons démontré que l’Hb et la Ngb sont capables de promouvoir la survie des astrocytes en condition de stress oxydatif, et ce en réduisant significativement la surproduction des ROS, la surexpression des gènes pro-inflammatoires (IL-6, IL-33, iNOS), le dysfonctionnement mitochondrial et la stimulation de l’activité de la caspase-3/7. Nous avons montré aussi que les effets anti-apoptotiques impliquent l’activation des voies de signalisation ERK-MAPK. En outre, nous avons vérifié les effets glioprotecteurs sur un modèle animal de stress oxydatif chronique, les souris KO TP53INP1, ainsi que sur un modèle animal d’hypoxie. / Oxidative stress plays a major role in the death of neuronal cells under various neuropathological conditions. However, reactive astrocytes, by producing neuroprotective and antioxidant factors, are able to protect neurons against oxidative stress. Therefore, protecting glial cells from harmful factors is essential to prevent nerve cell damage. Brain globins, in particular, neuroglobin (Ngb) and hemoglobin (Hb), expressed in neurons and glial cells, play an important role in the metabolism of oxygen. Recently, it has been demonstrated that these proteins exert neuroprotective effects in experimental models of neurodegenerative diseases. However, no glioprotective effect has been reported. The objectives of this thesis work are to demonstrate the protective effects of Hb and Ngb in cultured astrocytes in the presence of oxidative stress and to elucidate the intracellular mechanisms involved. We have demonstrated That Hb and Ngb are able to promote the survival of astrocytes under oxidative stress conditions by significantly reducing over-production of ROS, overexpression of pro-inflammatory genes (IL-6, IL-33, iNOS) Mitochondrial dysfunction and stimulation of caspase-3/7 activity. We have also shown that anti-apoptotic effects involve the activation of ERK-MAPK signaling pathways. In addition, we verified the glioprotective effects on an animal model of chronic oxidative stress, KO mice TP53INP1, as well as on an animal model of hypoxia.

La neuroglobine humaine (Ngb) est une globine découverte en 2000 dont la fonction principale demeure encore inconnue. Par comparaison avec l’hémoglobine (Hb) et la myoglobine (Mb), les globines les plus étudiées, la Ngb possède une séquence en acides aminés particulière. Il en résulte des caractéristiques structurales propres à la Ngb. L’hème, qui constitue le site actif de la Ngb, est hexacoordiné par l’histidine distale 64 et existe sous deux formes isomères A et B. La Ngb comprend également un pont disulfure Cys46-Cys55 intramoléculaire.La relation entre ces spécificités et d’éventuelles fonctions de la Ngb demeure cependant assez mal explorée. Notre objectif durant la thèse, était de mettre en évidence in vitro l’influence de différents éléments structuraux sur les propriétés et la réactivité de la Ngb. Pour ce faire, les mutations H64V, F106L, A90P et C46G ont été réalisées. Des études expérimentales à l’aide de spectrophotométrie UV-visible, de dichroisme circulaire et de RMN, ont été effectuées pour caractériser les mutants synthétisés, tester leur stabilité en fonction du pH et évaluer leur réactivité vis-à-vis de la fixation du ligand CN.Nous avons ainsi montré que la structure de la Ngb était influencée par la présence de l’histidine distale, du pont disulfure et de l’environnement de l’hème. L’étude, pour la première fois, des coefficients d’extinction molaire des protéines mutées a permis de souligner l’impact des acides aminés au voisinage de l’hème mais aussi du pont disulfure sur l’environnement électronique de l’hème. Nous avons aussi mis en évidence que le pont disulfure et les acides aminés mutés influaient sur la capacité de la forme isomère A de la Ngb à fixer le cyanure. La forme isomère B est en revanche peu impactée par ces deux paramètres. Cela soulève la question de l’existence et de la fonction des deux formes isomères de l’hème in vivo. / The physiological function of Human Neuroglobin (Ngb), discovered in 2000, is still unknown. Compared to other classical globins Haemoglobin and Myoglobin, Ngb has some structural specificities. Its haem, which is its reactive centre, is hexacoordinated by distal histidine 64 and exists under two isomer forms A and B. Moreover, Ngb possesses an intramolecular disulfide bridge between two cysteines 46 and 55.The relationship between its structural characteristics and its functions in vivo does not remain well-understood. The goal of this thesis was to underline the impact of some structural features on the Ngb properties and reactivity in vitro. Thus Ngb variants H64V, F106L, A90P and C46G were produced. Experimental studies were performed by UV-Visible spectrophotometry, circular dichroism and NMR. Variants were characterized : their stability as a function of pH were tested and their reactivity trough the CN binding reaction were evaluated.We have shown that the Ngb structure was strongly dependant on the presence of the distal histidine, the disulfide bridge and the haem environment. The first and unique determination of variants’ molar absorption coefficients underlined the influence of the haem vicinity and disulfide bridge on the electronic haem environment. We have brought some evidence that the disulfide bridge and the mutated amino acids have an impact on the isomer A Ngb ability to bind the cyanide whereas isomer B is poorly affected by those two parameters. This phenomenon raises the issue of the existence and function of the two isomer forms in vivo.

Stroke is the world's leading cause of adult disability, caused by lack of oxygen and nutrients to the brain due to a blood clot in a major artery. This leads to ischemic damage of neuronal cells that leads to paralysis, motor, and speech deficits. While most stroke therapies aim at removing or reducing the blood clots in the brain, few treatments target cell damage. Neuroglobin (NGB) is a protein in the brain that is able to aid in neuroprotection following oxidative stress. Hypoxia-Inducible Factor-1 (HIF-1) is a transcription factor that serves as a marker for cell recovery after hypoxia or low oxygen levels. Exosomes are microscopic extracellular vesicles that can help deliver proteins across the blood-brain barrier. This thesis focuses on finding a correlation between exosomal-delivered neuroglobin to ischemic cells and the regulation of HIF-1 in order to develop an innovative treatment using exosomes. The specific aims of this thesis are as follows: Aim 1: Package NGB in exosomes of healthy cell The XPAK-NGB plasmid will be used to transfect NGB DNA into wild-type human embryonic kidney (HEK-293 cell line) cells. Exosomes will be harvested from the spent media. The exosomes will be analyzed to ensure that the protein is packaged inside the exosomes. Aim 2: Determine the limit of hypoxic conditions and effects of NGB on damaged cells A literature review will be performed to determine the ideal concentration of H2O2 for the survival of neuronal cells. This will include the composition of hypoxia as well as the length of time that cells can be exposed to and remain viable. Aim 3: Correlate NGB concentration and HIF-1 concentration Another literature review will determine the specific markers of NGB and HIF-1.

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ~2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Le but de cette thèse est de mettre en relation les propriétés structurale, dynamique et fonctionnelle de la forme humaine d’une nouvelle protéine découverte dans le cerveau des vertébrés en 2000 : la Neuroglobine. Dans un premier temps, j’ai réalisé une étude théorique dans laquelle un mécanisme à deux voies menant à la forme pentacoordinée avec cystéines oxydées a été mis en avant. A travers ce mécanisme, un conformère de la Neuroglobine au sein duquel le groupe prosthétique hème a basculé au cœur de la structure protéique a été déterminé. A partir des structures de ce mécanisme, une étude sur la diffusion de petits ligands au sein des cavités internes de la protéine à l’aide de la méthode de métadynamique a mis en évidence que la formation du pont disufure intramoléculaire favorisait la poche de ligation. De plus un certain nombre de voies de sortie pour les ligands a pu être obtenu. Pour compléter ce premier aspect de la thèse, une étude des propriétés mécaniques, communes avec les autres globines, a montré l’importance de quatre résidus centraux, dit mécaniquement sensibles, qui régulent les canaux d’accès aux différentes poches internes de la protéine, appelé phénomène de respiration. Dans un second temps, je me suis intéressé à l’interaction de la Neuroglobine avec un petit ligand via une étude expérimentale par ITC. La première conclusion importante est que la cinétique de ligation est plus importante lorsque le pont disulfure est formé. De plus j’ai observé une diminution de la cinétique lors du passage Wild Type vers C120S puis réaugmentation de la cinétique lors du passage C120S vers C46G/C55S/C120. Afin de comprendre ce phénomène, une simulation de la Neuroglobine triplement mutée a été réalisée au cours de laquelle un réseau de deux liaisons hydrogènes a été mis en avant. Ce réseau change considérablement les voies d’entrée/sortie pour les ligands. Ainsi la mutation 120 ferme une/ou plusieurs voies de sortie alors que la mutation 46 ouvre la voie naturelle des globines. Le changement observé étant important, une étude par RMN de Ngb TM et WT cystéines réduites a montré qu’il y avait une différence de structure entre ces formes pas seulement au niveau des points de mutation mais sur l’ensemble de la structure. Ces nouveaux résultats mettent ainsi en évidence le rôle important des trois cystéines chez la Neuroglobine humaine. / In this PhD work, I tried to link together the different structural, dynamic and fonctional properties of a new human protein discovered in the mamals brain in 2000: the Neuroglobin. First of all, I established a new two ways mecanism in order to get the pentacoodinated oxydized cysteins state using theoritical method. One of this mecanism’s conformer shows an important heme sliding inside of the proteic structure. Furthermore with help of metadynamic method, I studied the small ligand diffusion and migration in the internal cavity network. I showed the higher ligand affinity when the disulfide bridge is bond and we proposed an important number of exit pathways. Then we developed a method to understand the mechanical properties of the globins and we found four residues mechanically sensitive which form together a control access pathway between internal cavities, called breath phenomenon. Secondly I used ITC method in order to characterize the interaction between the Neuroglobin and a small ligand. From this experiment we highlighted that the kinetic ligation is faster when the disulfide bridge is formed. Then I noticed a relative decrease of the velocity when the mutation C120S is operated followed by a relative increase of the velocity for the triple mutation C46G/C55S/C120 compared to the Wild Type data. To understand these results, I performed a molecular simulation of the triple mutation Neuroglobin form. During this trajectory, I discovered a structure with a two hydrogen bonds network, which significantly changes the ligand entry/exit pathways. The 120 mutation closes one/several exit pathways while the 46 mutation opens the natural globin exit pathway. Because of the considerable structural change observed in the triple mutation Neuroglobin form, I decided to produce NMR results. These last points reveal a relative structure difference between the Wild Type oxidized cysteins form and the triple mutation form not only on the mutation points but also on the global structure. All these new results highlight the essential role of the three cysteins in the human Neuroglobin.

The retina is the ocular tissue responsible for the detection of light. Its extensive demand for oxygen, coupled with a concomitant elevated supply, renders this tissue prone to both hypoxia and hyperoxia. In this thesis, we construct mathematical models of the retina, formulated as systems of reaction-diffusion equations, investigating its oxygen-related dynamics in healthy and diseased states. In the healthy state, we model the oxygen distribution across the human retina, examining the efficacy of the protein neuroglobin in the prevention of hypoxia. It has been suggested that neuroglobin could prevent hypoxia, either by transporting oxygen from regions where it is rich to those where it is poor, or by storing oxygen during periods of diminished supply or increased uptake. Numerical solutions demonstrate that neuroglobin may be effective in preventing or alleviating hypoxia via oxygen transport, but that its capacity for oxygen storage is essentially negligible, whilst asymptotic analysis reveals that, contrary to the prevailing assumption, neuroglobin's oxygen affinity is near optimal for oxygen transport. A further asymptotic analysis justifies the common approximation of a piecewise constant oxygen uptake across the retina, placing existing models upon a stronger theoretical foundation. In the diseased state, we explore the effect of hyperoxia upon the progression of the inherited retinal diseases, known collectively as retinitis pigmentosa. Both numerical solutions and asymptotic analyses show that this mechanism may replicate many of the patterns of retinal degeneration seen in vivo, but that others are inaccessible to it, demonstrating both the strengths and weaknesses of the oxygen toxicity hypothesis. It is shown that the wave speed of hyperoxic degeneration is negatively correlated with the local photoreceptor density, high density regions acting as a barrier to the spread of photoreceptor loss. The effects of capillary degeneration and treatment with antioxidants or trophic factors are also investigated, demonstrating that each has the potential to delay, halt or partially reverse photoreceptor loss. In addition to answering questions that are not accessible to experimental investigation, these models generate a number of experimentally testable predictions, forming the first loop in what has the potential to be a fruitful experimental/modelling cycle.