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Garantir la qualité des produits sanguins est crucial. Les lysats plaquettaires (LP) riches en facteurs de croissance (FC) s’imposent comme le complément idéal pour l’expansion ex vivo des cellules souches, et comme produit thérapeutique pour la régénération cellulaire. L’intérêt est croissant pour les microparticules (MPs) extracellulaires, mais l’expression de phosphatidylsérine à leur surface peut induire des effets thrombotiques et inflammatoires. L’autre risque transfusionnel, la transmission de virus, dont le virus de l’hépatite C (VHC), est maîtrisable par traitements de réduction virale par solvant/détergent (S/D), chauffage, ou nanofiltration. Nous avons étudié des technologies de sécurisation des produits sanguins: (a) élimination des MPs par nanofiltration sur filtres de 75 nm et (b) traitements S/D, chauffage à 56°C ou nanofiltration pour inactiver ou éliminer le VHC. Les informations ont été utilisées pour développer des LP utiles en médecine régénérative. L’un d’eux destiné à la neurorégénération, a été préparé en émettant l’hypothèse qu’un lysat de culot plaquettaire (LCP) enrichi en facteurs neurotrophiques et dépourvu de protéines plasmatiques se montrerait efficace contre les maladies neurodégénératives. Nos résultats montrent que la nanofiltration sur des filtres de 75 nm préserve la composition en protéines plasmatiques, et le pouvoir hémostatique. La nanofiltration retire les MPs et évite, in vitro, la génération de thrombine. Par ailleurs le traitement S/D à 31°C pour 30 minutes élimine le pouvoir infectieux du VHC. Pris globalement les traitements de nanofiltration et S/D apparaissent donc comme des méthodes de choix pour l’amélioration de la sécurité du plasma vis à vis de risques thrombogènes et infectieux. Nous avons ensuite préparé un LCP appauvri en protéines plasmatiques (dont le fibrinogène) et enrichi en un mélange pléiotrope physiologique de FC destiné à l’administration cérébrale. Les analyses par ELISA et par protéomique ont montré qu’un chauffage de 56°C pour 30 min réduisait le contenu en protéines et modifiait favorablement la composition relative en facteurs neurotrophiques. Par ailleurs le chauffage améliore l’action neuroprotectrice et, associé aux traitements S/D et de nanofiltration, contribue à l’inactivation du VHC. Ce LCP exerce une neuroprotection élevée dans des modèles de la maladie de Parkinson (MP) tout à la fois (a) in vitro (cellules LUHMES différentiées en neurones dopaminergiques et exposées au MPP+) et (b) in vivo (souris intoxiquées par MPTP). L’expression de la tyrosine hydroxylase (TH) dans la Substantia nigra pars compacta montre que l’administration intracérébroventriculaire (ICV) ou intranasale (i.n.) apparait comme une option thérapeutique possible des maladies neuro-dégénératives. Les études cellulaires In vitro sur LUHMES et NSC34 ont montré que l’inhibition spécifique des voies signalétiques relayées par AkT et ERK altère l’activité neuroprotectrice du LC. Des événements neuro-inflammatoires pouvant aggraver l’évolution des maladies neurodégénératives, nous avons vérifié que le LCP n’induit pas de marqueurs inflammatoires (COX-2, iNOS) chez des cellules microgliales BV2, et pouvait même diminuer celle de COX-2 après exposition à des lipopolysaccharides. De plus, nous avons identifié que le LCP contenait 1.7 x 1012 MP/mL d’une taille moyenne de 160 nm. Isolées, ces MPs pourraient exercer un rôle neuroprotecteur des cellules LUHMES exposées à des agents neurotoxiques. En conclusion, nos résultats montrent la faisabilité technique à préparer des lysats plaquettaires viro-inactivés pour des usages dans le domaine de la médecine régénérative, y compris comme agent neuroprotecteur du système nerveux central. / Ensuring quality and safety of blood products is crucial. Platelet lysates (PL) rich in growth factors (GFs) have emerged as ideal clinical-grade supplement for ex vivo expansion of mesenchymal stromal cells, and as therapeutic product promote cellular regeneration. Interest for platelet extracellular microparticles (MPs) is growing but expression of phosphatidylserine on their surface may cause thrombotic and inflammatory side effects. Another transfusional risk, transmission of viruses, including hepatitis C virus (HCV), can be fully controlled by dedicated viral reduction methods as solvent/detergent (S/D) or heat treatments, or nanofiltration. We have evaluated technologies to secure therapeutic blood products: (a) removal of MPs by 75nm-nanofiltration and (b) inactivation/removal of HCV by S/D or 56°C heat treatments, or nanofiltration. Data have been used to develop LP of interest for regenerative medicine. In particular, one, targeting neuroregenerative applications, has been prepared based on the hypothesis that a platelet pellet lysate (PPL) enriched in multiple neurotrophic growth factors and depleted of plasma proteins could exert potent neuroprotective actions in neurodegenerative disease models. Our data show that 75 nm-plasma nanofiltration preserved plasma protein biochemical profile, and hemostatic power. Nanofiltration removes MPs and avoids in vitro the generation of thrombin. In addition, the S/D treatment at 31°C for 30 minutes fully inactivates HCV infectivity. Therefore, altogether, nanofiltration and S/D emerge as choice procedures to improve the safety of plasma for thrombogenic and infectious risks. We have then prepared a PPL depleted of plasma proteins (in particular fibrinogen), and rich in a physiological pleiotropic mixture of neurotrophins for brain administration. ELISA and proteomics studies revealed that the heat-treatment at 56°C for 30 min decreased the protein content and favorably modified the relative composition in neurotrophic factors. Heat-treatment improved the neuroprotective activity and, together with S/D and nanofiltration contributed to HCV inactivation. This PPL exerted strong neuroprotective effects in Parkinson’s disease (PD) models (a) in vitro, using LUHMES cells exposed to MPP+ neurotoxin, and (b) in vivo, in mice intoxicated by MPTP neurotoxin. Expression of tyrosine hydroxylase (TH) in the Substantia nigra pars compacta indicated that brain delivery by intracerebroventricular (ICV) or intranasal (i.n.) administration may be a therapeutic option for disease-modifying strategies of neurodegenerative diseases. In vitro studies in LUHMES and NSC34 cells showed that specific inhibition of signal transduction pathways through AkT and ERK influenced PPL neuroprotective function. Since neuro-inflammation detrimentally affects neurodegenerative disorders, we verified that the PPL did not stimulate the release of inflammatory markers (e.g. COX-2, iNOS) by BV2 microglial cells in culture, and could even restrict COX-2 expression when cells were exposed to LPS. In addition, the PPL was found to contain 1.7 x 1012 MP/mL with a mean size of 160 nm. These MPs may exert neuroprotective activity on LUHMES cells exposed to neurotoxins. Altogether, our data demonstrate the technical feasibility of developing virally-safe customized platelet lysate preparations with specific applications for cell therapy and regenerative medicine, in particular as neuroprotective agents of the central nervous system.

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

<p>Valproic acid (<em>2-propylpentanoicacid</em>) has long been in use as an anticonvulsant and mood-stabilizer. Recently, VPA has been shown to inhibit the activity of histone deacetylases (HDACs), resulting in chromatin remodelling and changes in gene expression<em>.</em> Although the molecular mechanism for VPA action in the central nervous is not well understood, many signalling pathways have been suggested as targets for this HDAC inhibitor. For instance, VPA was found to induce differentiation in adult hippocampal neural progenitor cells via the β-catenin-Ras-ERK pathway. Also, VPA up regulated Bcl-2, a neurotrophic/neuroprotective protein, with association of extracellular signal-regulated kinase (ERK-1) and phosphatidylinositol 3- kinase (PI3) pathway activation. In this study, C17.2 neural stem cells were used to examine the effects of VPA on the expression of several neurotrophic factors including; cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF), glial cell-derived neurotrophic factor (GDNF), <em>brain-derived neurotrophic factor</em><em> (</em>BDNF). Other genes including; the orphan nuclear receptor-related factor1 (Nurr-1), the early growth response protein 1(Egr-1), and the sex determining region Y-box-2 (Sox-2) were examined. Histone H3 acetylation and the ERK1/2 pathway were examined as possible targets for VPA action. Treatment with clinically relevant concentrations of VPA (1mM, and 3 mM) induced a significant increase of CDNF protein concentrations. Also, increases in the mRNA expression of GDNF, Nurr-1, and Egr-1 were detected following 24 hours VPA treatment at clinically relevant concentrations. Moreover, an increase of histone H3 acetylation was noticed in C17.2 NSCs. These findings might support the role of VPA in neuronal differentiation and neuroprotection.</p> / Master of Science (MSc)

Die rasanten Fortschritte in der neonatalen Intensivmedizin haben zwar die Ueberlebenschancen von Fruehgeborenen enorm verbessert, aber auch viele Probleme und Fragen aufgeworfen. In dieser Arbeit wurde untersucht, ob Hyperoxie Einfluss nimmt auf die Expression von apoptotischen Genen, Wachstumsfaktoren und Zytokinen und so ueber verschiedene Mechanismen und Signalwege zu einem Ungleichgewicht der ueber das neuronale Ueberleben entscheidenden Faktoren fuehrt. 6-Tage alte Ratten wurden fuer bestimme Zeitabschnitte (2, 6, 12, 24, 48, 72 Stunden) einer 80%igen Sauerstoffkonzentration ausgesetzt. In dieser Arbeit konnte am unreifen Rattengehirn nachgewiesen werden, dass eine 80%ige Sauerstoffkonzentration in der Atemluft maximal nach 12 bis 24 Stunden zu einer ausgepraegten, diffusen apoptotischen Neurodegeneration im Gehirn fuehrt. Die Exposition mit hoher Sauerstoffkonzentration fuehrte im unreifen Gehirn zu einer deutlich verminderten Expression der Neurotrophinen, wie deren Signalproteine ERK 1/2 und Akt. Als spezifischer Nachweis fuer eine apoptotische Neurodegeneration wurden neben dem histologischen Verfahren auf molekularer Ebene apoptotische Gene untersucht. Unter Hyperoxie kam es zu einer erhoehten Expression des Todesrezeptors Fas und einer gesteigerten Aktivitaet von Caspase-3.Des Weiteren fand sich infolge der Hyperoxieexposition ein drastischer Anstieg der inflammatorischen Zytokine IL-1beta und IL-18. Es zeigt sich also, dass hohe Sauerstoffkonzentrationen in einer sehr vulnerablen Phase der Hirnentwicklung (Phase des rapiden Hirnwachstums) zu massiven Veraenderungen fuehren, welche den bisher ungeklaerten diffusen Neuronenuntergang bei Fruehgeborenen erklaeren koennten. Die vorliegenden Ergebnisse implizieren aeu§erste Vorsicht bei der therapeutischen Anwendung von Sauerstoff bei Fruehgeborenen, fuer die die postnatalen Konditionen, verglichen mit den intrauterinen Bedingungen, immer hyperoxisch sind und die noch ueber ein unreifes Antioxidationssystem verfuegen. / Infants born prematurely may develop neurocognitive deficits without an obvious cause. Oxygen, which is widely used in neonatal medicine, constitutes one possible contributing neurotoxic factor, because it can trigger neuronal apoptosis in the developing brain of rodents. Premature infants are exposed to partial oxygen pressures that are fourfold higher compared to intrauterine conditions, even if no supplemental oxygen is administered. Here is reported that short exposures to nonphysiologic oxygen levels can trigger apoptotic neurodegeneration in the developing brain. Vulnerability to oxygen neurotoxicity is confined to the first 2 weeks of life, a period characterized by rapid growth, which in humans expands from the sixth month of pregnancy to the third year of life. Hyperoxia caused decreased expression of neurotrophins, and inactivation of survival signalling proteins extracellular signal-regulated kinase (ERK1/2), and protein kinase B (Akt). In addition we hypothesized that two caspase-1-processed cytokines, interleukin (IL)-1beta and IL-18, are involved in oxygen-induced neuronal cell death. Six-day-old Wistar rats were exposed to 80% oxygen for various time periods (2, 6, 12, 24, 48, 72 hours). Neuronal cell death in the brain, as assessed by silver staining, peaked at 12 to 24 hours and was preceded by a marked increase in mRNA of IL-1beta, IL-18 and FAS and a decrease in mRNA and protein levels of neurotrophins and ERK1/2 and Akt Our findings reveal mechanisms that could potentially damage the developing brain of human premature neonates.

Οι νευροτροφίνες (ΝΤs), Nerve Growth Factor (NGF), Brain-Derived Neurotrophin Factor (BDNF), NΤ-3, ΝΤ-4, ΝΤ-5 και ΝΤ-6 ανήκουν σε μια οικογένεια πολυπεπτιδικών αυξητικών παραγόντων οι οποίοι απαιτούνται για την ανάπτυξη του νευρικού συστήματος στα σπονδυλωτά. Εμπλέκονται στην επιβίωση, στη διαφοροποίηση, στην ωρίμανση των νευρώνων, στη συναπτική πλαστικότητα, στη μάθηση, στη μνήμη, καθώς επίσης και στην έκφραση και ενεργότητα σημαντικών πρωτεϊνών, όπως ιοντικών καναλιών και νευροδιαβιβαστικών υποδοχέων. Οι λειτουργίες αυτές επιτελούνται μέσω της δέσμευσής τους σε δύο είδη μεμβρανικών υποδοχέων, της οικογένειας κινάσης-τυροσίνης TrkA, TrkB και TrkC (tropomyosinerelated kinase) και του pan-neurotrophin (με ικανότητα δέσμευσης με όλες τις νευροτροφίνες) υποδοχέα p75NTR που είναι μέλος των υποδοχέων Tumor Necrosis Factors (TNFs). Οι νευροτροφίνες εκφράζονται σε κύτταρα του Κ.Ν.Σ. και Π.Ν.Σ. αλλά και σε ιστούς-όργανα εκτός νευρικού συστήματος, όπως είναι η υπόφυση. Σκοπός της εργασίας ήταν να μελετήσουμε την έκφραση των υποδοχέων των νευροτροφινών με σύγχρονες μεθόδους ανοσοϊστοχημείας σε αδενώματα της υπόφυσης και να συσχετίσουμε την έκφρασή τους με τα κλινοκοπαθολογικά χαρακτηριστικά των ασθενών. Όλα τα αδενώματα της μελέτης που συμπεριλήφθησαν στη μελέτη (10ανδρών και 8 γυναικών) εμφάνισαν ανοσοϊστοχημική χρώση για τον υποδοχέα TrkA και συγκεκριμένα έντονη χρώση (+3) τα 9/18 (50%) των περιστατικών, μέτρια χρώση (+2) τα 8/18 (45%) των περιστατικών και ασθενή χρώση (+1) 1/18 (5%) των περιστατικών. Ο υποδοχέας TrkB εμφάνισε θετικότητα στο 83% (15/18) των περιπτώσεων. Τα 6/15 (40%) περιστατικά παρουσίασαν έντονη χρώση (+3), τα 4/15 (27%) περιστατικά μέτρια χρώση (+2) και τα 5/15 (33%) περιστατικά ασθενή χρώση (+1). Ανοσοϊστοχημική χρώση για τον υποδοχέα TrkB παρατηρήθηκε επίσης στα αγγεία 4/15 (27%) των αδενωμάτων. Τα 11/18 (61%) των αδενωμάτων παρουσίασαν ανοσοθετικότητα για τον TrkC και συγκεκριμένα τα 3/11 (27%) περιστατικά εμφάνισαν μέτρια χρώση (+2) και 8/11 (73%) περιστατικά ασθενή (+1). Χρώση για τον υποδοχέα TrkC εντοπίστηκε σε αγγεία σε 4/11 περιστατικά (36%). Τέλος έκφραση για τον p75 υποδοχέα δεν παρατηρήθηκε σε κανένα αδένωμα. Με δεδομένο ότι οι υποδοχείς TrkB και TrkC εκφράζονται στα αγγεία των αδενωμάτων, μελετήθηκε η έκφραση των υποδοχέων των νευροτροφινών σε σχέση με την αγγειογένεση, ένας μηχανισμός που αφορά άμεσα την πρόγνωση και την ανταπόκριση στην αντίστοιχη θεραπεία των όγκων. Μελετήθηκε η έκφραση του CD31(platelet endothelial cell adhesion molecule) και του VEGFR3 (Vascular Endothelial Growth Factor Receptor 3). Ο παράγοντας VEGFR3 συμβάλλει επίσης και στην ανάπτυξη λεμφαγγείων στο στρώμα του όγκου επάγοντας την ανάπτυξή του. Η εκτίμηση της ανοσοεντόπισης για τον CD31 και τον VEGFR3 για κάθε νεόπλασμα έγινε κατόπιν επιλογής τριών αγγειοβριθέστερων περιοχών, την καταμέτρηση των αγγείων σε κάθε περιοχή και τον υπολογισμό του μέσου όρου (MCV Microvessel Count). Για τον παράγοντα VEGFR3 το 89% των περιστατικών ήταν θετικά εμφανίζοντας ένα εύρος MCV της τάξης των 2 έως 32,67, ενώ για τον παράγοντα CD31 το 100% των αδενωμάτων ήταν θετικά με MCV της τάξης των 4,67 έως 53,67. Δεν παρατηρήθηκε συσχέτιση του MCV με την έκφραση των υποδοχέων των νευροτροφινών. Οι υποδοχείς των νευροτροφινών ενώ εκφράζονται στη φυσιολογική υπόφυση συμμετέχοντας στην ανάπτυξη και στην επιβίωση των κυττάρων, δεν «σιωπούν» στα αδενώματά της. Το ερώτημα που γεννάται είναι αν δρουν ως παράγοντες διατήρησης της καλοήθειας ή αν συμβάλλουν στην ογκογένεση και στην μετέπειτα εξέλιξη των νεοπλασμάτων της υπόφυσης. Περαιτέρω μελέτες απαιτούνται για την διερεύνηση του ρόλου των νευροτροφινών μέσω των υποδοχέων τους στα αδενώματα υπόφυσης, στον άνθρωπο. / -

Anxiety disorders are the most prevalent psychiatric conditions within primary care, affecting up to 29% of people across their lifetime. Generalized Anxiety disorder (GAD) is frequently comorbid with Major Depressive Disorder (MDD), resulting in greater functional impairment. Gut microbiota have been shown to modulate brain chemistry and function, possibly also playing a role in the genesis of anxiety. Bacteria are also able to produce, or interact with the host metabolism of neuroactive substances, including classical neurotransmitters and trace amines, like octopamine, which although found in trace concentrations in the mammalian brain, can affect CNS function. Specifically, trace amines can affect catecholamine release, reuptake and biosynthesis, and modulate dopamine and serotonin metabolism. We investigated whether microbiota from patients with GAD with no signs of immune activation can alter behaviour in gnotobiotic mice and whether this is accompanied by changes in metabolites within the gastrointestinal tract. Germ-free NIH Swiss mice (n=35) were colonized with microbiota from either a GAD patient (n=18) with severe anxiety, comorbid depression, and low serum and fecal octopamine, or an age and sex-matched healthy control (HC) (n=17). Three weeks post- colonization, mouse behaviour was assessed by standard psychometric tests. Emotionality z-scores were calculated to provide a robust integrated behavioural assessment. Microbiota profiles were assessed by 16S rRNA based Illumina, fecal β-defensin-3 level was measured by ELISA. After sacrifice, mouse brain BDNF and GDNF expression was assessed by immunofluorescence, and gene expression in the hippocampus, amygdala, and olfactory bulbs was assessed by Nanostring. Stool and cecum metabolites were measured in all colonized mice by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). There were no differences in fecal β-defensin levels between mice colonized with GAD microbiota as compared to mice colonized with HC microbiota. However, GAD mice exhibited greater anxiety and depressive-like behavior compared to HC mice in the digging and tail suspensions tests. Behavioural z-scoring across all six standard psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice. Mice colonized with microbiota from a GAD patient had distinct bacterial profiles from mice colonized with HC microbiota. Compared to HC mice, GAD mice had lower levels of dopamine, octopamine and acetylcholine in cecum contents. Furthermore, GAD mice had higher expression of BDNF in the amygdala, lower expression of BDNF in the hippocampus, and lower expression of GDNF in the midbrain. GAD mice also had lower expression of CCR2 in the hippocampus, higher Cnlp/CAMP in the amygdala and olfactory bulb, and higher Nfkb1 in the olfactory bulb compared to HC mice. Our results suggest that microbiota from a selected patient with GAD has the ability to induce anxiety and depressive-like behavior, by mechanisms independent of immune system, likely by altered production of biogenic amines and neurotransmitters. / Thesis / Master of Science (MSc)