A series of Fe2+ spin crossover (SCO) complexes [Fe(5/6)]2+ employing hexadentate ligands (5/6) with cis/trans-1,2-diamino cyclohexanes (4) as central building blocks were synthesised. The ligands were obtained by reductive amination of 4 with 2,2′-bipyridyl-6-carbaldehyde or 1,10-phenanthroline-2-carbaldehyde 3. The chelating effect and the rigid structure of the ligands 5/6 lead to exceptionally robust Fe2+ and Zn2+ complexes conserving their structure even in coordinating solvents like dmso at high temperatures. Their solution behavior was investigated using variable temperature (VT) 1H NMR spectroscopy and VT Vis spectroscopy. SCO behavior was found for all Fe2+ complexes in this series centred around and far above room temperature. For the first time we have demonstrated that the thermodynamics as well as kinetics for SCO can be deduced by using VT 1H NMR spectroscopy. An alternative scheme using a linear correction term C1 to model chemical shifts for Fe2+ SCO complexes is presented. The rate constant for the SCO of [Fe(rac-trans-5)]2+ obtained by VT 1H NMR was validated by Laser Flash Photolysis (LFP), with excellent agreement (1/(kHL + kLH) = 33.7/35.8 ns for NMR/LFP). The solvent dependence of the transition temperature T1/2 and the solvatochromism of complex [Fe(rac-trans-5)]2+ were ascribed to hydrogen bond formation of the secondary amine to the solvent. Enantiomerically pure complexes can be prepared starting with R,R- or S,S-1,2-diaminocyclohexane (R,R-trans-4 or S,S-trans-4). The high robustness of the complexes reduces a possible ligand scrambling and allows preparation of quasiracemic crystals of [Zn(R,R-5)][Fe(S,S-5)](ClO4)4·(CH3CN) composed of a 1 : 1 mixture of the Zn and Fe complexes with inverse chirality. / Dieser Beitrag ist aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Ο ρόλος του χαλκού είναι πολύ σημαντικός για τη σωστή λειτουργία της κυτοχρωμικής c οξειδάσης (CcO), και συνεπώς για την κυτταρική αναπνοή στους ευκαρυωτικούς και προκαρυωτικούς οργανισμούς. Η συγκρότηση της CcO στον ενδομεμβρανικό μιτοχονδριακό χώρο είναι μια πολύπλοκη διαδικασία, εξαρτώμενη από πλήθος συνεργών πρωτεϊνών, υπεύθυνων για τη λειτουργική αναδίπλωση των υπομονάδων του ενζύμου και τη μεταφορά αίμης και ιόντων Cu σε αυτές. Ενώ οι πρωτεΐνες που ενέχονται στη διαδικασία είναι μάλλον γνωστές, οι μηχανισμοί μεταφοράς και ενσωμάτωσης των μεταλλικών ιόντων στα δυο ενεργά κέντρα της CcO, CuA και CuB, παραμένουν ανεξερεύνητοι. Το CuA κέντρο είναι ένα διπυρηνικό κέντρο χαλκού, του οποίου ο ρόλος εντοπίζεται στη μεταφορά e- από το κυτόχρωμα c στο καταλυτικό κέντρο CuB της CcO. Η σωστή συγκρότηση του CuA κέντρου είναι κρίσιμης σημασίας για την καταλυτική δράση του ενζύμου. Αρκετές πρωτεΐνες έχουν χαρακτηριστεί σαν ενεργοί παράγοντες στη μεταφορά ιόντων Cu στο CuA κέντρο, ο ακριβής, ωστόσο, μοριακός μηχανισμός και ρόλος της κάθε πρωτεΐνης είναι άγνωστος. Στους προκαρυωτικούς οργανισμούς, δυο οικογένειες πρωτεϊνών έχουν προταθεί για την εμπλοκή τους στη συγκρότηση του CuA. Η πρώτη περιλαμβάνει πρωτεΐνες που δεσμεύουν ιόντα Cu1+ με ένα συντηρημένο μοτίβο δέσμευσης H(M)x10Mx21HxM (υποθετικές πρωτεΐνες Hyp1) ενώ η δεύτερη περιλαμβάνει τις πρωτεΐνες της οικογένειας Sco, των οποίων ο ρόλος στον μηχανισμό του CuA κέντρου σαν θειορεδοξίνες ή χαλκομεταφορείς, παραμένει ασαφής. Στην παρούσα εργασία αποδείχθηκε ότι μια νέα περιπλασματική πρωτεΐνη (TtHyp1 ή PCuAC) εισάγει επιλεκτικά ιόντα Cu1+ στην Cox2 υπομονάδα της ba3-CcO του Thermus thermophilus προς σχηματισμό του φυσιολογικού διπυρηνικού TtCuA κέντρου, καθώς και ότι η Sco πρωτεΐνη του συγκεκριμένου οργανισμού (TtSco1) δεν μεταφέρει μεταλλικά ιόντα στο CuA, αλλά δρα σαν θειο-δισουλφιδική αναγωγάση ρυθμίζοντας τη σωστή οξειδωτική κατάσταση των κυστεϊνικών καταλοίπων του CuA. Οι πρωτεΐνες PCuAC, TtSco1 και TtCuA εκφράστηκαν, απομονώθηκαν και μελετήθηκαν τα βιοχημικά χαρακτηριστικά τους, η ικανότητα δέσμευσης μεταλλικών ιόντων και οι μεταξύ τους αλληλεπιδράσεις. Επιπλέον η PCuAC χαρακτηρίστηκε δομικά με φασματοσκοπία NMR στην απο και Cu(I) μορφή της. Ο ρόλος των προκαρυωτικών Sco διερευνήθηκε περαιτέρω με μελέτες γονιδιακής ανάλυσης και την έκφραση και τον προκαταρκτικό χαρακτηρισμό μιας νέας πρωτεΐνης, PpSco1/cytc της Pseudomonas putida, πρωτεΐνης αποτελούμενης από δυο επικράτειες, Sco1 και cytc, συνδέοντας το ρόλο των Sco πρωτεϊνών με τη θεωρια περί θειρεδοξινικής τους δράσης. Καινοτομία στην παρούσα εργασία αποτέλεσε η μεθοδολογική προσέγγιση της πολλαπλής κλωνοποίησης των γονιδίων-στόχων με μια νέα τεχνολογία κλωνοποίησης (Gateway) συνδυασμένης με τοποειδική ένθεση σε πολλαπλούς πλασμιδιακούς φορείς και η ανάπτυξη high throughput τεχνικών για πολλαπλές δοκιμές έκφρασης – απομόνωσης. Η συγκεκριμένη μελέτη παρέχει νέα δεδομένα για το μηχανισμό τη συγκρότησης του CuA κέντρου της προκαρυωτικής CcO, υποστηρίζοντας ένα νέο μοντέλο για τη συγκεκριμένη διαδικασία και παράλληλα συνδράμει στην αποκρυπτογράφηση του πολύπλοκου ρόλου των Sco πρωτεϊνών. / Copper is essential for the correct assembly and function of the cytochrome c oxidase (CcO), thus for the efficient cellular respiration in both eukaryotes and prokaryotes. CcO assembly in the inner mitochondrial membrane space is a multi complicated procedure, depended on a number of co-factors and their synergic action. These co-factors are proteins commissioned with the correct folding of the enzyme subunits and the transport/incorporation of heme moieties and Cu ions to them. While the proteins involved in this multistep procedure are rather known, the mechanisms of metal ion delivery and incorporation within the two active centers of CcO, CuA and CuB, still remain uncharted. The CuA center is a binuclear copper center, whose part in the respiratory chain is spoted in electron transport from the active cytochrome c to the catalytic CuB center of CcO. Efficient CuA assembly is crucial for the catalytic action of the entire enzyme. Several proteins have been characterized as essential factors for the transport of Cu ions to the CuA center; however their exact molecular mechanism of action still remains obscure. In prokaryotes, two protein families have been suggested to be involved in the CuA assembly. The first includes proteins that bind Cu1+ ions through a potential conserved motif H(M)x10Mx21HxM (hypothetical proteins, Hyp1), while the second includes proteins of the Sco family, whose exact role in CuA assembly as thioredoxins or copper chaperones is widely debated. In this work, it is propesed that a new periplasmic protein (TtHyp1 or PCuAC) selectively inserts Cu1+ ions in the Cox2 subunit of the ba3-CcO of Thermus thermophilus resulting the formation of the physiological binuclear TtCuA center, as well as that the Sco protein of the organism (TtSco1) is not able to transfer metal ions to the CuA center; instead it acts rather like a thio-disulfide reductase adjusting the proper redox state of the CuA cysteine residues. Proteins PCuAC, TtSco1 and TtCuA were over-expressed, purified and subjected to biochemical characterization, while their Cu binding capability and their inter se interactions were studied through NMR and UV spectroscopy. In addition, PCuAC was structurally characterized through NMR in its apo and Cu(I) form. The role of Sco proteins was further investigated through genome based analysis and the expression and biochemical characterization of a new protein, PpSco1/cytc from Pseudomonas putida, a unique bacterial protein consisted on two domains, a Sco1 and a cytc domain, presumptively connecting the role of Sco proteins with the suggested theory of thioredoxin action. A novelty in this work was the methodological aspect of the multiple cloning of the target genes with a new cloning technology (Gateway) combined with site specific recombination into multiple expression plasmid vectors and the development of a high throughput technique for parallel expression/purification tests. The infra work provides new insights to the CuA center assembly molecular mechanism of the prokaryotic CcO, supporting a new model for the particular procedure and also subscripts for the decipherment of the complicated role of Sco proteins.

The Sco proteins, present in all kind of organisms, are regarded as one of the key players in the cytochrome c oxidase (COX) assembly. However, experimental and structural data, such as the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. Our current studies in S. cerevisiae have strongly suggested an antioxidant role to Sco2 protein (ySco2p). While the single deletion of SCO2 does not result in a distinctive phenotype, the concomitant deletion of superoxide dismutase 1 (SOD1) leads to an increased sensitivity to oxidative stress generating agents (paraquat, menadione, plumbagin) compared to the respective single mutants. Since S. cerevisiae is a good model to functionally characterize genes from more complex organisms, identification of such a phenotype has paved the way to test whether the Sco2 homologues from other organisms are able to substitute for the function of ySco2p. The Sco homologues from Homo sapiens, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster and Kluyveromyces lactis were integrated into the genome of the double deletion mutant. The functional complementation was tested by both growth and biochemical ROS assays. All homologues except for K. lactis K07152 and A. thaliana HCC1 were able to complement the phenotype, indicating their role in antioxidant defense. Interestingly, pathogenic human SCO2 point mutations failed to restore this function. The observation of non-functional homologues despite of the high sequence similarity to ySco2p strengthened our hypothesis on the importance of conserved aminoacid(s) for the defensive role. For this purpose, selected homologues were aligned and the conservation was judged not only based on identity but also similarity (e.g. charge, hydrophobicity). Interestingly, alignment results have pointed out an aminoacid site (located 15 aminoacids downstream of CxxxC motif) that a positively charged lysine is found only in the non-functional homologues. Subsequent mutagenesis analyses verified the functional importance of this aminoacid site (gain and loss of functions) and revealed the detrimental effect of positive charge on antioxidant function. In order to explain the observed functional change, further effort will be put into the calculations of the electrostatic potential and identifications of protein-protein interactions.:Contents List of figures x List of tables xii Abbreviations xiii 1 Introduction 1 1.1 ROS production 1 1.2 Oxidative stress 2 1.3 Antioxidant response 3 1.4 The thioredoxin fold: From structure to function 6 1.5 Sco proteins 7 1.5.1 Structural similarity of Sco proteins to antioxidant enzymes 8 1.5.2 Current knowledge about Sco proteins of S. cerevisiae 9 1.6 Background studies 10 1.7 Using yeast as a model 11 1.7.1 Cross-species complementation studies 11 1.7.2 Yeast model for human mitochondria studies 12 1.8 Aim of the study 12 2 Materials & Methods 14 2.1 Materials 14 2.1.1 Chemicals and Reagents 14 2.1.2 Equipments 16 2.1.3 Kits 17 2.1.4 Antibodies 18 2.1.5 Plasmid 18 2.1.6 Primers 19 2.1.7 S. cerevisiae strains 22 2.1.8 Media 22 2.2 Methods 24 2.2.1 Cultivation of S. cerevisiae cells 24 2.2.1.1 Culture conditions 24 2.2.1.2 Preparation of glycerol stocks 24 2.2.2 Molecular Biology Methods 24 2.2.2.1 S. cerevisiae genomic DNA isolation 24 2.2.2.2 RNA isolation 25 2.2.2.2a Cultured mammalian cells (HEK293) 25 2.2.2.2b Drosophila melanogaster 25 2.2.2.3 RNA purity and concentration determination 25 2.2.2.4 Reverse transcription 25 2.2.2.5 Polymerase chain reaction 25 2.2.2.5a Standard PCR 25 2.2.2.5b Overhang PCR 26 2.2.2.5c Overlap extension PCR 27 2.2.2.5d Site-directed mutagenesis by overlap extension PCR 27 2.2.2.6 DNA agarose gel electrophoresis 28 2.2.2.7 DNA gel extraction and clean-up 29 2.2.2.8 DNA sequencing 29 2.2.2.9 Southern blotting 29 2.2.2.9a DNA preparation 29 2.2.2.9b Blotting 30 2.2.2.9c Preparation of a DIG-labelled probe 30 2.2.2.9d Hybridization of the DIG-labelled probe to DNA 30 2.2.2.9e Detection of hybridized DIG-labelled URA3 probe 31 2.2.2.10 Yeast transformation 32 2.2.2.11 Growth assay 32 2.2.3 Protein methods 33 2.2.3.1 Isolation of crude mitochondria from yeast 33 2.2.3.2 SDS-PAGE 33 2.2.3.3 Protein transfer 34 2.2.3.4 Colloidal Coomassie gel staining 34 2.2.3.5 Protein detection 35 2.2.3.6 Stripping the membrane and reprobing 35 2.2.4 Biochemical methods 35 2.2.4.1 Methylene Blue staining 36 2.2.4.2 Quantification of ROS 36 2.2.4.2a Amplex Red staining 36 2.2.4.2b Lipid peroxidation assay 36 2.2.5 Bioinformatics 37 2.2.6 Statistical Analysis 37 3 Results 40 3.1 Selection of homologues by bioinformatic analysis 40 3.2 Generation of recombinant strains 42 3.3 Confirmation of site-specific integration by check PCR 44 3.4 Verification of single site integration by Southern Blotting 44 3.5 Analysis of the functional homology between selected homologues and ySCO2 45 3.5.1 Complementation assay in solid media 45 3.5.2 Complementation assay in liquid media 47 3.6 Determination of cell viability 48 3.7 Quantification of ROS 51 3.7.1 Quantification of extracellular H2O2 51 3.7.2 Quantification of lipid peroxidation 53 3.8 Investigation of the expression and subcellular localization of homologues 55 3.9 Investigation of the impact of pathogenic hSCO2 mutations on its antioxidant role 58 3.10 Mutational analysis of ySCO2 60 3.11 Identification of functionally important residues 61 3.12 Prediction of salt bridges 65 3.13 Alanine mutagenesis 66 4 Discussion 68 4.1 Functional homology between the selected homologues and ySCO2 68 4.1.1 A. thaliana homologues, HCC1 & HCC2 68 4.1.2 H. sapiens homologues, hSCO1 & hSCO2 69 4.1.3 D. melanogaster homologue, SCOX 70 4.1.4 Yeast homologues, K07152 & SpSCO1 70 4.2 The localization and expression pattern of homologues 71 4.3 The impact of pathogenic hSCO2 mutations on its antioxidant role 72 4.4 Mutational analysis of ySCO2 73 4.5 Attempts to understand the underlying reason(s) behind charge-related functional change 74 4.6 Potential mechanisms associated with the antioxidant action of ySco2p 78 5 Summary 81 6 References 84

Le travail présenté dans ce manuscrit concerne la conception et les études magnéto-structurales de nouveaux systèmes de Fe(II) à transition de spin (TS), en particulier ceux faisant intervenir les tétraazamacrocycles fonctionnalisés et les ligands de type 4-R-1,2,4-triazole. L’objectif principal est l’étude de l’effet de substitution ou de solvants (ou mélange de solvants) sur le comportement élastique de ces matériaux originaux. Dans un premier temps, nous avons étudié l’effet de solvant et contre-ions sur les propriétés de commutation, dans une série des complexes [Fe(L2)](X).nH2O (L2 = 1,8-bis(2’-pyridylméthyl)-1,4,8,11-tétraaza-cyclotétradécane, X = 2(tcm)- = 2[C(CN)3]-, n = 2 (1) ; X = [Ni(CN)4]2-, n = 1 (2) ; X = (BF4)-, n = 1 (3)). Cette étude a clairement élucidé l’effet crucial des interactions hydrogène, à travers les contre-ions et les molécules de solvants, sur la coopérativité observée dans la série des trois sels. Dans un second temps, l’utilisation des ligands de type 4-R-1,2,4-triazole nous a permis l’étude de deux systèmes commutables : (i) le premier concerne un complexe trinucléaire de formule [Fe3(furmetrz)6(tcnsme)6] (furmetrz = 4-(furan-2-ylmethyl)-1,2,4-triazole ; (tcnsme)- = 1,1,3,3-tétracyano-2- thiométhylpropenure), pour lequel les études magnétiques ont mis en évidence une transition thermique en deux étapes et un effet LIESST avec une T(LIESST) de 58 K. La comparaison de ce dérivé à d’autres systèmes similaires, mais présentant une transition de spin en une seule étape, révèle que la présence de deux étapes est favorisée par de fortes interactions le long de l’axe du trimère ; (ii) le second système concerne l’étude de plusieurs séries de matériaux polyfonctionnels exhibant la transition de spin et la photoluminescence. Le résultat le plus marquant concerne la synthèse et l’étude du complexe mononucléaire [Fe(naphtrz)6](tcnsme)2.4CH3CN (naphtrz = N-(1,2,4-triazol-4-yl)-1,8-naphthalimide) pour lequel les études magnétiques et optiques ont montré l’existence des deux propriétés, d’une manière synergétique. Ce résultat prouve la possibilité de contrôler, sans ambiguïté, les propriétés optiques par des excitations extérieures telles que la température, la pression, la lumière, … Au-delà de l’aspect fondamental, ce travail ouvre aussi des perspectives très originales pour l’utilisation de ces matériaux comme capteurs ou étalons de température. / This work is dedicated to the design and magnetostructural studies of new Fe(II) spin crossover (SCO) systems, in particular those involving the functionalized macrocycle and the 4-R-1,2,4-triazole triazole ligands.The principal objective concerns the study of the substitution and solvent effects on the SCO characteristics. In the first step, we have studied the solvent and the counter-ion effects, on the SCO behavior, in the series [Fe(L2)](X).nH2O (L2 = 1,8-bis(2’-pyridylméthyl)-1,4,8,11-tétraaza-cyclotétradécane, X = 2(tcm)- = 2[C(CN)3]-, n = 2 (1) ; X = [Ni(CN)4]2-, n = 1 (2) ; X = (BF4)-, n = 1 (3)), based on the macrocycles ligands. We have clearly shown in this study that the strong cooperative effects are mediated by the water solvent molecules and the counter-ions involved in the hydrogen bonding in the crystal packing. In the second step, the use of the 4-R-1,2,4-triazole fuctionalized triazole ligands led us to two original switchable systems: (i) the first one concerns the trinnuclear complex of formulae Fe3(furmetrz)6(tcnsme)6] (furmetrz = 4-(furan-2-ylmethyl)-1,2,4-triazole; (tcnsme)- = 1,1,3,3-tetracyano-2- thiométhylpropenide), for which the magnetic study reveals a two-step SCO transition and a LIESST effects with a T(LIESST) of 58 K. Examination of the intermolecular interactions in this complex and other parent trinuclear systems exhibiting complete one-step spin transition, revealed that the presence of the two-step behavior is clearly favored by strong inter-trimer interactions along the trimer axis; (ii) the second system the design and syntheses of to several series of polyfunctional materials exhibiting SCO and luminescent behaviors. In this new way, the most significant result concerns an original discrete Fe(II) complex of formulae [Fe(naphtrz)6](tcnsme)2.4CH3CN (naphtrz = N-(1,2,4-triazol-4-yl)-1,8-naphthalimide), for which the magnetic and photoluminescent studies showed that this material exhibits synergetic SCO and luminescent behaviors. This proves the possibility to control the luminescent properties through simple external perturbations such as temperature, pressure, light irradiation,… In addition, beyond the fundamental aspect, this work open new innovative perspectives for the use of such original materials as sensors or standards of temperature.

No description available.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

「上海合作組織」是第一個以中國城市為名的永久性政府間組織，以中文及俄文為正式語言，成員國包括中國、俄羅斯及地處中亞的哈薩克、吉爾吉斯、塔吉克、烏茲別克。而「上海合作組織」的前身是建立於1996年的「上海五國」機制，2001年中，烏茲別克加入「上海五國」機制，同年6月15日，六國元首共同發表「上海合作組織成立宣言」，宣佈在「上海五國」機制基礎上成立「上海合作組織」，當時中共藉著這個機制與俄羅斯及中亞四國開始展開邊境地區信任和裁軍的談判，而這也是「上海合作組織」軍事合作的開端。 本論文以「上海合作組織」軍事合作為研究主題，首先主要探討的目的為從「上海五國」到「上海合作組織」，其軍事合作形成的背景有那些主要因素、其演進的歷程及其內容，其次「上海合作組織」軍事合作的主要內容有那些，接著探討「上海合作組織」軍事合作發展至今，不論外部或內部有那些限制因素影響軍事合作，最後逐一分析「上海合作組織」的軍事合作對全球、區域及台海兩岸的安全情勢未來的發展。 研究發現隨著「上海合作組織」的成立，中共在中亞地區的影響力逐漸加大，中共在中亞日益提昇的力量也形成對俄羅斯的另一種挑戰。2007年6月27日「上海合作組織」六個成員國簽署「上海合作組織成員國關於舉行聯合軍事演習的協定」，使聯合軍演朝向定期化、常態化與制度的趨勢發展，而「上海合作組織」軍事合作發展至今，雖然有許多內外的限制因素，但是其範圍及影響層面卻日漸擴大，對全球、亞太地區或台海兩岸的安全情勢都產生重大的影響，不論兩岸和平談判如何發展，我們都要密切注意「上海合作組織」軍事合作對我軍事、外交等層面所造成的威脅。 / The Shanghai Cooperation Organization (SCO) is the first permanent inter-government organization named by one of the Chinese cities. Its official language is Chinese and Russian, and its members include China, Russian Federation, Republic of Kazakhstan, Republic of Kyrgyzstan, Republic of Tajikistan and Republic of Uzbekistan. "Shanghai Five" mechanism, established in 1996, was the forerunner of SCO. In 2001, Republic of Uzbekistan joined the "Shanghai Five" mechanism, and in the same year of June 15, the leaders of the six countries announced a joint statement－"the founding declaration of SCO", which declared SCO was established on the basis of the "Shanghai Five" mechanism . By using this mechanism, China started the negotiation, which is about a mutual trust of boarding area and disarmament issues with Russia and four center Asia countries, and this mechanism started the military cooperation in SCO. This thesis mainly focuses on the military cooperation of SCO. First, it will be discussed that the purpose of the organization from the "Shanghai Five" to SCO, including what are the primary factors of forming the military cooperation and its courses of evolution and contents. Second, we talk about what are the main contents of the military cooperation, and the SCO’s external or internal limitation which may influence the military cooperation. Finally, we analyze its future development of security situation in global, regional, and the two sides of the Taiwan Strait areas step by step. The research discovered, with the foundation of SCO, that the China’s influence in center Asia is increasing, which will form another challenge to Russia. On June 27, 2007, the six members of SCO signed “an agreement of holding joint military maneuvers among SCO members” to have them held periodically, normally, and systematically. From now, although there are a lot of limitations, the influence of the military cooperation is increasing and it has played a key role in global, Asia Pacific and the two sides of the Taiwan Strait areas. No matter how the peace negotiation between the two sides of the Taiwan Strait areas will go, we must keep a close eye on the fact that the military cooperation could cause Taiwan’s military and diplomacy to be under threat.

Central Asia has been the focal point of intense geopolitical power struggle throughout history. At the dawn of the 21st century, Central Asia has undergone major changes as the European Union and the China-led Shanghai Co-operation Organization have emerged as two normative powers, both seeking to influence the patterns of security governance in the region. This study aims to delve deep into ‘the black boxes’ of the EU’s and China’s foreign policies toward five CA republics. It starts from the premise that the bulk of research on Eurasian politics tend to concentrate mostly on realist and traditional geopolitical doctrine, which seem to have failed to properly explain the normative and ideational transformations that have taken place in the region as a result of the presence of these two emerging normative agents. By interweaving both realist and constructivist theories of International Relations (IR) into a new all-encompassing analytical framework, termed “meta-geopolitics”, the thesis seeks to trace and examine how geopolitical as well as normative components of the EU and Chinese regional strategies have affected the contemporary power dynamics in the post-Soviet space. I argue that, in contrast to the geopolitical struggle during the 19th and 20th centuries, a clash of normative powers is brewing in the region between China, under the aegis of the SCO, and the EU. The research also concludes that China has relatively been in a better position in comparison to the EU to render its policies as feasible, effective and legitimate to the Central Asian states.