Rozprawy doktorskie na temat „Filamine C”

Des variants du gène FLNC ont été impliqués dans le développement de cardiomyopathies (CM), toutefois les mécanismes de physiopathologie ne sont pas entièrement élucidés. Ce travail a comporté la caractérisation génétique d’une cohorte clinique puis le développement d’un modèle cellulaire et d’un modèle Drosophile pour confirmer la pathogénicité de variants faux sens et d’évaluer les conséquences fonctionnelles des mutants FLNC. L’étude de 1150 patients atteints de CM a permis d’établir la prévalence du gène FLNC entre 1 et 8 %. De plus, les variants tronquants ont été associés préférentiellement à des CM dilatées (CMD) avec un risque rythmique accru et les variants faux sens (régionalisés dans le ROD2 de la protéine) à des CM hypertrophiques (CMH). Chez la drosophile, la perte de fonction de l’orthologue du gène FLNC (ARN interférence) a montré une dilatation cardiaque et une diminution de la fraction d’éjection chez l’adulte, associées à une désorganisation des sarcomères et du réseau d’actine. Les 3 variants faux sens sélectionnés, identifiés dans des CMH, et introduits par CRISPR/Cas9 dans des Drosophiles n’ont pas montré d’effet fonctionnel ou histologique significatif. L’étude de lignées produisant une protéine tronquée a permis d’établir que le domaine C-terminal de la filamine n’était pas requis pour la fonction cardiaque chez la Drosophile. Un clone hiPSC édité par CRISPR/Cas9 portant une délétion homozygote du site d’épissage de l’exon 42 qui aboutit à un saut en phase de l’exon a été différencié en cardiomyocytes. Les cardiomyocytes amplifiés ont permis de former des pseudo-tissus cardiaques battant dont l’analyse a montré une modification du phénotype rythmique qui correspondrait à un phénotype de CMD. En conclusion, ce travail a permis de montrer l’importance du gène FLNC dans le développement des CM et de développer 2 nouveaux modèles pour étudier les mécanismes de pathogénicité et développer des thérapeutiques ciblées
Variants of FLNC gene have been implicated in the development of cardiomyopathies (CM), however the pathophysiological mechanisms are not fully understood. This work involved the genetic characterization of a clinical cohort and then the development of a cell model and a Drosophila model to confirm the pathogenicity of missense variants and to assess the functional consequences of FLNC mutants. The study of 1150 patients with CM found the prevalence of FLNC gene to be between 1 and 8%. In addition, the truncating variants were preferentially associated with dilated CMs (DCM) with an increased rhythmic risk, and the missense variants (regionalized in the ROD2 of the protein) with hypertrophic CMs (MHC). In Drosophila, loss of function of the ortholog of FLNC gene (RNA interference) showed cardiac dilation and a decrease of the fractionnal shortening in adults, associated with disorganization of the sarcomeres and the actin network. The 3 selected missense variants, identified in MHCs, and introduced by CRISPR / Cas9 in Drosophila did not show any significant functional or histological effect. Study of lines producting a truncated protein revealed that the C-terminal domain of filamine was not required for heart function in Drosophila. A CRISPR / Cas9 edited hiPSC clone carrying a homozygous deletion of the exon 42 splice site that results in exon phase skiping was differentiated into cardiomyocytes. The amplified cardiomyocytes formed beating cardiac pseudo-tissues, and their analysis showed a change in rhythmic phenotype corresponding to DCM patients pattern. In conclusion, this work made it possible to show the importance of the FLNC gene in the development of CM and to develop 2 new complementary models to study the mechanisms of pathogenicity and develop targeted therapies

The serotonin receptor 2A (5-HT₂[A]) is a member of the G-protein coupled receptor family and is of interest due to its role in physiological functions such as smooth muscle contraction, platelet aggregation, thermoregulation, learning and memory. More importantly, 5-HT₂[A] has also been implicated in CNS disorders including schizophrenia, depression and anxiety. A yeast two-hybrid screen had previously been carried out to identify proteins that interacted with 5-HT₂[A] and therefore may modulate intracellular function. The cytoskeletal actin-binding protein filamin-C was identified as a possible 5-HT₂[A] interacting partner. The aim of the research in this thesis was to further investigate the potential interaction between 5-HT₂[A] and filamin-C and to investigate functional roles for the interaction. A fragment of human filamin-C, aa 2162-2725, was shown to interact with the C-terminus of human 5-HT₂[A] using two in vitro techniques, the yeast-two hybrid system and a GST capture assay. The region of filamin-C needed to bind to 5-HT₂[A] was narrowed to the start of repeat 20, aa 2251, through to aa 2424 at the beginning of repeat 22 and comprises 182 residues. The 5-HT₂[A] region needed to bind to filamin-C was ascertained via yeast two-hybrid to be 31 amino acids between 394-423. Work was performed to determine whether FLNC mRNA was expressed in neural and glial cells and whether FLNC and HTR2A mRNA were co-expressed in any cells. FLNC mRNA was identified in seven out of eight neural and glial cell lines and western blot analysis confirmed this finding at the protein level. Two cell lines, U-118MG and A172, were found to contain both HTR2A and FLNC mRNA. Co-immunoprecipitation experiments showed endogenous filamin-C bound to endogenous 5-HT₂[A] and this complex could be precipitated using anti-filamin-C antibody. Additionally, a GST-5-HT₂[A] fusion complex was found to bind to endogenous filamin-C from U-118MG cells. Immunofluorescent labelling of cells was used to study filamin-C and 5-HT₂[A] proteins in vivo. U-118MG cells showed staining for 5-HT₂[A] around the membrane of the cell, as well as in the cytoplasm, whereas filamin-C staining occurred in the cytoplasm. Co-localisation analysis identified some areas of overlap between 5-HT₂[A] and filamin-C in the cytoplasm of U-118MG cells. The functional role for the 5-HT₂[A]/filamin-C colocalisation was investigated. It was postulated that filamin-C may be involved in the internalisation of 5-HT₂[A]. To test this hypothesis, an in vivo model system was used to investigate whether disruption of the filamin-C/5-HT₂[A] interaction affects internalisation of the receptor. The key preliminary findings of this study, which used expression of a competitor peptide, to disrupt and co-interact, suggested that the filamin-C/5-HT₂[A] interaction is not essential for the internalisation of receptors in response to ligand binding. However, this interaction was important for delivery or maintenance of 5-HT₂[A] to the cell membrane, and expression of the competing peptide caused an accumulation of cytoplasmic 5-HT₂[A].

Background. Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart muscle function that is necessary for both normal contraction and for increased contractility in response to inotropic stimuli(1-7). Effects of cMyBP-C on contraction are due to dynamic interactions of cMyBP-C with both actin and myosin, but the mechanism(s) by which cMyBP-C binding to these ligands is modulated are only partly understood. Recently, calmodulin (CaM) was shown to bind to cMyBP-C in the regulatory M-domain(8, 9) near a conserved actin binding site(10). Here we investigated whether CaM competes with actin for binding to cMyBP-C and thus whether CaM affects cMyBP-C function. Methods. Recombinant N’-terminal domains of cMyBP-C were used in pull-down assays, co-sedimentation binding assays, and actin activated myosin ATPase assays to determine effects of CaM binding on cMyBP-C. Results. In accordance with previous reports, we found that CaM binds to N’-terminal domains of cMyBP-C in the presence of Ca2+ (Ca2+/CaM) with a binding affinity comparable to cMyBP-C binding to actin (3-10 μM). We further show that Ca2+/CaM reduces cMyBP-C apparent binding affinity for actin, consistent with the competition between Ca2+/CaM and actin for binding to cMyBP-C. Ca2+/CaM also reversed the inhibitory effects of cMyBP-C N’-terminal domains on actin activated myosin ATPase rates, consistent with reduced cMyBP-C interactions with actin. However, apo-CaM (calcium-free calmodulin) did not influence the ability of cMyBP-C to activate actomyosin ATPase rates at low Ca2+. Phosphorylation of cMyBP-C by PKA significantly increased its binding to both Ca2+/CaM and apo-CaM. Phosphorylated cMyBP-C was also a less potent regulator of cross-bridge cycling in ATPase assays. Conclusions. These data demonstrate that Ca2+/CaM competes with actin for binding to cMyBP-C and selectively reverses the inhibitory effects of cMyBP-C on actomyosin interactions. However, apo-CaM does not compete with cMyBP-C binding to actin and does not affect the ability of cMyBP-C to activate the thin filament at low Ca2+. Ca2+/CaM may serve as additional regulatory mechanism in addition to phosphorylation of cMyBP-C to regulate its function. These data suggest that Ca2+/CaM is a novel modulator of cMyBP-C function that can dynamically tune cMyBP-C effects on contraction potentially as [Ca2+]i rises and falls during the time course of a single heart beat.

Background: Therapy resistance is a great challenge in cancer treatment. Among numerous factors, cell adhesion to extracellular matrix is a well-known determinant of radiochemo-resistance. It has been shown that targeting focal adhesion proteins (FAPs), e.g. β1 integrin, enhances tumor cell radio(chemo)sensitivity in various entities such as head and neck squamous cell carcinoma (HNSCC), lung carcinoma, glioblastoma, breast carcinoma and leukemia. Previous studies demonstrated a functional crosstalk between specific FAPs and DNA repair processes; however, the molecular circuitry underlying this crosstalk remains largely unsolved. Hence, this study in HNSCC aimed to identify alternative FAPs associated with DNA damage repair mechanisms and radioresistance. Materials and Methods: A novel 3D High Throughput RNAi Screen (3DHT-RNAi-S) using laminin-rich extracellular matrix (lrECM) was established to determine radiation-induced re-sidual DNA double strand breaks (DSBs; foci assay) and clonogenic radiation survival. In the screen, we used UTSCC15 HNSSC cells stably expressing the DSB marker protein 53BP1 tagged to pEGFP. Validations were performed in 10 additional HNSCC cell lines (Cal33, FaDu, SAS, UTSCC5, UTSCC8, UTSCC14, UTSCC15, UTSCC45 and XF354fl2) grown in 3D lrECM. Immunofluorescence staining, immunoblotting, chromatin fractionation were utilized to evaluate protein expression, dynamics and kinetics post irradiation. Investigations of molecular mechanisms of DNA repair and radio(chemo)resistance employed DSB repair reporter assays for non-homologous end joining (NHEJ) and homologous recombination (HR), cell cycle analysis, chromatin fractionation levels evaluation and kinase activity profiling (PamGene) upon protein knockdown in combination with/-out X-ray exposure. Foci assay and clonogenic survival assay were performed after single or multiple knockdowns of synemin and associated proteins such as DNA-PKcs and c-Abl. Protein-protein interactions between synemin and associated proteins were determined using immunoprecipitation and proximity ligation assay. Mutant/depletion constructs of synemin (ΔLink-Tail, ΔHead-Link, Synemin_301-961, Synemin_962-1565, S1114A and S1159A) were generated in order to identify essential synemin’s sites controlling DNA repair functions. Results: Among the targets found in the 3DHT-RNAi-S, synemin was one of the most promising FAP candidates to determine HNSCC cell survival and DNA damage repair. Synemin silencing radiosensitized HNSCC cells, while its exogenous overexpression induced radio-protection. Radiation induced an increased synemin/chromatin interaction and a marked ac-cumulation of synemin in the perinuclear area. Intriguingly, synemin depletion elicited a 40% reduction in NHEJ activity without affecting HR or Alt-EJ. In line, ATM, DNA-PKcs and c-Abl phosphorylation as well as Ku70 expression strongly declined in synemin depleted and irra-diated cells relative to controls, whereas an opposite effect was observed under synemin overexpression. Single, double and triple depletion of synemin, DNA-PKcs and c-Abl resulted in a similar radiosensitizing effect and DSB levels as detected upon single knockdown of synemin, describing its upstream role. In kinome analysis, tyrosine kinases showed signifi-cantly reduced activity after synemin silencing relative to controls. Furthermore, immunoprecipitation assays revealed a protein complex formed between synemin, DNA-PKcs and c-Abl under pre- and post-irradiation conditions. This protein complex dispersed when ATM was pharmacologically inhibited, implying synemin function to be dependent on ATM kinase activity. By means of the different mutation/deletion constructs of synemin, the phosphorylation site at serine 1114 located on the distal portion of synemin’s tail was identified as essential protein-protein interaction site for synemin’s function in DNA repair. Conclusions: The established 3DHT-RNAi-S provides a robust screening platform for identifying novel targets involved in therapy resistance. Based on this screen and detailed mechanistic analyses, the intermediate filament synemin was discovered as a novel important determinant of DNA repair, tyrosine kinase activity and radiochemoresistance of HNSCC cells. These results further support the notion that DNA repair is controlled by cooperative interactions between nuclear and cytoplasmic proteins.

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase. Crown Copyright (C) 2016 Published by Elsevier Ltd. All rights reserved.

Ceramic additive manufacturing is gaining popularity with methods like selective laser sintering (SLS), binder jetting, direct ink writing and stereolithography, despite their disadvantages. Laser sintering and binder jetting are too expensive, while direct ink writing lacks resolution and stereolithography lacks scalability. The project aims to combine one of the most versatile, affordable, and readily available 3D printing methods: fused filament fabrication (FFF) with polymer derived ceramics to produce cellular ceramics to overcome the disadvantages posed by the other methods. The process uses a two-step approach. The first step is to 3D print the part using a polymer FFF 3D printer with a thermoplastic polyurethane filament and the second step is to impregnate the part in a polysilazane preceramic polymer and then pyrolyze it in an inert environment up to 1200C. The resulting product is a high-resolution cellular ceramic of the composition SiOC(N). This type of cellular ceramic can find an application in several fields such as scaffolds for bone tissue regeneration, liquid metal filtering, chemical and gas filtering, catalytic converters and electric applications. The process can provide an affordable alternative to the products used in these fields currently.

O complexo troponina-tropomiosina regula a contração muscular esquelética e cardíaca. A ligação do cálcio nos sítios regulatórios localizados no domínio N-terminal da troponina C (TnC) induz uma mudança conformacional que remove a ação inibitória da troponina I (TnI) e inicia a contração muscular. Nós usamos fragmentos recombinantes da TnI e uma série de mutantes da TnC para estudar as interações estruturais e regulatórias das diferentes regiões da TnI com os domínios da TnC, TnT e actina-tropomiosina. Nossos resultados indicam que a TnI é organizada em regiões que apresentam funções estruturais e regulatórias e que se ligam de modo antiparalelo com os correspondentes domínios estruturais e regulatórios da TnC. Estudos funcionais mostram que a região inibitória (aminoácidos 103-116) em combinação com a região C-terminal da TnI (TnI103-182) pode regular a atividade ATPásica da acto-miosina de maneira dependente de Ca2+. A regulação não é observada com a região inibitória em combinação com a região N-terminal (TnI116) Estudos de ligação mostram que a região N-terminal da TnI (TnI1-98) interage com o domínio C-terminal da TnC na presença e na ausência de Ca2+ e também interage com a TnT. A região inibitória/C-terminal da TnI (TnI103-182) interage com o domínio N-terminal da TnC de maneira dependente de Ca2+. Baseados nestes resultados, propomos um modelo para a mudança conformacional induzida pelo Ca2+. Neste modelo, a região N-terminal da TnI está ligada fortemente com o domínio C-terminal da TnC na presença ou na ausência de Ca2+. As regiões inibitórias e C-terminal da TnI ligam-se à actina-tropomiosina na ausência de Ca2+ e nos domínios N-terminal e C-terminal da TnC na presença de Ca2+.
The troponin-tropomyosin complex regulates skeletal and cardiac muscle contraction. Calcium binding to the regulatory sites in the N-terminal domain of troponin C (TnC). induces a conformational change which removes the inhibitory action of troponin I (TnI) and initiates muscular contraction. We used recombinant TnI fragments and a series of TnC mutants to study the structural and regulatory interactions between different TnI regions and the domains of TnC, TnT and actin-tropomyosin. Our results indicate that TnI is organized into regions with distinct structural and regulatory functions which bind, in an antiparallel manner, with the corresponding structural and regulatory domains of TnC. Functional studies show that a fragment containing the inhibitory and C-terminal regions of TnI (TnIl03-182) can regulate the actomyosin ATPase in a Ca2+- dependent manner. Regulation was not observed with a fragment containing the N-terminal and inhibitory regions (TnIl-116). Binding studies show that the N-terminal region of TnI (TnI1-98) interacts with the C-terminal domain of TnC in the presence of Ca2+ or Mg2+. The inhibitory/C-terminal region of TnI (TnI103-182) binds to the N-terminal domain of TnC in a Ca2+-dependent manner. Based on these results, we propose a model for the Ca2+ -induced conformational change. In this model the N-terminal region of TnI is bound strongly to the C-terminal domain of TnC in the presence or absence of Ca2+. The inhibitory and C-terminal regions of TnI bind to actin-tropomyosin in the absence of Ca2+ and to tne N- and C-terminal domains of TnC in the presence of Ca2+.

Three novel mutations (G159D, L29Q and E59D/D75Y) in cardiac troponin C (CTnC) associate their clinical outcomes with a given cardiomyopathy. Current paradigms propose that sarcomeric mutations associated with dilated cardiomyopathy (DCM) decrease the myofilament calcium sensitivity while those associated with hypertrophic (HCM) cardiomyopathy increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the calcium regulation of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. This required the development of new calculator programs to solve complex ionic equilibria to more accurately buffer and expand the free calcium range of our test solutions. In accordance with the DCM paradigms, our result show that G159D and E59D/D75Y CTnC decrease the myofilament calcium sensitivity and force generating capabilities which would likely increase the rate of muscle relaxation and weaken the contractile force of the myocardium. Alternatively, the lack of myofilament change from L29Q CTnC (associated with HCM) may explain why the only proband is seemingly unaffected. Notably, the changes in the calcium sensitivity of tension (in fibers) do not necessarily occur in the isolated CTnC and vice versa. These counter-intuitive findings are justified through a transition in calcium affinity occurring at the level of cardiac troponin (CTn) and higher, implying that the true effects of these mutations become apparent as the hierarchal level of the myofilament increases. Despite these limitations, the regulated thin filament (RTF) retains its role as the calcium regulatory unit and best indicates a mutation's ability to sensitize (+) or desensitize (-) the muscle to calcium. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse (+ or -) these aberrant changes in myofilament sensitivity. Therefore, we have developed an RTF mediated High Throughput Screening assay to identify compounds in libraries of molecules that can specifically modulate the calcium sensitivity of cardiac contraction. The knowledge gained from these studies will help us and others to uncover new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases.

Pour migrer efficacement dans différents tissus, les cellules doivent détecter et s'adapter aux variations des propriétés mécaniques de leur environnement. Dans ce processus d’adaptation, les adhérences focales (AF) renforcent leur lien avec la matrice extracellulaire et le cytosquelette d'actine. En réponse à la force, l'association de la taline et de la vinculine pourrait renforcer l'ancrage de l'actine aux AF en contrôlant l'assemblage de l'actine par un mécanisme inconnu. Des études antérieures ont montré que la vinculine contient un seul domaine de liaison à l'actine (ABD) qui lie les filaments d’actine, coiffe les extrémités barbées des filaments d’actine et nuclée des filaments d'actine. La taline contient également trois ABD mais leur capacité à réguler l'assemblage de l'actine n'était pas connue avant ce projet. L'objectif global de ce projet de thèse était de déterminer les mécanismes précis par lesquels le complexe taline-vinculine contrôle l'assemblage de l'actine en réponse à la force. Dans une première partie de ce projet de thèse, préalable à l'étude du complexe taline-vinculine, j'ai terminé la caractérisation de la taline. J'ai démontré que le domaine ABD1 de la taline bloque l'allongement des extrémités barbées des filaments d'actine observés en microscopie à fluorescence (TIRFM), alors que ABD2 et ABD3 n'affectent pas la dynamique de l'actine. Dans la deuxième partie de ce projet, j'ai déterminé l'activité du complexe taline-vinculine. Parce que la force est nécessaire pour déclencher l'association de la vinculine à la taline, et parce que les deux protéines sont auto-inhibées, il a jusqu'à présent été difficile de déterminer la capacité du complexe taline-vinculine à réguler la polymérisation de l'actine. Par conséquent, nous avons d'abord conçu des mutants de taline et de vinculine qui s'associent de manière constitutive en un complexe stable. En combinant des études cinétiques en spectroscopie de fluorescence, des tests de liaison à l’actine et l’observation de filaments d’acine uniques en microscopie TIRF, nous avons déterminé les activités de ces mutants et de leurs complexes sur la dynamique de l'actine. Notre étude a d'abord révélé que les trois activités de la vinculine sont contrôlées par des contacts auto-inhibiteurs spécifiques. Nous montrons également que des suppressions d'hélice le long de la taline exposent les sites voisins de liaison à la vinculine, imitant ainsi l'étirement mécanique de la taline. L'association des mutants de taline aux mutants de vinculine forme un complexe qui nuclée des filaments coiffés à leur extrémité barbée. La caractérisation d'une série de complexes, dans lesquels la vinculine et la taline sont amputés de divers ABD, révèle la contribution de chaque protéine dans ce mécanisme. En conclusion, nos données suggèrent un mécanisme pour le renforcement de l'ancrage de l'actine dans les AF en réponse à la force
To migrate efficiently in different tissues, cells must sense and adapt to variations of the mechanical properties of their environment. In this adaptive process, focal adhesions (FAs) strengthen their link with the extracellular matrix and the actin cytoskeleton. The force-dependent association of the actin binding proteins talin and vinculin could reinforce actin anchoring to FAs by controlling actin assembly though an unknown mechanism. Previous studies showed that vinculin contains a single actin-binding domain (ABD) which binds to actin filaments, caps actin filament barbed-ends and nucleates actin filaments. Talin also contains three ABDs but their ability to regulate actin assembly was not known before this project. The global objective of this PhD project was to determine the precise mechanisms by which the force-dependent talin-vinculin complex controls actin assembly. In a first part of this PhD project, as a prerequisite to the study of the talin-vinculin complex, I finished the characterization of talin. I demonstrated that the N-terminal ABD1 of talin blocks the elongation of actin filament barbed ends observed in fluorescent microscopy (TIRFM), whereas ABD2 and ABD3 do not affect actin dynamics. In the second and main part of this project, I determined the activity of the talin-vinculin complex. Because force is required to trigger vinculin association to talin, and because both proteins are autoinhibited, it has so far been difficult to determine the ability of the talin-vinculin complex to regulate actin polymerization. Therefore, we first designed talin and vinculin mutants that associate constitutively into a stable complex. By combining fluorescence spectroscopy, binding assays and single filament observation in TIRF microscopy, we determined the activities of these mutants and their complex on actin dynamics. Our study first revealed that the three activities of vinculin are controlled by specific auto-inhibitory contacts. We also show that helix deletions along the rod domain of talin expose neighboring vinculin-binding sites, mimicking the mechanical stretching of talin. The binding of these talin and vinculin mutants forms a complex that nucleates filaments capped at their barbed ends. The characterization of a series of complexes, in which vinculin and talin are deleted from various ABDs, reveals the contribution of each protein in this mechanism. Altogether our data suggest a mechanism for the force-dependent reinforcement of actin anchoring in FAs

碩士
國立東華大學
材料科學與工程研究所
87
The synthesis of metastable diamond via liquid metal process at low pressures combines some of the diamond growth mechanisms and methods of both HTHP(high temperature and high pressure) and low pressure CVD. It could be the third major method, which could produce diamond at large-scale and at low cost. The work is to study the metastable diamond synthesis using various powder mixtures as the starting material in pure H2 and 0.8﹪CH4 in H2 at low pressures, to understand the importance of temperature and reduction processes, to enhance the growth rate, and to understand the effect of solubility of carbon in metal. A hot filament chemical vapor deposition (HFCVD) reactor was used to excite and disengage the gases, and to heat starting powder mixtures. The powder mixtures included various combinations of graphite, diamond, Co, Ni and Mn metal powders. Samples were heated to the range of metal or alloy melting point, and heated for 5 hrs. The XRD patterns of samples with diamond seeds, Co and Ni metal powders processed in CH4/H2 showed an small increases in diamond peak intensities, and their corresponding SEM photos also indicated the growth of diamond seeds into single-crystals and poly-crystals. The size of these crystals are 2~3 times larger than grown on Si substrate under comparable CVD conditions. In this results, the synthesis rate of metastable diamond via liquid metal process still in the range of that via gas phase process. If we use graphite powder under 99.2 H2 and 0.8﹪CH4 , the synthesis rate showed decreases. In condition of metal/graphite/diamond seeds/pure H2 (liquid metal process), the solubility of C and H2 for synthesis rate increasing has positive affection.

碩士
國立清華大學
分子與細胞生物研究所
100
Mitochondria are essential for survival of eukaryotic cells since they are responsible for ATP synthesis, calcium buffering and apoptosis. Thus, specialized transport and anchoring mechanisms are necessary to distribute and position mitochondria in response to local needs for ATP and calcium buffering. Mitochondrial transport and positioning are particularly challenging in neurons because of the unique geometry and metabolic needs of neurons. In neurons, mitochondria are transported from the soma, where mitochondria biogenesis takes place, to ATP-demanding sites as synaptic terminals and growth cones. Mitochondria remain at these sites until they are transported back to the soma for recycling or degradation. This long-range bidirectional transport mainly depends on microtubules and microtubule-based motor proteins as kinesins and dynein, while short-range mitochondrial transport and docking mainly rely on actin and actin-based motors myosins. In contrast to actin and microtubules, intermediate filaments (IFs) do not possess polarity or associated motor proteins. Therefore, how IFs take part in mitochondrial positioning is relatively unclear. Previous studies suggest that IFs may serve as docking sites for mitochondria, or affect mitochondrial transport through microtubule-based motors. In this study, we investigate the functions of IFB-1, a C. elegans IF protein, in the mitochondrial transport system of C. elegans amphid neurons. Amphids are sensory organs consisting of a pair of sensilla running along the lateral sides of the worm head. We established wild-type and ifb-1 mutant worms carrying fluorescently-labeled mitochondria in the amphid neurons, and recorded time-lapse images from these worms and their isolated neuronal cells. We found that mutations of IFB-1 lead to larger pools of stationary mitochondria, suggesting a role of IFB-1 in the balance of stationary phases and motile phases of mitochondrial transport. In addition, loss of one isoform of IFB-1 reduces mitochondrial transport velocities and frequencies of changes of directions, but increases pausing frequencies as well as moving persistencies. To explain these complex effects of IFB-1 on mitochondrial motility, we propose a model in which IFB-1 influences mitochondrial transport through affecting kinesin or kinesin-associated proteins.