Thèses sur le sujet « PRX6 »

L’osteoartrite (OA) è una delle malattie reumatiche con più alta incidenza nel mondo moderno e rappresenta la pricipale cause di disabilità. L’OA è data da un disquilibrio tra degradazione e riparazione della cartilagine, a favore della degradazione, con un incremento dell’attivita degli enzimi catabolici come le matrici metalloproteinasiche. I condrociti sono responsabili della riparazione e della biosintesi degli elementi che compongono la ECM. Diversi studi supportano l’ipotesi che il diabete è uno dei fattori che causano l’osteoartrite. Sebbene non si conoscono ancor ai meccanismi molecolari che permettano l’associazione tra diabete e OA. Abbiano analizzato la crescita cellulare, i livelli dei ROS e l’apoptosi di condrociti posti in terreno con differenti concentrazioni di glucosio. I risultati mostrano che i condrociti preferiscono la concentrazione 2.5 mM di glucosio che è stata utilizzata come concentrazione normoglicemica; mentre 25 mM di glucosio è stata utilizzata come concentrazione iperglicemica. I livelli di ROS e la morte cellulare aumentano in condrociti cresciuti in alto glucosio, anche il citoscheletro si presenta disorganizzato in cellule C28/I2 cresciute in queste condizioni. Questo correla con lo stato di attivazione della GTPasi RalA, la cui attivazione è ridotta in condizioni iperglicemiche. Infatti questa GTPasi è coinvolta nella regolazione della riorganizzazione citoscheletrica. Inoltre sono stati effettuati esperimenti utilizzando medium contenenti ITS (Insulina, transferrina e selenio) che pruomuove il differenziamento a condrocita; mentre l’interleuchina-1β per simulare l’ambiente osteoartritico. Anche in uesto caso i livelli di RalA GTP diminuiscono in cellule cresciute in 25 mM glucosio e stimolate con IL-1β. Inoltre in queste condizioni diminuiscono ache i livelli di p-ERK1/2. Sono stati valutati anche i livelli di NF-κB, iNOS e LC3II. I risultati dimostrano che l’alto glucosio blocca l’autofagia nei condrociti. Inoltre sono stati valutati anche i pathways attivati dall’alto glucosio in condrociti primari umani dopo 24 h di trattamento. I risultati mostrano che la fosforilazione di ERK1/2, p38, Akt e p65 è alterata in condrociti cresciuti in condizioni iperglicemiche tale evento è correlato con un aumento di MMP-13. Per analizzare meglio il ruolo dei ROS nei condrociti ho lavorato per 6 mesi nel laboratorio del Dr. Loeser alla scuola di medicina presso l’università della north Carolina a Chapel Hill. Ho lavorato su PRX6 che è coinvolto nella detossificazione dai ROS. Lo stato di ossidazione di PRX6 è stato valutato nei condrociti trattati con diversi stimoli come H2O2, Fn-f, menadione (men) and DMNQ. Dopo questi esperimenti abbiamo voluto osservare se PRX6 potesse influenzare i pathway delle MAPK in cellule trattate con IGF-1, menadione, combinazione tra IGF-1 e menadione e con Fn-f. è stata valutata la localizzazione di PRX6 che si è visto essere sia nucleare che citoplasmatico. Inoltre ho lavorato in collaborazione con Prof. Laura Cipolla e Prof. Maddalena Collini per sviluppare e caratterizzare nuovi idrogel fatti di gelatina usando come agente cross-linkante lo squarato.
Osteoarthritis (OA) is the most common rheumatic disease in the world and represents the first cause of disability in the world. OA results from the loss of balance between degradation and repair inside cartilage, in favor of degradation, with increased activity of catabolic enzymes such as matrix metalloproteinases and decreased production of ECM proteins. Chondrocytes are responsible for the repair and biosynthesis of elements of the extracellular matrix. Experimental findings support the hypothesis that diabetes is an independent risk factors for OA. However, correct molecular mechanisms underlying the diabetes-associated OA phenotype is still largely unknown. Firstly chondrocytes cell growth, ROS levels and apoptosis were analyzed using different glucose concentration. Results shown that chondrocytes prefer 2.5 mM of glucose which was used as normal glucose concentration and 25 mM of glucose was used as high glucose concentration. ROS levels and cell death increase in chondrocytes growth in high glucose environment. Also cytoskeletal network is more disorganized in C28/I2 cells growth at high glucose concentration, this correlates with different RalA-GTP levels which is involved in the regulation of cytoskeletal organization. Experiments were performed even using medium supplemented by ITS (Insulin-transferrin-selenium) to promote chondrocyte differentiation and IL-1β was used to simulate osteoarthritic cartilage environment. Ral A-GTP levels are lower in cells grown in 25 mM of glucose and stimulated with IL1β. Levels of p-ERK1/2 decrease in cells grown at high glucose concentration and in cells stimulated with IL1β. Furthermore, NF-κB, iNOS and LC3II levels were evaluated. Results demonstrate that high glucose media block autophagic process in chondrocytes. Effect of glucose concentration on human primary chondrocytes cells was evaluated after only 24 h to understand which signaling pathways is activated by high glucose environment. Phosphorylation of ERK1/2, p38, Akt and p65 is altered in chondrocytes growth at high glucose concentration and this correlates with an increase secretion of MMP-13. To better analyze the role of ROS levels in the chondrocytes I worked for 6 months in the Dr. Loeser Lab at the School of Medicine in the University of North Carolina at Chapel Hill; one of the best lab in the cartilage biology field. I worked on PRX6 which is involved in the recovery from H2O2. Oxidation state of PRX6 was evaluated in chondrocytes treated with different stimuli, like H2O2, Fn-f, menadione (men) and DMNQ. After this experiment, we wanted to see if PRX6 could impact the MAPK signaling pathways in cells treated with IGF-1, menadione, combination of menadione and IGF-1 and with Fn-f. Localization of PRX6 was analyzed using nuclear and cytoplasm extraction. Then I worked in collaboration with Prof. Laura Cipolla and Prof. Maddalena Collini to develop and characterized a new gelatin-based hydrogel using Diethylsquarate as crosslinker.

Lung cancer is a leading cause of cancer-related mortality irrespective of gender. The Sulfiredoxin (Srx) and Peroxiredoxin (Prx) are a group of thiol-based antioxidant proteins that plays an essential role in non-small cell lung cancer. Understanding the molecular characteristics of the Srx-Prx interaction may help design the strategies for future development of therapeutic tools. Based on existing literature and preliminary data from our lab, we hypothesized that the Srx plays a critical role in lung carcinogenesis and targeting the Srx-Prx axis or Srx alone may facilitate future development of targeted therapeutics for prevention and treatment of lung cancer. First, we demonstrated the oncogenic role of Srx in urethane-induced lung carcinogenesis in genetically modified FVB mice. The Srx-null mice showed resistance to urethane-induced lung cancer. Second, we demonstrated the Srx and Prx sites important for Srx-Prx interaction. The orientation of this arm is demonstrated to cause some steric hindrance for the Srx-Prx interaction as it substantially reduces the rate of association between Srx and Prx. Finally, we carried out virtual screening to identify molecules that can successfully target Srx-Prx interaction. Multiple in-silico filters were used to minimize the number of chemicals to be tested. We identified ISO1 as an inhibitor of the Srx-Prx interaction. KD value for Srx-ISO1 interaction is calculated to be 42 nM. Together, these data helps to identify an inhibitor (ISO1) of the Srx-Prx interaction that can be further pursued to be developed as a chemotherapeutic tool.

The peroxiredoxuis (Prxs) are a ubiquitous family of antioxidant enzymes that regulate intracellular levels of H2O2 where they are implicated in both tissue protection against oxidative stress and H2O2-niediated signalling pathways (Wood et al., 2003). This thesis describe our results on the structure-function studies of PrxIII, a mitochondrial typical 2-Cys peroxiredoxin. Bovine PrxIII was cloned previously in our laboratory; however PrxIII requires its cognate partners, mammalian mitochondrial thioredoxin (Trx2) and mammalian mitochondrial thioredoxin reductase (TRR2), to reconstitute the complete antioxidant defence system. To establish a direct in vitro assay for PrxIII, Trx2 and TRR2 were cloned, overexpressed and purified in this study. As TRR2 is a selenocysteine (SeCys) protein, a suitable selenocysteine insert sequence (SECIS) for the translation of its penultimate SeCys codon was introduced by incorporating it into the reverse primer for PCR. A combination of different approaches was used for the successful overexpression of active TRR2. Overexpression in modified rich LB media at 15°C in the presence of low IPTG concentrations gave good overexpression of soluble enzyme. Moreover, the addition of the SECIS at the C-terminal of the insert, in the presence of 1 nM Na2SeO3 and co-expression of the SelABC plasmid ensured an optimal supply of the relevant tRNA, tRNA synthase and elongation factor for translation of the UGA SeCys codon. Assays showed that NADPH-linked oxidation needed the presence of all three enzymes to reduce H2O2. PrxIII was also shown to reduce other organic peroxides, although with lower activity. Cys47 and Cysl68, but not Cys66, proved to be crucial for peroxiredoxin activity. Interestingly, at high H2O2 concentrations in the non- physiological range, TRR2 also had the capacity to reduce H2O2 directly in an NADPH-dependent manner. PrxIII is also shown to be susceptible to overoxidation and loses peroxidase activity at increased H2O2 levels in the range 50μM to 1mM. This was monitored by SDS-PAGE analysis of partially or fully overoxidised forms of H2O2-mediated PrxIII and PrxIII pathway assays. Gel filtration chromatography was used to determine under which conditions the PrxIII dodecamer would dissociate into dimers. The results show that redox state, protein concentration and the N-terminal His-tag all affect the oligomerization of PrxIII. The crystal structure of the PrxIII C168S mutant from bovine mitochondria has been determined at a resolution of 3.3 angstroms. The structure reveals that the toroid is composed of 12 (not 10) monomers with a 6(2,2) symmetry. Each ring has an external diameter of 150 angstroms and encompasses a central cavity 70 angstroms in width. Surprisingly, two PrxIII rings are mechanically interlocked in the crystal to form a protein catenane. Interestingly, the catenated form represents only a small proportion (3-5%) of the total PrxIII population, as observed by electronic microscopy studies at dilute concentration (10-50μg/ml). Preliminary analytical ultracentrifugation data suggest that 2-ring catenane formation is concentration dependent. A general model illustrating catenane formation arising from polar contacts between two basic dimeric units is described. It is not clear whether the catenated form of PrxIII has any physiological function. However, the observation that Prxs can protect cells from heat shock in a peroxidase-independent process might provide new insights into possible novel functions.

This thesis focus on the analysis of spin-lattice NMRD relaxation profilesmeasured in various complex systems such as proteins, zeolites and ionicliquids. Proton, deuterium and fluoride T1-NMRD relaxation profiles wereobtained from a fast-field cycling (FFC) instrument. It is found that alsopossible to obtain NMRD profiles from the molecular dynamics (MD)simulation trajectories. NMRD Profiles were analyzed by using differentrelaxation models, such as the Solomon-Bloembergen-Morgan (SBM) theoryand the Stochastic Liouville (SL) theory. Paper I described the hydration of protein PrxV obtained from a MDsimulation, and compared with the picture emerges from an analysis byusing a generally accepted relaxation model [appendix C]. The result showsthat the information from NMRD analysis is an averaged picture of watermolecules with similar relaxation times; and the MD simulations containsinformation of all types of interested water molecules with differentresidence times. In paper II NMRD profiles have been used to characterize the hydration ofthe oxygen-evolving complex in state S1 of photosystem II. NMRDexperiments were performed on both intact protein samples and Mndepletedsamples, and characteristic dispersion difference were foundbetween 0.03 MHz to 1 MHz; approximately. Both the SBM theory and theSL theory have been used to explain this dispersion difference, and it isfound that this is due to a paramagnetic enhancement of 1-2 water moleculesnearby ~10 Å from the spin center of the Mn4CaO5 cluster. The result showsthe reorientation of the molecular cluster is in μs time interval. Whencompare these two theories, the SL theory presented a better interpretationbecause parameters obtained from the SBM theory shows they didn’t fulfilthe presupposed perturbation criterion (the Kubo term). Paper III deals with the water dynamics in the restricted/confined spaces inthe zeolite samples (H-ZSM-5 and NH4-ZSM-5) and obtained by proton anddeuterium spin-lattice NMRD profiles. The results show that the spin-latticeNMRD can be used to characterize various zeolites. The temperature has aweak effect on the relaxation rate R1, but the change of different counter ionsmay change the hydration and the translational diffusion pores and givedifferent R1. Proton and fluoride NMRD profiles and MD simulations were both used tostudy the dynamics of BMIM[PF6] in paper IV. Results indicate the reorientation of the molecules are in the ns time regime, and the effectivecorrelation time obtained from 1H and 19F are the same. From the MDsimulation it is found the reorientation of [PF6]- ions is much faster (in ps)compare with BMIM+ ion which moves in the ns time range. With previous results, the FFC NMRD profiles are indeed very informativetools to study the molecular dynamics of complex systems. The MDsimulation can be used as a complementary method to obtain detailedinformation. By combine these two methods, it provide a more colorfulpicture in the study of protein hydration and liquid molecular dynamics.

The PROTEOLYSIS 6 (PRT6) branch of the N-end rule pathway is a well-characterized negative regulator of flooding and low oxygen tolerance in plants. This study investigated the role of this pathway in adaptation to salinity stress in Arabidopsis and maize via physiological and molecular characterization of Arabidopsis prt6-1 and maize prt6 MU insertion mutants, respectively. Our study demonstrated that the loss of function mutation of prt6 in Arabidopsis activated hormonal and transcriptional responses associated with adaptation to salinity stress, enhancing high salt tolerance at seed germination, seedling, and adult plant stages. Our data also indicated that salinity tolerance conferred by the prt6 mutation is attributed to increased mRNA abundance of key transcriptional factors in ABA-dependent (AREB/ABFs) and independent (DREBs) pathways, together with the dominant expression of downstream dehydrins. Furthermore, this study revealed that the prt6 mutation enhances ethylene and brassinosteroid responses, resulting in restricted Na+ accumulation in roots and shoots as well as increased expression of dehydrin genes such as RD29A and RD29B. Maize prt6 mutant plants, contrary to our observation in Arabidopsis, showed lower seed germination, primary root elongation, and shoot biomass growth along with increased malondialdehyde (MDA) accumulation under high salt. Moreover, maize prt6 mutants exhibited reduced grain yield and yield-related components under high salt. These results indicate that PRT6 functions as a negative regulator for salinity tolerance in Arabidopsis, whereas this gene plays a positive role in salinity tolerance in maize. In wheat, we compared two genotypes with contrasting nitrogen-use-efficiency (NUE), VA08MAS-369 and VA07W-415, to dissect physiological and molecular mechanisms underlying NUE regulation. Our agronomic data revealed that line 369 maintained yield and yield-related parameters and exhibited greater NUE indexes relative to line 415 under N deficient conditions. Furthermore, our analyses suggested that the significantly higher nitrogen use efficiency (NUE) in line 369 could be attributed to the greater N uptake efficiency in this genotype. In fact, line 369 was able to maintain the development of root systems under N limitation. Consistently, genes encoding high-affinity nitrate transporters such as TaNRT2.1 and TaNRT2.2 were expressed more abundantly in the roots of line 369 than line 415 at limited N. Overall, the results of this study characterized physiological and molecular phenotypes associated with high N uptake efficiency in line 369. This is useful information for the development of new wheat accessions with improved NUE.
Doctor of Philosophy
In coastal areas, sea-level rise increases the chances of saltwater intrusion into cultivable lands, making a hostile environment for crop growth and production by imposing flooding and salinity stresses simultaneously. Identification of central regulators that regulate the adaptation to both flooding and salinity is a critical step for the development of new crop genotypes with enhanced tolerance to these stresses. Previous studies have characterized the function of the PROTEOLYSIS 6 (PRT6) gene in adaptation to flooding stress in plants. This study assessed whether this gene is involved in adaptation to salinity stress in Arabidopsis and maize by evaluating the growth and survival of their respective prt6 mutants under high salt. Consistent with the flooding tolerance data, our study showed that the PRT6 gene also functions as a negative regulator of salinity stress tolerance in Arabidopsis. The prt6 mutation in Arabidopsis activated the key transcriptional and hormone response pathways associated with adaptation to both salinity/osmotic stress and sodium toxicity, expressed as enhanced tolerance to excess salt at seed germination, seedling, and adult plant stages. In maize, disruption of the PRT6 gene decreased seed germination, primary root elongation, and shoot biomass growth under high salt, which is opposite to our observations in Arabidopsis. Additionally, the maize mutant plants encountered more oxidative stress, as demonstrated by the higher accumulation of malondialdehyde (MDA) under high salt. Moreover, maize prt6 mutants exhibited reduced grain yield under high salt. Overall, these results indicate that disruption of the PRT6 gene confers increased tolerance to high salt in Arabidopsis, whereas it conversely reduced salinity tolerance in maize. In wheat, we compared two genotypes with distinct nitrogen use efficiency (NUE), VA08MAS-369 and VA07W-415, to determine critical traits involved in NUE regulation. Our study showed that grain yield and yield-related parameters were significantly higher in line 369 than line 415 under low N. Moreover, high NUE in line 369 was attributed to efficient N uptake in this genotype under limited N. Our root architecture analysis demonstrated that line 369 was able to maintain root depth, volume, and thickness even under N limitation. Consistently, line 369 highly induced expression of genes associated with nitrogen transport at low N. Altogether, this study identified key traits involved in high NUE in wheat, facilitating the breeding of new wheat genotypes with enhanced NUE.

La photosynthèse oxygénique peut être le lieu de formation des Formes Réactives de l’Oxygène (FROs). Ces espèces réactives peuvent altérer toutes les macromolécules de la cellule, générant ainsi un stress oxydatif. Les cyanobactéries étant les premiers organismes à avoir émis de l’oxygène sur terre, elles ont développé très tôt au cours de l’évolution des mécanismes de défense pour lutter contre le stress oxydatif. Nous nous sommes intéressés à l’étude de la réponse de la cyanobactérie filamenteuse Anabaena sp. PCC 7120 à ce stress. Nous avons mis en évidence l’implication d’un opéron, dit pkn, dans cette réponse adaptative. Nous avons notamment caractérisé la fonction de la peroxiredoxine PrxQ-A dans la réduction des FROs de type peroxyde. Nous avons également caractérisé une protéine codée par le dernier gène de cet opéron comme étant une cystéine désulfurase. Cette classe d’enzymes est impliquée dans la mobilisation du soufre dans divers processus comme la biosynthèse de la thiamine, la biosynthèse de la molybdoptérine, la thiolation des ARNt ou encore la biogénèse des centres [Fe-S]. L’éventuelle relation fonctionnelle entre les différentes protéines codées par l’opéron pkn et le rôle qu’elles jouent dans la réponse de ce micro-organisme au stress oxydatif offrent de nouvelles perspectives à ce travail
Oxygenic photosynthesis may generate of reactive oxygen species(ROS). These reactive species can damage all the macromolecules of the cell, inducing an oxidative stress. Cyanobacteria were the first organisms producing oxygen on earth, they have developed very early in the evolution various defenses to protect themselves against deleterious effects of ROS. We are interested in studying the response of the filamentous cyanobacterium Anabaena sp. PCC7120 to oxidative stress. During this wrok, we have highlighted the involvement of an operon, the pkn operon, in this adaptive response. We have characterized the function of peroxiredoxin Prx QA in reducing peroxide. We also characterized a protein encoded by the last gene of this operon as a cysteine desulfurase. They are pyridoxal phosphate-depending enzymes involved into the mobilization of sulfur to various processes such as biosynthesis of thiamine, the biosynthesis of molybdopterin, tRNA thiolation and also the biogenesis of [Fe-S] clusters. The possible relationship between the different proteins encoded by this operon and their role in the response of Anabaena sp. PCC 7120 to oxidative stress offer interesting perspectives

As an important hemodynamic mechanism, pressure autoregulation protects the brain against inappropriate fluctuations in cerebral blood flow subject to changing cerebral perfusion pressures. In acute neurological illnesses, including traumatic brain injury in children, impaired autoregulation is associated with a worse prognosis. It also has important clinical implications for managing blood pressure and intracranial pressure. Two common methods of measuring pressure autoregulation reported in the adult literature have been rarely reported in children. This pilot study aimed to examine the relationship between two autoregulatory indices, namely PRx (pressure reactivity index) and ARI (autoregulatory index) in children with severe TBI. The study also examined their relationship with the response of clinically relevant variables such as intracranial pressure (ICP), brain oxygenation (PbtO2) and local cerebral blood flow (loCBF) to dynamic testing. The study is a retrospective cohort study of prospectively collected data conducted at the Red Cross Children Hospital. We analyzed the results of 18 patients in 28 tests of autoregulation to determine the static state of autoregulation by calculating the autoregulatory index (ARI). These tests were done by controlled elevation of blood pressure to evaluate changes in transcranial Doppler-derived flow velocity of the middle cerebral artery. Concomitant recordings were made of ICP, PbtO2, and loCBF. Secondly, we also calculated the PRx as a moving correlation co-efficient between slow changes in blood pressure and ICP. Two time epochs of PRx were examined in relation to the static tests: 1 hour before and after the test, and 12 hours before and after the test. The results included 28 tests done for ARI and 27 calculations for PRx epochs; all tests had ICP and PbtO2 data and 23 had loCBF. PRx and ARI showed no significant relationship between them. However, there was a significant relationship between ARI and ΔICP (p=0.04), i.e. when autoregulation was weak the change in ICP with a change in blood pressure was greater; and between PRx and ΔPbtO2 (p=0.04). There was a trend in correlation analysis between loCBF and PRx but not in the linear mixed effects model In conclusion, the study showed no correlation between the two autoregulatory indices, PRx and ARI, probably because they assess different aspects of autoregulation. However, significant relationships exist between ARI and ΔICP as well as PRx and ΔPbtO2, which generate interesting hypotheses about autoregulation and have clinical implications. Both autoregulatory indices have benefits and limitations. Further studies on such relationships, taking into consideration a larger sample group, inclusion of unstable patients, and utilization of the same range in BP for calculating the indices, are recommended.

L'éthylène comme la stratification au froid et le GA3, stimule la germination des graines d'Arabidopsis thaliana (Col-0) ayant une dormance primaire, à 25 °C et à l'obscurité. L'élimination de la dormance au froid n'exige pas ETR1, EIN4 et EIN2, alors que l'effet du GA3 nécessite ETR1. L'effet stimulateur de l'éthylène est associé à une réduction de l'expression des gènes impliqués dans les voies de signalisation des GAs (DELLAs) et de l'ABA (ABI5). Les graines du mutant prt6 affectées dans la voie "N-end rule" de la protéolyse, sont insensibles à l'éthylène, montrant que PRT6 intervient dans l'action de l'éthylène, et cette insensibilité résulte aussi d'une interrelation avec la balance ABA/GAs. D'autre part, les facteurs de transcription de la réponse à l'éthylène du groupe VII, (ERF VIIs), substrats de la voie "N-end rule", interviennent dans cette insensibilité. La sensibilité à l'éthylène de prt6rap2.2rap2.3rap2.12 et l'insensibilité de prt6hre1hre2, permet de montrer que les 3 RAPs et les 2 HREs interviennent respectivement en aval et en amont de PRT6. L'éthylène induit une diminution de l'expression des 3 RAPs dans Col, mais maintient ou induit celle-ci dans prt6. De plus, l'expession de HRE2 augmente dans prt6, mais diminue dans prt6rap2.2rap2.3rap2.12, suggérant que les 3 RAPs peuvent réguler l'expression de HRE2. De plus, l'éthylène modifie différentiellement le protéome de Col et de prt6 avec respectivement 587 et 30 protéines significatives. L'analyse des classes fonctionnelles a permis d'identifier la réponse à hypoxie comme processus biologique spécifique de prt6, mais leur insensibilité à l'éthylène est indépendante des ROS et de l'intensité respiratoire
Ethylene as chilling and GA3, was able to improve the germination of primary dormant seeds of Arabidopsis thaliana (Col-0) at 25 °C in darkness. Chilling did not require EIN4, ETR1 and EIN2 involved in ethylene signaling to break seed dormancy while GA required ETR1.The improving effect of ethylene in seed germination is EIN4 independent, and is associated with a decrease in ABA/GA ratio and a down-regulation of DELLAs and ABI5 genes related to GA and ABA signaling, respectively. The mutant affected in the proteolytic N-end rule pathway, prt6, was insensitive to ethylene in seed germination evidenced that PRT6 was involved in dormancy release by ethylene, and this insensitivity was related to a crosstalk with ABA/GAs. The substrates of the N-end rule pathway, ERFVIIs (HRE1, HRE2, RAP2.2, RAP2.3, and RAP2.12), might result in the insensitivity with an increased germination in prt6rap2.2rap2.3rap2.12 rather than in prt6hre1hre2, which also indicated that the 3 RAPs acted downstream of PRT6, while the 2 HREs acted upstream of PRT6. Ethylene reduced the transcript expression of the 3 RAPs in Col-0, but the 3 RAPs were maintained or induced by ethylene in prt6. Besides, HRE2 was up-regulated in prt6 seeds, but it was down-regulated in prt6rap2.2rap2.3rap2.12, suggesting that the 3 RAPs might stimulate the expression of HRE2. Ethylene differently changed the seed proteome of Col and prt6 with 587 and 30 significant proteins, respectively. The functional class scoring analysis identified one biological process, response to hypoxia, which was distinct in prt6, however the insensitivity of prt6 to ethylene was independent of ROS production or respiration intensity

Understanding the recruitment of multipotent skeletal progenitor cells and the factors that influence their differentiation would be helpful in providing a means for harnessing the regenerative capacity of skeletal progenitor cells in bone tissue engineering. In order to track the recruitment of skeletal stem cells in fracture healing, transgenic mice containing a Tamoxifen-inducible Cre recombinase that had been placed under the control of a 2.4 kb Prx1 promotor were used to induce conditional expression in periosteal skeletal stem cells that express the Prx1 gene. In order to initially see the cells expressing Prx1, a green fluorescent protein gene (GFP) had also been put downstream to the Prx1 promotor. We then crossed these Prx1CreER-GFP transgenic mice with a second strain containing the Beta-galactosidase gene that becomes constitutively expressed after recombination by the Cre recombinase. The enzymatic activity of Beta-galactosidase was then used to generate a colormetric staining reaction that was used to visualize the cells in which recombination had occurred based on a blue staining product. The recombination activity should only be present in Prx1 expressing cells and their progeny. The goal of the present study was to assess several different approaches to optimize the Beta-galactosidase enzymatic staining protocol and to visualize the Prx1-expressing cells during fracture healing. These studies further examined those populations of cells in the fracture calluses that became labeled and arose from the stem cell populations that had expressed Prx1 at post-operative day 7 and 14. The optimization of a staining method for histology will allow this study to track Prx1 cell fates in a fracture model both in response to specific drug treatments, mechanical loading of the fracture during healing and under pathological conditions that effect healing.

碩士
國防大學理工學院
化學工程碩士班
100
Doping of Pr (5 and 10% loading) on Ce0.5Co0.5O2 catalyst, used for steam reforming of ethanol (SRE), was prepared by incipient wetness impregnation method. The topic focused on the effect of the reduction pretreatment under 250 and 400 ℃ (assigned as H250 and H400). All samples were characterized by XRD, TPR and TEM. Catalytic performance of SRE was tested from 250 to 500 ℃ in a fixed-bed reactor under H2O/EtOH molar ratio of 13 and 22,000 h-1 GHSV. In the preliminary test, we found that the activity can be enhanced on increasing the Pr loading. Also, the pretreatment of reduction can influence the distribution of products, i.e. the byproduct of acetone is higher than 30 mol% under H250, while, the CO byproducts are lower than 2 mol% under H400. According to the results, we suggested that the 10Pr/Ce0.5Co0.5O2-H400 is a preferential SRE catalyst. The products can be varied with the increasing of reaction temperature (TR), i.e. the acetaldehyde is oxidized to acetone and then, decomposes into CO2 and methyl group. Further, the steam reforming of methyl and water gas shift reaction accompany as the TR exceeds 375 ℃. At 475 ℃, the yield of hydrogen approached 5.38.

INTRODUCTION: Damage to the musculoskeletal system through disease, injury, or ageing can have long-lasting, and detrimental effects on one’s overall well-being. By understanding the processes by which the different tissues of the musculoskeletal system function and communicate, we can apply it to a variety of medical interventions that will benefit the patient population. These include reducing the prevalence of injury-inducing ectopic bone formation in muscle and slowing the degeneration of muscle and bone tissue associated with aging. A major focus is the relationship between muscle and bone tissue, specifically the stem cell populations found in each tissue type. Two genes that are thought to mark stem cell populations associated with muscle and bone tissue are Pax7 and Prx1, respectively. OBJECTIVES: Establish the capability and define optimal conditions to culture primary stem cells isolated from the muscle tissue of the reporter animals that fluorescently tag the Pax7 and Prx1 cell populations. Manipulate culture medium conditions to characterize the differentiation potential for multiple lineages, osteogenic, adipogenic, and myogenic. Lastly, assess whether there is more adipogenic cell differentiation in older animal cell cultures. METHODS: The tamoxifen inducible Pax7^tm1(cre/ER2)Gaka/J and Prx1^CreER-GFP were both crossed with B6.Cg-Gt(ROSA)26sor/J to create the tamoxifen inducible Pax7/Ai14 and Prx1/Ai14 reporter mice. These animals were then crossed to the B6,129S7-Rag1^tm1Mom/J , creating the Pax7/Ai14/Rag and Prx1/Ai14/Rag reporter mice strains. This transgenic mouse model made it possible to fluorescently identify the Pax7 and Prx1 population of cells isolated from the muscle tissue and characterize the differentiation potential to different cell lineages in vitro. Cells were harvested from both male and female mice that received two tamoxifen injections. Cells were then cultured in various culture media conditions. Determination of specific cell densities, culture conditions, and differentiation time points were determined by manipulating these variables, and assessing the levels of proliferation and differentiation. Multiple assays were run in order to quantify and identify the different cell lineages that were grown in culture under different culture conditions. RESULTS: Cells grown on gelatin coated dishes at densities of 2.2x10^4 to 2.2x10^5 showed optimal performance in proliferation and differentiation. Cells grown in Growth Media containing Chick Embryo Extract (GM) and without (GM-) produced myogenic and adipogenic cell types that were positive for Prx1 expression. Prx1 positive cells grown in the Osteo-Inductive Media (GMOI) produced osteogenic cell types validated through tetracycline uptake. Pax7 expression was low in all culture media conditions. Finally, adipogenic cells were present in both younger and older animals. The adipogenic cells for both populations showed positive Prx1 expression. Younger animals showed a larger relative expression of Plin1 in qRT-PCR analysis. CONCLUSIONS: Although Prx1 is thought to be associated with bone tissue, Prx1 positive cells are located within the muscle and able to be cultured. This muscle derived Prx1 cell population is able to differentiate toward the myogenic, adipogenic, and osteogenic cell lineages. By altering the specific components of culture conditions such as extracellular matrices, seeding density, and media constituents, it is possible to force a particular lineage differentiation for Pax7 and Prx1 muscle derived stem cells. Further studies are needed to elucidate the differentiation potential of Prx1 cells isolated from animals of various ages. Additional in vivo studies are needed to understand the mechanisms surrounding the Prx1 and Pax7 population of cells with their roles in healing and regeneration in response to degeneration and trauma.

碩士
國立虎尾科技大學
電子工程系碩士班
102
In this study, BaY2ZnO5:Pr3+ phosphor was selected as the host material. The first part Pr3+ ions co-doped BaY2ZnO5 phosphor were synthesized using a sol-gel method with metal oxides and calcined at 1200 °C for 8 h in air. The second part studies different added chelating and pH with BaY2ZnO5:Pr3+ phosphors. The third part studies on the Structure and Photo-Luminescence for Ba(Y1.98Pr0.02)ZnO5 doped with fluxes Na2CO3 ion phosphors. The XRD results shows that the BaY2ZnO5 phase with an orthorhombic structure can be formed when the calcination temperature increases from 700 to 1200 °C and calcination time increases from 2 to 10 h, respectively. The optimum condition for calcination is 1200 °C /8 h. The XRD results indicated that there are secondary phase formed when the Pr3+ concentrations increase from 0.5 mol% to 2 mol%. The SEM results showed that there are no obvious differences on surface morphology for BaY2ZnO5 doped with various Pr3+ ion concentrations. The dominant transitions of BaY2ZnO5:Pr3+ phosphor are 3P0→3H4, 3P1→3H4, 3P0→3H5, 1D2→3H4, 3P0→3H6, 3P0→3F2 at lower Pr3+ concentration. The intensities of the emission from 3P0 and 3P1 decrease with increasing the Pr3+ concentration, and which is due to the concentration quenching . The BaY2ZnO5:Pr3+ phosphor emit red luminescence when the Pr3+ concentration is 0.5 mol% to 2 mol% . The Critical distance RC=0.181 A. The maximum PL intensity is obtained for BaY1.98Pr0.02ZnO5 and has a CIE chromaticity coordinates of (0.066, 0.0.675). The SEM observations demonstrated the morphological transformation of Ba(Y1.98Pr0.02)ZnO5 powders by altering the pH value of the synthesis solution. That is, due to the formation of the highly-crystallized structure, the final product prepared at pH=3.16 shows the highest intensity among them. The ph=3.16 exhibit apparent strong excitations in the blue-purple visible region and emission bands in green region with maximum peaks at around 515 nm, corresponding to the characteristic 3P1→3H4 transition of Pr3+. The XRD patterns of Ba(Y1.98Pr0.02)ZnO5 added with 0.5~3 wt.% fluxes Na2CO3.The matches well with the standard JCPDS-Card. The SEM results indicate that the surface morphology of Ba(Y1.98Pr0.02)ZnO5 added with Na+ ion concentrations exhibits small grain sizes. When the added concentrations increase continuously, the grain sizes were decreased but uniform than the others. The optimal added concentration was measured to be 1 wt.% for Na+ in Ba(Y1.98Pr0.02)ZnO5.The CIE chromaticity coordinates (0.089, 0.684) .

Signal transduction pathways play a critical role in plant growth and reproduction by perceiving extracellular signals, leading to a cellular response. FERONIA (FER) is a transmembrane receptor kinase found on the plasma membrane in the model plant Arabidopsis thaliana and plays critical roles in growth, development, and fertilization. FER works upstream of master molecular switch RAC/ROP GTPase to regulate signaling into the cytoplasm. LORELEI-Like Glycosylphosphatidylinositol (GPI)-Anchored Protein 1(LLG1) is a GPI-anchored protein and co-receptor of FER on the plasma membrane. LLG1 is responsible for chaperoning FER from the endoplasmic reticulum (ER) to its functional location on the plasma membrane. Rapid Alkalinization Factor 1 (RALF1) is a small, secreted growth-regulatory peptide that interacts with FER, regulating signaling activity. This interaction, among other, regulates the activity of a downstream plasma membrane proton ATPase (AHA2) which impacts cell growth. Additionally, published pulldown data indicates LLG1, FER, and RALF1 complex together. My data suggests that LLG1, in addition to localizing and chaperoning FER, binds directly to RALF1. My results show that this RALF1-LLG1 interaction is required for proper RALF1 mediated signaling through FER. Data also indicates that FER and LLG1 regulate RALF1 location on the plasma membrane. Additionally, RALF1 binds the MALA domain of FER. Another aspect of my thesis focuses on LURE1. LURE1 is a secreted cysteine-rich, defensin like protein which guides incoming pollen tubes to the ovule in a process called pollen tube guidance. LURE1 guides pollen tubes by binding with pollen-specific receptor kinase 6 (PRK6), located on the plasma membrane of the incoming pollen tubes, to facilitate proper fertilization. My data also shows that the ovule derived signaling molecule nitric oxide (NO), also regulated by FER, negatively impacts the property of LURE1, causing it to fall out of solution and aggregate. Furthermore, the negative impact of NO on LURE1 disrupts the binding affinity of LURE1 to PRK6. Together with data from my lab showing pollen tube arrival at the ovule triggers NO production in a FER dependent manner, my findings provide a biochemical explanation for why pollen tubes do not target fertilized ovules.