Дисертації з теми "CP26"

Utilisation de molécules dinuclaires à l'état solide. Caractéristiques physicochimiques des molécules de départ et conditions d'élaboration des dépôts. Utilisation de ces matériaux dans le revêtement anticorrosion.

Current ethanol oxidation catalysts in direct ethanol fuel cells (typically platinum-based) suffer from low conversion and are susceptible to CO poisoning. Therefore we determined to find viable alternative catalysts for ethanol oxidation based on iridium using density functional theory to model bimetallic alloy (111) surfaces. Iridium was alloyed with another transition metals M in an overlayer (one layer of metal M on top of bulk iridium) or subsurface configuration (M is inserted under the first layer of iridium). Complete oxidation of ethanol is limited by the breaking of strong C-C bonds, so any catalyst must lower the barriers for C-C bond breaking. We modeled the reaction CH+CO →CHCO.Segregation energies were calculated and the subsurface configuration was the most stable configuration in the vast majority of alloy cases. CO adsorption was also studied and a lower CO adsorption energy was found in many alloy cases compared to pure Pt (, providing encouraging results about the possibility of reducing CO poisoning. Activation energies were lowered for the vast majority of the alloys used in an underlayer structure, reinforcing our interest in the underlayer structures or “subsurfaceâ€� alloys. Finally, we found, based on the CO adsorption energies, activation energies of the C-C breakage reaction, and metal cost, three important catalyst descriptors, a number of promising catalysts for the ethanol oxidation reaction. The most interesting alloys all adopted the underlayer structure Ir/M/Ir. With M = Ta, Hf, Nb, V, Zr, they demonstrated enhanced reactivity and high CO tolerance, having the advantage of reducing the cost of the catalyst, potentially substituting expensive platinum group metals by more affordable components.

Dans leur environnement naturel, les plantes doivent continuellement s’adapter pour survivre aux diverses stimuli auxquels elles sont confrontées. La salinité est responsable de fortes pertes de rendements et une meilleure compréhension des mécanismes capables d’induire la tolérance de la plante est nécessaire. Au niveau cellulaire, les stress biotique et abiotique perçus induisent la production de messagers secondaires ce qui active des voies de signalisation faisant intervenir différentes familles de protéines kinases dont les protéines kinases dépendantes du calcium, les CDPKs. Lorsqu’elles sont activées, elles vont phosphoryler des substrats permettant la mise en place des réponses spécifiques au stress perçu.CPK5 et CPK6 sont des régulateurs positifs de la tolérance au stress salin et également de la résistance aux bactéries, notamment par l’induction de gènes de réponses. L’identification de 25 nouveaux substrats putatifs par une approche phosphoprotéomique pourraient permettre une meilleure compréhension du rôle de CPK5 et CPK6 dans la réponse aux stress. Parmi les candidats étudiés dans ce manuscrit, six ont été validées in vitro, notamment deux ubiquitine ligases E3 dont la mutation d’un site conservé inhibe la phosphorylation par CPK5 et CPK6. Bien que l’étude des mutants knock-out n’ait pas permis de leur attribuer un rôle dans la tolérance à la salinité, certains pourraient agir en aval de CPK5/6 dans la réponse aux pathogènes
Plants are subjected to several environmental stimuli and must adapt to survive. A better understanding of the mechanisms involved in plant tolerance is necessary to find solution to the salt stress that is responsible for important yield loss. At the cellular level, the perception of biotic and abiotic stress leads to the accumulation of secondary messengers that activate protein kinases involved in different signaling pathways as calcium-dependent protein kinases (CDPKs). Once they are activated, CDPKs can phosphorylate substrates which are able to induce appropriate responses to the stimuli.CPK5 and CPK6 are positive regulators of salt stress and biotic stress, notably through the induction of response genes. To better understand their roles in plant stress response, a phosphoproteomic approach was conducted and could identify 25 new putative substrates for CPK5. Six of the studied candidates were validated in vitro for phosphorylation by CPK5 and CPK6. Among them, two ubiquitin E3 ligases are not phosphorylated anymore by CPK5 and CPK6 when mutated on a conserved phosphosite. Unfortunately, the knock-out mutants of the substrates in salt condition did not show any phenotype. However some of them are known to play a role in response to pathogen and could act downstream of CPK5 and CPK6

Orientadora: Marcia Aparecida Silva Graminha
Banca: Adelino Vieira de Godoy Netto
Banca: Luiz Ricardo Orsini Tosi
Banca: Pio Colepicolo Neto
Banca: Silvia Reni Bortolin Uliana
Resumo: As leishmanioses são doenças mundialmente distribuídas, encontradas nas áreas tropicais e subtropicais do mundo e que são caudas por protozoários parasitos do gênero Leishmania spp. A pesar dos inúmeros problemas associados aos tratamentos disponíveis (como alta toxicidade dos fármacos, limitada eficácia e casos de resistência haverem surgido), ainda estás doenças são negligenciadas pelas industrias farmacêuticas e pelos governos. Na procura por novos fármacos com amplo espectro de ação e baixa toxicidade, há evidências que sugerem que complexos de metais de transição podem atuar em diversos compartimentos ou organelas dos protozoários, além de apresentar baixa toxicidade no hospedeiro mamífero. No presente trabalho, realizou-se a avaliação leishmanicida in vitro de seis compostos ciclopaladados, [Pd(dmba)(µ-Cl)]2 (CP1), [Pd(dmba)(µ-N3)]2 (CP2), [Pd(dmba)(µ-NCO)]2 (CP3), [Pd(dmba)Cl(isn)] (CP4), [Pd(dmba)(N3)(isn)] (CP5) e [Pd(dmba)(NCO)(isn)] (CP6) e seus correspondentes ligantes livres, Hdmba: N,N-dimetilbenzilamina e isn: isonicotinamida. O composto CP2 inibiou o crescimento das formas promastigotas de Leishmania amazonensis (IC50 = 13,2 ± 0,7 µM), reduz a proliferação das formas amastigotas intracelulares (IC50 = 10,2 ± 2,2 µM) e apresentou um baixo efeito citotóxico frente a macrófagos peritoneais (CC50 = 506,0 ± 10,7 µM). Dados in vitro da atividade anti-T. cruzi e anti-T. brucei, parasitos causadores das doenças de Chagas e do sono, respectivamente, também demonstraram que os compostos ciclopaladados apresentam amplo espectro de ação constituindo-se em excelentes candidatos para o tratamento das doenças negligenciadas em estudo. O composto CP2 apresentou-se para as formas amastigotas intracelulares de L. amazonensis pelo menos 50 vezes mais seletivo e 200 vezes mais seletivo para as amastigotas de T. cruzi vs. células de mamífero...
Abstract: Leishmaniasis are diseases globally distributed in tropical and subtropical areas of the world and Leishmania spp. are the etiological agents of the diseases. Numerous problems associated with available treatments of the disease are still unsatisfactory because currently available drugs are highly toxic, little effectiveness and drug resistance cases have emerged. Furthermore, leishmaniasis are a neglected disease by pharmaceutical industries and governments. In the search for new drugs with a broad spectrum of action and low toxicity, there is evidence to suggest that transition metal complexes can act in several compartments or organelles of protozoa, as well as to present low toxicity in the mammalian host. In this work, we evaluated the leishmanicidal and trypanocidal in vitro activity of six cyclopalladated compounds: [Pd(dmba)(µ-Cl)]2 (CP1), [Pd(dmba)(µ-N3)]2 (CP2), [Pd(dmba)(µ-NCO)]2 (CP3), [Pd(dmba)Cl(isn)] (CP4), [Pd(dmba)(N3)(isn)] (CP5), [Pd(dmba)(NCO)(isn)] (CP6) and the free ligands, Hdmba: N,N- dimethylbenzylamine e isn: isonicotinamide. The cyclopalladated complexes CP2 inhibited the growth of the promastigote forms of Leishmania amazonensis (IC50 = 13,2 ± 0,7 µM), reduced the proliferation of intracellular amastigote forms (IC50 = 10,2 ± 2,2 µM) and showed a low cytotoxic effect against peritoneal macrophages (CC50 = 506,0 ± 10,7 µM). In vitro assays against T. cruzi and T. brucei, parasites that cause Chagas disease and sleeping sickness, respectively, demons... (Resumo completo, clicar acesso eletrônico abaixo)
Doutor

The work presented in this thesis was aimed to investigate the structure-function relationship of several photosynthetic Light-Harvesting Complexes (LHCs) including Chlorophyll Protein 29 (CP29) and Light-Harvesting Complex II (LHCII) of green plants, and Fenna-Matthews-Olson (FMO) complex of green sulfur bacterium Chlorobium tepidum. Based on the exciton calculations, a model was proposed to the electronic excited states (EES) of both CP29 and LHCII complexes by incorporating a considerable part of the current information offered by structure determination, mutagenesis analysis and spectroscopy in the modeling. The essential parameters for characterizing the excited states, Qy dipole orientations and site energies were assigned by suggesting a model that can explain both the key features of the linear (polarized) absorption spectra and the time scales of the energy transfer processes in CP29 and LHCII. The idea of offering structural information through setting connection between the spectroscopy and the spectral simulations were supported by the presented results on CP29 and LHCII. New spectroscopic measurements (absorption, linear dichroism (LD) and circular dichroism), carried out at 4 K on the FMO complex were presented, and also the LD spectrum was corrected for the degree of orientation of the sample, in order to provide comparison of not only the shape but also the size of the simulated and experimental spectra. The EES structure of the FMO complex was studied by simulating the measured optical spectra with more realistic model than the previously applied models. Simulations have been carried out with a computer program based on exciton model, which includes inhomogeneous, homogeneous and lifetime broadenings explicitly.

El presente plan de acción busca fortalecer a los docentes en la planificación de estrategias de expresión oral que los lleve a lograr en los estudiantes la lectoescritura al nivel de su desarrollo, es evidente el impacto negativo que causa la inadecuada planificación de los aprendizajes, ya que se trabaja con estrategias tradicionales. Los docentes carecían de un trabajo colegiado, lo que no les permitía compartir y reflexionar de las experiencias que cada una de ellas planteaba en el desarrollo de sus estrategias con los estudiantes en el aula. Los objetivos de la investigación apuntan a implementar un sistema de estrategias para el desarrollo de la expresión oral en los estudiantes, desarrollar un trabajo colaborativo con las docentes en la planificación de los proyectos que busque el descubrimiento de la lectoescritura en los estudiantes y buscar la metodología que permita superar las deficiencias en la planificación de las sesiones de aprendizaje. Emilia Ferreiro en su aporte afirma que el niño diferencia el dibujo de la escritura comienza a representar por escrito lo que quiere comunicar, empleando al principio signos arbitrarios; a medida que se apropia del código escrito convencional su escritura cambia hasta emplear las letras del alfabeto. Estas formas sucesivas de representación escrita se denominan los niveles de construcción de la escritura. Estos niveles son: Pre silábico, silábico, silábico–alfabético y alfabético. Llegando a la conclusión que los estudiantes de preescolar tienen que ir descubriendo y apoderarse del mundo escrito con naturalidad, en forma libre y a través del método lúdico, respetando su desarrollo.
Trabajo académico

Doctor of Philosophy
Department of Chemistry
Duy H. Hua
Three research projects were carried out and they are described below. The synthesis of substituted phenolic compounds including halogenated di- and trihydroxybenzenes, aminophenols, and substituted di-tert-butylphenols are described. Redox potentials of the synthesized molecules along with various known laccase substrates were measured, and an inverse relationship between the oxidation potential and the efficiency of oxidation by laccase of halogenated hydroxybenzenes and aminophenols is demonstrated. The synthesized substituted phenols were found to be substrates but not inhibitors of laccase. We discovered a new class of di-tert-butylphenols compounds that inhibits the growth of mosquito larvae at low concentrations. Compound 17, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl) phenol caused greater than 98% mortality of third-instar larvae of Anopheles gambiae in the concentration of 0.18 µM. These compounds do not inhibit laccases. It appears that they affect a new target of the mosquito that is different from those of currently existing pesticides. Two anti-Alzheimer molecules, CP2 and TP70, discovered in our laboratory were studied for their pharmacokinetics and distribution. The distribution of CP2 and TP70 in mouse brain region and various tissues of mice were examined. HPLC analysis revealed that CP2 treatment in primary neurons accumulates in mitochondria fraction. Similarly, the amount of CP2 in the brain tissue from wild type and APP/PS1 mice treated with 25 mg/kg/daily for 2 months also have the highest concentration in the mitochondria fractions in the hippocampus. The results show that CP2 and TP70 can penetrate the blood brain barrier and accumulate in the tissue in significant amounts. Pharmacokinetics and bioavailability of compound TP70 were determined. Area under the curve and bioavailability value F were calculated, and data show that TP70 has a good PK profile and bioavailability. For the preparation of a novel tripeptidyl norovirus 3C-like protease (3CL[superscript]pro) inhibitor, the P3 unnatural amino acid, (S)-3-hydroxyphenylalanine was synthesized. The P3 is designed to increase the polarity with the addition of the alcohol group. After combining the P3 unnatural amino acid with the P1 and P2 to form the novel tripeptidyl compound, a study comparing the relations between the structure and its activity (SAR) will confirm whether prediction is correct in our pursuit for an antiviral therapeutic drug in the form of a protease inhibitor.

Two-charged-particle correlations are measured as a function of pseudorapidity and azimuthal angle difference in pp collisions at √s = 13, 2.76 and 5.02 TeV with the ATLAS detector at the Large Hadron Collider. A long-range structure in the two-dimensional function centered at ∆φ = 0 and extending over a large range of ∆η referred to as the “ridge” is seen in the three data sets. A template fitting method is implemented to extract the Fourier harmonics of the flow and gives the dependence of the harmonics on the charged-particle multiplicities. In this method a rescaled correlation function from peripheral events representing the recoil component plus a cosine modulation representing the ridge is used to describe the whole one-dimensional correlation function. Different multiplicity intervals for the peripheral events are used to extract the harmonics. The results presented show that vn,n from correlation functions can be factorized into the products of single particle vn. Significant contributions from v₂, v₃ and v₄ are obtained and their dependences on multiplicity and transverse momentum are studied. It is also shown that there is significant vn even in the lowest multiplicity bins. In addition, the second harmonics v₂ in pp do not have a significant dependence on both the multiplicity and collision energy. Results of pp and pPb at the same energy are compared with each other in both multiplicity and pT distributions. Both chᵗʳᵏ−chᵗʳᵏ and chᵗʳᵏ-muon correlations are measured in pPb collisions at √sNN = 8.16 TeV. Long-range correlations are studied through template fitting procedure. chᵗʳᵏ-v₂ increases with the number of reconstructed charged tracks at low multiplicity and saturates at high multiplicity. Muon-v₂ is considerably smaller than chᵗʳᵏ-v₂ and only has a weak dependence on event multiplicity. Factorization in both cases works pretty well. Two-charged-particle correlation functions are also measured in Xe+Xe events at √sNN = 5.44 TeV. In the most central collisions direct Fourier decomposition is preferred to avoid negative recoil component that might appear in the template fitting method. vn reaches its maximum value in the mid-centrality region and becomes smaller at both low and high centralities. Results are compared with Pb+Pb events at √sNN = 5.02 TeV showing that vn obtained from these two systems have similar values and behaviors.

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other CP2 family members, CRTR-1 expression is regulated developmentally. Major sites of expression in the embryo include the pluripotent inner cell mass (ICM) of the pre-implantation blastocyst and the developing kidney. It is also expressed in embryonic stem (ES) cells, which are derived from the ICM of blastocysts, and is downregulated as these cells differentiate into early primitive ectoderm-like (EPL) cells. This expression pattern suggests that CRTR-1 plays a role in early pluripotent populations. This thesis aims to characterize the transcription factor CRTR-1 at the molecular level and analyses the role of sumoylation on CRTR-1 function to develop a better understanding of the molecular role of CRTR-1 in ES cells. Luciferase reporter assays show that CRTR-1 is able to regulate the activities of other CP2 family members: CP2, NF2d9 and altNF2d9. It enhances CP2- and NF2d9-mediated activation but suppresses altNF2d9-mediated activation. To map the functional domains in the CRTR-1 protein, transactivation studies using CRTR-1 deletion mutants fused to the GAL4 DNA binding domain and a GAL4-responsive reporter system were performed. These studies map repressor activity to amino acids 48-200, but fail to identify a transactivation domain within the CRTR-1 protein. In order to understand the mechanisms by which CRTR-1 regulates the transcriptional activities of CP2 family members, a number of approaches are taken, including co-immunoprecipitation to show that CRTR-1 interacts with other CP2-like proteins, EMSA which demonstrate that CRTR-1 forms DNA binding complexes with CP2 family members, and subcellular protein localisation studies which reveal the ability of CRTR-1 and other family members to shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. In addition, the subcellular localisation of CRTR-1 appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells. Co-expression of CRTR-1 with CP2 or NF2d9 results in the re-localisation of CRTR-1 to the cytoplasm in ES cells. The sumoylation enzymes Ubc9 and PIAS1 have previously been identified as CP2-interacting proteins (Kang et al., 2005a). Given the identification of two potential sumoylation sites within CRTR-1, FK³⁰ QE and LK⁴⁶ ⁴AE, and the ability for sumoylation to regulate transcription factor function, the possibility that CRTR-1 is regulated by sumoylation is investigated in this thesis. Immunoprecipitation experiments show that CRTR-1 is modified by SUMO-1 and that lysine 30 is the critical residue for this modification. Mutation of lysine 30 to alanine, which abolishes CRTR-1 sumoylation, results in enhancement of transactivation by CRTR-1, suggesting that sumoylation of CRTR-1 blocks maximal activation. Unexpectedly, however, overexpression of Ubc9, PIAS1, or SUMO-1 results in enhancement of CRTR-1 transcriptional activity, indicating that a more complex mechanism of regulation of CRTR-1 activity is likely. This thesis presents several novel properties of CRTR-1 and other CP2 family members, including the ability of CRTR-1, previously characterized as a repressor, to activate transcription. It is also the first demonstration that CP2 proteins are regulated by sumoylation and that they shuttle between the nucleus and cytoplasm via a CRM1-dependent mechanism.
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Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other CP2 family members, CRTR-1 expression is regulated developmentally. Major sites of expression in the embryo include the pluripotent inner cell mass (ICM) of the pre-implantation blastocyst and the developing kidney. It is also expressed in embryonic stem (ES) cells, which are derived from the ICM of blastocysts, and is downregulated as these cells differentiate into early primitive ectoderm-like (EPL) cells. This expression pattern suggests that CRTR-1 plays a role in early pluripotent populations. This thesis aims to characterize the transcription factor CRTR-1 at the molecular level and analyses the role of sumoylation on CRTR-1 function to develop a better understanding of the molecular role of CRTR-1 in ES cells. Luciferase reporter assays show that CRTR-1 is able to regulate the activities of other CP2 family members: CP2, NF2d9 and altNF2d9. It enhances CP2- and NF2d9-mediated activation but suppresses altNF2d9-mediated activation. To map the functional domains in the CRTR-1 protein, transactivation studies using CRTR-1 deletion mutants fused to the GAL4 DNA binding domain and a GAL4-responsive reporter system were performed. These studies map repressor activity to amino acids 48-200, but fail to identify a transactivation domain within the CRTR-1 protein. In order to understand the mechanisms by which CRTR-1 regulates the transcriptional activities of CP2 family members, a number of approaches are taken, including co-immunoprecipitation to show that CRTR-1 interacts with other CP2-like proteins, EMSA which demonstrate that CRTR-1 forms DNA binding complexes with CP2 family members, and subcellular protein localisation studies which reveal the ability of CRTR-1 and other family members to shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. In addition, the subcellular localisation of CRTR-1 appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells. Co-expression of CRTR-1 with CP2 or NF2d9 results in the re-localisation of CRTR-1 to the cytoplasm in ES cells. The sumoylation enzymes Ubc9 and PIAS1 have previously been identified as CP2-interacting proteins (Kang et al., 2005a). Given the identification of two potential sumoylation sites within CRTR-1, FK³⁰ QE and LK⁴⁶ ⁴AE, and the ability for sumoylation to regulate transcription factor function, the possibility that CRTR-1 is regulated by sumoylation is investigated in this thesis. Immunoprecipitation experiments show that CRTR-1 is modified by SUMO-1 and that lysine 30 is the critical residue for this modification. Mutation of lysine 30 to alanine, which abolishes CRTR-1 sumoylation, results in enhancement of transactivation by CRTR-1, suggesting that sumoylation of CRTR-1 blocks maximal activation. Unexpectedly, however, overexpression of Ubc9, PIAS1, or SUMO-1 results in enhancement of CRTR-1 transcriptional activity, indicating that a more complex mechanism of regulation of CRTR-1 activity is likely. This thesis presents several novel properties of CRTR-1 and other CP2 family members, including the ability of CRTR-1, previously characterized as a repressor, to activate transcription. It is also the first demonstration that CP2 proteins are regulated by sumoylation and that they shuttle between the nucleus and cytoplasm via a CRM1-dependent mechanism.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Mouse CP2 is the founding member of a group of highly conserved proteins in mouse, human, chicken and Drosophila collectively referred to as the CP2 family of transcription factors. CP2 was originally identified in murine erythroleukemia (MEL) cell nuclear extracts as an activator of the mouse a-globin gene, binding a promoter element overlapping the CCAAT box. In addition Io CP2, the family includes mouse NF2d9, human LBP-1c and LBPla, homologues of mouse CP2 and NF2d9 respectively, LBP-9, chicken CPZ and Drosophila melanogaster CP2. Mammalian CP2-related proteins have generally been shown to be activators of transcription and expressed ubiquitously. There is little knowledge of factors that regulate their activity. This thesis describes CRTR-I, a novel member of the CP2 family, originally identified as a transcript differentially expressed between mouse ES and early primitive ectoderm-like (EPL) cells in vitro. In vivo expression analysis showed CRTR-I to be expressed by the pluripotent inner cell mass cells of the blastocyst, but not in the later pluripotent cells of the primitive ectoderm. Analysis during later stages of development and in the adult mouse showed CRTR-1 expression to be developmentally and spatially regulated. Greatest levels of CRTR-I expression were detected in the embryonic and adult kidneys with CRTR-I specifically expressed in the branching ureteric buds of the developing kidney with expression becoming restricted to the epithelial lining of the distal convoluted tubules during later kidney development and in the adult mouse. These sites of CRTR-I expression suggest distinct biological roles for CRTR-I function in the maintenance of pluripotency in the early stage embryo and in the development and function of the kidney. Conservation in nucleotide and amino acid sequence defined CRTR-I as a novel member of the mouse CP2 family of transcription factors. Consistent with this, CRTR-1 was shown to interact with all other members of the mouse CP2 family, forming protein complexes competent to bind a CP2 family consensus DNA response element. However, unlike other CP2 family members, CRTR-I was shown to act as a transcriptional repressor with the ability to repress transcription localised to a novel 52 amino acid N-terminal repression domain. Furthermore, the ability of CRTR-I to repress transcription was shown to be dominant over CP2 mediated transcriptional activation. CRTR-I is therefore distinct from other family members in two respects, CRTR-I expression is spatially and temporally regulated and CRTR-I acts as a dominant transcriptional repressor of CP2 family mediated transcriptional activation. This thesis also describes the identification, isolation and functional characterisation of an alternatively spliced isoform of CP2, altCP2. Similar to CRTR-1, altCP2 appears to be differentially expressed and was demonstrated to act as a dominant repressor of CP2 family mediated transcriptional activation by formation of heteromultimers with other CP2 family proteins that cannot bind DNA. Together, altCP2 and CRTR-1 provide mechanisms to achieve spatially and temporally regulated activity of ubiquitously expressed CP2 family transcriptional activators. Possible mechanisms regulating the cellular localization and transcriptional regulatory ability of CP2 family members were investigated by identification of nonrelated binding proteins. Yeast-2-hybrid analysis identified Ubc9, PIAS1 and FKBP4 as CRTR-I binding proteins. Ubc9 and PIASl function as regulators of post-translational modification by sumoylation. These have been shown to regulate the cellular localization and transcriptional regulation of other transcription factors, and were shown here to interact with other mouse CP2 family members. In contrast the immunophilin FKBP4 was determined to be a CRTR-I specific binding protein suggesting that altemate mechanisms regulate CRTR-I and other family members. Identification of CP2 family member specific binding proteins suggest mechanisms for independent regulation of CP2 family members through distinct cellular pathways. Finally, identification of the sumoylation enhancer PIAS3 and transcription factors Rexl and YYI as CRTR-I binding proteins provide further mechanisms for the regulation of CRTR-1 activity through the control of cellular localization, transcriptional regulation and promoter specificity and together suggest mechanisms for the regulation of CRTR-I activity through signaling pathways important for pluripotence and kidney development.
Thesis (Ph.D.) -- University of Adelaide, Dept. of Molecular Biosciences, 2003

La fosforilazione reversibile di proteine tilacoidali negli organismi fotosintetici è un meccanismo per far fronte a condizioni di luce variabili. Nelle monocotiledoni, contrariamente a quanto avviene nelle dicotiledoni, è stato dimostrato che la fosforilazione dell’antenna minore CP29, in seguito ad esposizione ad alte intensità luminose, induce un incremento in NPQ e diminuisce la produzione di specie reattive dell’ossigeno (ROS). La chinasi del complesso di antenne maggiori (LHCII), denominata STN7, e la relativa fosfatasi TAP38/PPH1, non partecipano in questo meccanismo, come in precedenza dimostrato nel nostro laboratorio, suggerendo l’ipotesi che un set di chinasi/fosfatasi differente fosse coinvolto nella regolazione di questo fenomeno. Recentemente, abbiamo analizzato un mutante knockout OsSTN8, gentilmente concessoci dal laboratorio di CH Lee, dimostrando che in aggiunta alla fosforilazione del core del Fotosistema II (PSII), anche quella di CP29 era soppressa, così provando che STN8 è la chinasi coinvolta nella fosforilazione di CP29 nelle monocotiledoni. Per meglio analizzare l’attività di OsSTN8 abbiamo trasformato linee mutanti di A. thaliana, dove il meccanismo di fosforilazione di CP29 in alta luce è assente, data la presenza di una libreria di mutanti molto vasta e dalla facilità con cui questa specie è geneticamente manipolabile rispetto a riso. I mutanti di A. thaliana stn8 e stn7stn8 complementati con OsSTN8 mostravano un recupero della fosforilazione del PSII core, come dimostrato tramite analisi Western blot. In aggiunta, la chinasi di riso fosforilava anche CP29 in condizioni di alta luce, contrariamente a quanto osservato nelle linee wild type. Misure di NPQ sono state eseguite sulle linee trasformate al fine di valutare l’effetto della fosforilazione dell’antenna minore sulla fotoprotezione. Un lieve incremento è stato osservato nelle linee trasformate con la chinasi di riso, indicando un possibile contributo di P-CP29 nella fotoprotezione in condizioni di di alta luce. Per meglio discernere il contributo di P-CP29 dalla fosforilazione del PSII core, mutanti Atlhcb4 sono stati co-trasformati con OsSTN8 e CP29 di riso o Arabidopsis, sia nella forma nativa che mutata al sito di fosforilazione Thr-83, ossia il sito identificato in riso come target della chinasi STN8. Un 6X-histag è stato addizionato alle proteine espresse per facilitare i processi di purificazione e permette analisi spettroscopiche delle forme fosforilate e non di CP29. Linee transgeniche sono state recentemente ottenute e misure fisiologiche saranno eseguite, sia in vivo che in vitro a seguito della purificazione della proteina. In A. thaliana la fosfatasi PBCP (Photosystem II Core Phosphatase) è stata dimostrata essere responsabile della defosforilazione del PSII core, controbilanciando l’attività di STN8. La proteina ricombinante OsPBCP da noi ottenuta era in grado di defosforilare in vitro sia le proteine del core che CP29 presenti nei tilacoidi e nelle preparazioni di complessi isolati da gradiente di saccarosio. Alla luce di questi risultati, abbiamo dimostrato che STN8 e CP29 sono, rispettivamente, la chinasi e fosfatasi coinvolte nella fosforilazione di CP29 nelle monocotiledoni, e OsSTN8 conserva la sua attività quando espressa in una dicotiledone come Arabidopsis thaliana.
Reversible phosphorylation of thylakoid proteins in photosynthetic organisms is a way to cope with changing light conditions. It has been demonstrated that in monocots, as opposed to dicots, upon high light exposure the minor antenna CP29 is phosphorylated enhancing NPQ and reducing singlet oxygen production. The major light-harvesting complex II (LHCII) kinase STN7 and its related phosphatase PPH1/TAP38 have been proven not to be involved in this mechanism in monocots, indicating that a different set of kinases/phosphatases act in regulating this acclimatory response. Recently, we have analyzed an OsSTN8 knockout mutant, kindly provided by the laboratory of CH Lee, in which we determined that in addition to that of the PSII core proteins, CP29 phosphorylation was suppressed as well, thus proving that STN8 is the kinase involved in CP29 phosphorylation in monocots. To further investigate OsSTN8 activity we transformed A.thaliana mutant lines, where CP29 phosphorylation is absent in high light, given the availability of mutant libraries and the ease with which this species is manipulable compared to rice. A.thaliana stn8 and stn7stn8 mutants transformed with OsSTN8 restored phosphorylation of the PSII core proteins, as confirmed through immunoblot analysis. Furthermore, the kinase was able to phosphorylate CP29 under high light conditions, as opposed to the wild type strain. Non-Photochemical Quenching (NPQ) measurements were performed on the transformed lines to assess the effect of the minor antenna phosphorylation on photoprotection, showing a mild increase in NPQ. To better understand the individual contribution of CP29 phosphorylation in transgenic Arabidopsis apart from that of PSII core phosphorylation in high light, knockout lines for Lhcb4 of A.thaliana were co-transformed in order to express OsSTN8 and CP29 either from rice or A.thaliana, both in its native and mutated form at Thr-83, site of phosphorylation in rice. A 6X-Histag was added for improved purification in order to conduct spectroscopic analyses on phosphorylated and unphosphorylated forms of CP29. Transgenic lines were recently obtained and physiological analyses will be performed in the near future, both in vivo and in vitro through purification of the protein. In A.thaliana the phosphatase PBCP was determined to be involved in PSII core dephosphorylation and counteract the effect of STN8. Our recombinant OsPBCP was capable of dephosphorylating in vitro both PSII core proteins and CP29, in thylakoids as well as isolated complexes from a sucrose gradient. In light of these results, we have determined that STN8 and PBCP are respectively the kinase and phosphatase involved in CP29 phosphorylation in monocots, and OsSTN8 retains its activity when expressed in a dicot such as Arabidopsis thaliana.

Nickel Nanostrands (NiNS) are nano-particles that are highly branched and have a high aspect ratio. These particles show promise as excellent additives to composites when electrical conductivity is desired. Unfortunately, there is very little research done on dispersing powdered NiNS in various polymer matrices. This thesis covers the research in dispersing NiNS in three separate polymer systems, and related composite processing and characterization. An aromatic polyimide (CP2) is first used as a thermoplastic matrix and attempts to incorporate NiNS via an in-situ processing technique concurrent with in-situ polymerization are detailed. Epoxy is then used as a representative thermoset where the NiNS are dispersed in the resin before a hardener is added. The last polymer tested is thermoplastic Polyvinylidene Fluoride (PVDF). NiNS are introduced to this polymer in a solution mixture. Once dispersed, the PVDF solution is heated until the solvent evaporates leaving a PVDF melt containing NiNS, which is subsequently cooled. Samples of all three polymer nano-composites are created and dispersion is observed with an optical microscope. Using DSC, DMA and dielectric spectroscopy, thermal, mechanical and electrical properties are measured and analyzed. Results for the CP2 nano-composites showed that during the cure phase, the NiNS settled to the bottom of the films resulting in a non-dispersed composite. This result highlighted the difference between NiNS and other more conventional nano-particles, namely that the NiNS are larger and heavier, therefore are not 'locked into' a dispersed state by the polymer chains. Several techniques were investigated for dispersing NiNS in the epoxy matrix. A method without solvent was shown to be the most effective and resulted in a well-dispersed nano-composite that showed increases in electrical conductivity and dielectric constant as NiNS concentration increases. Enhancement in storage modulus was observed above the composite's Tg as well. PVDF nano-composites also showed good dispersion and a general increase in electrical properties. Below Tg, storage modulus decreases at first before a slight recovery with increasing NiNS. Beyond Tg, the opposite effect is observed. FTIR measurements for the PVDF were also taken and showed no significant changes in the polymer morphology with additions of NINS.