Dissertations / Theses on the topic 'Shadow protein'

Prion protein (PrP) is best known for its involvement in prion diseases. A normal, dynamic isoform of prion protein (PrP^C) transforms into a pathogenic, compact isoform (PrP^Sc) during prion disease pathogenesis. The PrP^Sc, acting as a template upon which PrP^C molecules are refolded into a likeness of itself, accumulates in the brain neurones and causes disease. It is the only known component of prions, proteinaceous infectious particles. Both prion protein isoforms have the same primary amino acid structure and are encoded by the same prion protein gene (PRNP). PRNP determines susceptibility/disposition to prion diseases and their phenotypes.¶The normal function of PRNP is elusive. The Prnp knock-out mice with disrupted ORF show only very subtle phenotype. A number of hypotheses were proposed on the function of mammalian PRNP. The extracellular, GPI-anchored, glycosylated mammalian PrP^C expressed in a heterogenous set of cells could: transport copper from extracellular to intracellular milieu, buffer copper from synapse, contribute to redox signalling, act neuroprotectively, mediate cell-cell contacts, affect lymphocyte activation, participate in nucleic acid metabolism, be a memory molecule, and be a signal-transduction protein.¶ Experimental evidence demonstrated a redundancy between the PRNP and another, unknown gene. The critical issue therefore is to discover new genes homologous with PRNP, candidates for this redundancy. Using unpublished data, a sequence of zebrafish cDNA sequenced by Prof. Tatjana Simonic’s group (University of Milan, Italy), I discovered a new paralogue of PRNP. By searching manually, and in a targeted fashion, data deposited in public biological databases, I compiled support for the new human gene Shadow of prion protein (SPRN) including the direct evidence, homology-based evidence and ab initio gene prediction. The protein product called Shadoo (shadow in Japanese) is an extracellular, potentially glycosylated and GPI-anchored protein of a mature size of 100-odd amino acids. It is conserved from fish (zebrafish, Fugu, Tetraodon) to mammals (human, mouse, rat), and exhibits similarity of overall protein features with PrP. Most remarkably, the Sho is the first human/mammalian protein apart from PrP that contains the middle hydrophobic region that is essential for both normal and pathogenic properties of PrP. As this region is critical for heterodimerization of PrP, Sho may have potential to interact with PrP and is a likely candidate for the Protein X. Mammalian SPRN could be predominantly expressed in brain (Tatjana Simonic Lab, University of Milan, Italy).¶ Using the same approach to search public databases, I found, in addition, a fish duplicate of SPRN called SPRNB, and defined a new vertebrate SPRN gene family. Further, I also expanded a number of known fish genes from the PRNP gene family. The total number of the new genes that I discovered is 11. With the representatives of two vertebrate gene family datasets in hand, I conducted comparative genomic analysis in order to determine evolutionary trajectories of the SPRN and PRNP genes. This analysis, complemented with phylogenetic studies (Dr. Lars Jermiin, University of Sydney, Australia), demonstrated conservative evolution of the mammalian SPRN gene, and more relaxed evolutionary constraints acting on the mammalian PRNP gene. This evolutionary dialectic challenges widely adopted view on the “highly conserved vertebrate” PRNP and indicates that the SPRN gene may have more prominent function. More conserved Sprn could therefore substitute for the loss of less conserved, dispensable Prnp in the Prnp knock-out mice. Furthermore, the pathogenic potential of PRNP may be a consequence of relaxed evolutionary constraints.¶ Depth of comparative genomic analysis, strategy to understand biological function, depends on the number of species in comparison and their relative evolutionary distance. To understand better evolution and function of mammalian PRNP, I isolated and characterized the PRNP gene from Australian model marsupial tammar wallaby (Macropus eugenii). Marsupials are mammals separated from their eutherian relatives by roughly 180 million years. Comparison of the tammar wallaby and Brazilian opossum PrP with other vertebrate PrPs indicated patterns of evolution of the PrP regions. Whereas the repeat region is conserved within lineages but differs between lineages, the hydrophobic region is invariably conserved in all the PrPs. Conservation of PrP between marsupials and eutherians suggests that marsupial PrP could have the same pathogenic potential as eutherian PrPs. Using the marsupial PRNP gene in comparison with the PRNP genes from eutherian species in which prion diseases occur naturally (human, bovine, ovine) or experimentally (mouse), I defined gene regions that are conserved mammalian-wide and showed the utility of the marsupial genomic sequence for cross-species comparisons. These regions are potential regulatory elements that could govern gene expression and posttranscriptional control of mRNA activity. These findings shed new light on the normal function of mammalian PRNP supporting best the signal-transduction hypothesis. The normal function of PRNP may be triggering of signalling cascades which contribute to cell-cell interactions and may act anti-apoptotically. Yet, in the heterogenous set of cells expressing PrP^C these pathways will contribute to a number of cell-specific phenotypes, such as the synaptic plasticity and activation of lymphoid cells.

La protéine Shadoo (Sho) appartient à la famille des. Protéines prion. Les protéines Sho et PrP partagent de nombreuses similitudes, allant de leur co-localisation anatomo-histologique à une forte similarité de séquence. L'inhibition de l'expression de Sho durant l'infection à prion, ainsi que l'interaction entre ses deux protéines observée par double hydride, laissent suggérer une relation étroite entre ses deux protéines pendant la phase de réplication du prion. L'objectif de ce travail de thèse est d'explorer le rôle de la protéine Sho dans la dynamique conformationnelle de PrP, ainsi que l'effet d'une telle interaction sur le phénomène de réplication. La caractérisation de la structure secondaire de montre que Sho est une protéine naturellement désordonnée entre les pH 3. 5 et 8. Le spectre RMN de Sho correspond au spectre typique des protéines non repliées, même-si Sho semble adopter une structure organisée localement au niveau du segment C-terminal. D'autre part, l'utilisation de technique de biochimies et de biophysiques nous ont permis de démontrer que Sho et PrP formaient un complexe 1 :1 avec un Kd de l'ordre du micromolaire. Cette interaction a pour conséquence une altération du repliement de PrP. L'utilisation de la forme tronquée de PrP, mimant le fragment Cl, nous a permis d'observer une liaison allostérique avec un nombre de hill de 4, une tétramérisation. La co-polymérisation semble avoir pour conséquence une augmentation du pouvoir infectieux des particules prion en culture de cellules. Nos résultats montre que la protéine Sho affecte la réplication du prion, soit en agissant comme une holdase, soit en interférant avec le rôle protecteur du fragment Cl
Shadoo (Sho) protein belongs to prion protein family. From the histo-anatomical co-localization and sequence similarities, it is suspected that Sho and PrP may be functionally related. Down regulation of Sho expression during prion pathology in one hand, and direct interaction between Sho and PrP, revealed by two-hybrid assay, suggests a relation between Sho and prion replication. The objective of this PhD thesis was to explore involvement of Sho in conformational dynamics of PrP and its effect on conversion process involved in prion replication. Secondary structure characterization of Sho revealed that Sho is a disordered protein between 3. 5

Pequenos e médios laticínios têm grandes dificuldades no planejamento da produção para maximizar o lucro. Além disso, a forma atual de precificar o leite cru no Brasil desestimula o produtor a melhorar sua qualidade, pois valoriza mais seu volume do que seus componentes sólidos. A composição do leite cru é muito importante para os laticínios, pois ela afeta diretamente o rendimento de produção e a qualidade dos produtos lácteos. Técnicas de otimização, como programação linear (PL), ajudam a resolver problemas relacionados à decisão do mix de produtos, além de fazer análise econômica dos recursos. Em razão disso, foi desenvolvido em PL um modelo para maximização da margem de contribuição total (MCT = receita total das vendas - custos e despesas diretas totais) e precificação do leite cru através da determinação do mix ótimo de produtos lácteos. O modelo foi simulado em cenários diários de alta (Janeiro) e baixa (Julho) disponibilidade de matéria prima comparando as soluções ótimas com resultados reais de um laticínio do sudeste do Brasil. Foram realizadas análises de sensibilidade dos componentes nutritivos de dois tipos de leite cru de diferentes composições (LC1, leite cru proveniente de um fornecedor, e LC50, leite cru proveniente de 50 fornecedores) para determinar seus preços-sombra. Foram observados preços-sombra para o quilograma da caseína em Janeiro e Julho e para o litro do LC50 em Julho. A MCT ótima e os preços calculados de ambos os tipos de leite cru foram maiores em Janeiro devido à maior disponibilidade de matéria prima. Em ambos os cenários, os preços calculados de ambos os tipos de leite cru foram maiores que os praticados pelo laticínio e o lucro antes de juros, impostos, depreciação e amortização (LAJIDA) diário real foi maior que o ótimo. A embaladora foi um recurso limitante atuante na produção do queijo Minas Frescal nos mixes ótimos. A relação entre margem de contribuição unitária (MCU = preço unitário de venda - custo e despesa diretos unitários) e quantidade necessária de matéria prima por unidade de produto e a disponibilidade de recursos são determinantes na definição do mix de produtos lácteos e da MCT do laticínio. A precificação do leite cru pelo modelo proposto remunera o produtor em função da quantidade de seus componentes. O laticínio pode maximizar seu LAJIDA planejando melhor seu mix de produtos com PL e remunerando melhor seus fornecedores em função da qualidade do leite cru.
Small and medium-sized dairies face great difficulties in production planning to maximize profit. In addition, the current way of pricing the raw milk in Brazil discourages producers to improve its quality because its volume values more than its solid components. The raw milk composition is very important for the dairies because it directly affects the yield and quality of dairy products. Optimization techniques, such as linear programming (LP), aid solve problems related to the decision of the product mix and perform economic analyzes of resources. For this reason, a model was developed in LP to maximizing total contribution margin (TCM = total sales revenues - total variable costs and expenses) and pricing the raw milk by determining the optimal mix of dairy products. The model was simulated in two daily scenarios of high (January) and low (July) availability of raw material comparing the optimal solutions with actual results from a dairy plant in Southeastern Brazil. Sensitivity analyzes of the nutritional components of two kinds of raw milk of different compositions (RM1 and RM50) were performed to determine their shadow-prices. Shadow-prices were observed for the kilogram of casein in January and July and for the liter of RM50 in July. The optimal TCM and the calculated prices of both types of raw milk were higher in January due the increased availability of raw materials. In both scenarios, the calculated prices of both types of raw milk were higher than those paid by the dairy plant and the actual daily earnings before interests, taxes, depreciation and amortization (EBITDA) were greater than the optimum ones. The wrapper was an active limiting resource in the production of Frescal Minas cheese in optimal mixes. The relationship between unit contribution margin (UCM = unit sales price - unit variable cost and expense) and required amount of raw material per unit of output and resource availability are crucial in defining the mix of dairy products and the dairy TCM. The proposed raw milk pricing model pays the producer by the quantity of its components. The dairies can maximize their EBITDA planning their product mixes with LP and remunerating their suppliers based on the quality of raw milk.

[EN] DELLA proteins are plant-specific transcriptional regulators known to relay environmental information to the transcriptional networks to modulate growth and development accordingly. The view of DELLAs as signalling hubs is justified by two characteristics: first, they control the activity of a large number of transcriptional factors (TFs) and other transcriptional regulators through physical interaction; and, second, they are degraded by the 26S proteasome in response to the phytohormone gibberellin (GA), whose metabolism is very sensitive to environmental stimuli (e.g. light, temperature, salt stress). However, at least two observations indicate that this mechanistic framework is still incomplete: (i) warm temperature destabilizes the GA-insensitive DELLA rga-¿17, indicating that DELLAs cannot be destabilized only by changes in GA levels; and (ii) when found at the chromatin, DELLAs are localized not only in gene promoters, but also in gene bodies, suggesting that DELLAs may regulate transcription through interactions with proteins other than TFs. In this Thesis, we provide evidence that shows how a different E3 ubiquitin ligase controls the stability of DELLAs in a GA-independent manner, and how DELLAs regulate gene expression by directly interacting with the basal transcriptional machinery. In the first chapter, using a combination of genetic, physiological, and molecular approaches we demonstrate that DELLAs are targeted to proteolytic degradation by the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). We show that COP1 interacts with the DELLAs GAI and RGA in vitro and in vivo, and that it promotes their polyubiquitination. We propose that COP1 represents a major pathway to degrade DELLAs in response to shade or to warm temperature. In the second chapter, we describe the interaction between DELLAs and the transcription elongation complex Polymerase-Associated Factor 1 (Paf1c). We show that Paf1c is required for the genome-wide deposition of monoubiquitinated H2B (H2Bub), a mark necessary for the progression of RNA polymerase II (RNAPII), and that this function is largely dependent on the presence of DELLAs. Likewise, impaired Paf1c or DELLA function results in a similar alteration in the accumulation and distribution of RNAPII in the Paf1c-target genes. We propose that DELLAs would exert this action by modulating the recruitment of Paf1c to the chromatin. These two new mechanisms underscore the importance of DELLAs as a central node in the environmental signalling network and should be considered in any potential application of DELLAs as biotechnological targets.
[ES] Las proteínas DELLA son reguladores transcripcionales específicos de plantas que transmiten información ambiental a las redes transcripcionales que modulan el crecimiento y el desarrollo. La propuesta de que las DELLAs actúan como "hubs" en redes de señalización se justifica por dos razones: primero, controlan la actividad de un gran número de factores de transcripción (FTs) y otros reguladores transcripcionales mediante interacción física; y segundo, son degradadas por el proteosoma 26S en respuesta a la fitohormona giberelina (GA), cuyo metabolismo es muy sensible a los estímulos ambientales (p. ej. luz, temperatura, estrés salino). Sin embargo, al menos dos observaciones sugieren que la información relativa a estos mecanismos no es completa: (i) las temperaturas altas desestabilizan incluso a rga-¿17, una versión de DELLA insensible a GAs, lo que indica que la estabilidad de las DELLAs no depende sólo de cambios en los niveles de GAs; y (ii) cuando se encuentran en la cromatina, las DELLAs no solo se posicionan en los promotores, sino también a lo largo de las regiones codificantes, lo que sugiere que las DELLAs podrían regular la transcripción mediante interacciones con otras proteínas diferentes a FTs. En esta Tesis, proporcionamos evidencia sobre una nueva E3 ubicuitina ligasa que controla la estabilidad de las DELLAs de una manera independiente a las GAs, y cómo las DELLA regulan la expresión génica interaccionando directamente con la maquinaria de transcripción basal. En el primer capítulo, usando una combinación de aproximaciones genéticas, fisiológicas y moleculares, demostramos que las DELLAs son marcadas para su degradación proteolítica por la E3 ubicuitina ligasa CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). Mostramos que COP1 interacciona al menos con las DELLAs GAI y RGA in vitro e in vivo, y que promueve su poliubicuitinación. Proponemos que COP1 representa una vía importante de degradación de DELLAs en respuesta a sombra y temperaturas altas. En el segundo capítulo, describimos la interacción entre las DELLAs y el complejo de elongación transcripcional Polymerase-Associated Factor 1 (Paf1c). Mostramos que, como en animales, Paf1c se requiere para la deposición a nivel genómico de la H2B monoubiquitinada (H2Bub), una marca necesaria para la progresión de la RNA polimerasa II (RNAPII), y que esta función depende en gran medida de la presencia de DELLAs. Asimismo, la reducción de la función de las DELLAs provoca defectos equivalentes a los de la pérdida de función de Paf1c en cuanto a la acumulación y distribución de la RNAPII en los genes diana de Paf1c. Proponemos que las DELLAs podrían por tanto regular la transcripción modulando el reclutamiento de Paf1c a la cromatina. Estos nuevos mecanismos inciden en la importancia de las DELLAs como nodos centrales en las redes de señalización al ambiente y podrían ser considerados como dianas biotecnológicas en aproximaciones futuras.
[CA] Les proteïnes DELLA són reguladors transcripcionals específics de les plantes conegudes per transmetre informació mediambiental a les xarxes transcripcionals per modular el creixement i desenvolupament. La visió actual de DELLAs com a "hubs" de senyalització es justifica per dues característiques: en primer lloc, controlen l'activitat d'un gran nombre de factors transcripcionals (FTs) i d'altres reguladors transcripcionals mitjançant la interacció física; i, en segon lloc, es degraden pel proteosoma 26S en resposta a la fitohormona giberel.lina (GA), el metabolisme de la qual és molt sensible als estímuls ambientals (per exemple, la llum, la temperatura, l'estrès salí). No obstant això, almenys dues observacions indiquen que aquest marc mecanicista encara és incomplet: (i) la temperatura càlida desestabilitza la DELLA rga-¿17, que és insensible a GAs, indicant que les DELLA no es desestabilitzen només per canvis en els nivells d'aquesta fitohormona; i (ii) quan es troben a la cromatina, les DELLA no només es localitzen en els promotors dels gens, sinó també a la regió que es transcriu, cosa que suggereix que poden regular la transcripció mitjançant interaccions amb proteïnes diferents de FTs. En aquesta tesi, proporcionem evidències que mostren com una ubiquitina E3 lligasa diferent controla l'estabilitat de les proteïnes DELLA de manera independent de GAs, i com les DELLA regulen l'expressió gènica interactuant directament amb la maquinària transcripcional basal. En el primer capítol, mitjançant una combinació d'enfocaments genètics, fisiològics i moleculars, demostrem que les DELLA s'envien a la degradació proteolítica mitjançant la ubiquitina E3 lligasa CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). Mostrem que la COP1 interacciona amb els DELLA GAI i RGA in vitro i in vivo, i que afavoreix la seva poliubiquitinació. Proposem que COP1 representa la via principal per degradar les DELLA en resposta a l'ombra o a la temperatura càlida. Al segon capítol, es descriu la interacció entre les DELLA i el complex d'allargament de transcripció Polymerase Associated Factor 1 (Paf1c). Mostrem que Paf1c es requereix per a la deposició a tot el genoma de H2B monoubiquitinada (H2Bub), una marca necessària per a la progressió de l'ARN Polimerasa II (RNAPII), i que aquesta funció depèn en gran part de la presència de DELLA. De la mateixa manera, quan la funció Paf1c o DELLA està deteriorada, es produeix una alteració similar en l'acumulació i distribució de RNAPII als gens diana de Paf1c. Proposem que les DELLA realitzen aquesta acció modulant el reclutament de Paf1c a la cromatina. Aquests dos nous mecanismes posen de manifest la importància de les proteïnes DELLA com a node central de la xarxa de senyalització ambiental i haurien de ser considerats en qualsevol aplicació potencial de DELLA com a objectius biotecnològics.
La realización de esta Tesis Doctoral ha sido posible gracias a un contrato predoctoral para la formación de doctores (BES-2014-068868) y a la ayuda a la movilidad predoctoral para la realización de estancias breves (EEBB-I-16-11070) del Ministerio de Economía y Competitividad, y a una beca EMBO Short-Term (number 8047).
Blanco Touriñán, N. (2020). New mechanisms of DELLA protein regulation and activity in Arabidopsis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/149477
TESIS

Potassium ion channels play a key role in the generation and propagation of action potentials. The S4-S5 linker peptide (L45) is believed to be responsible for the anesthetic/alcohol response of voltage-gated K+ channels. We investigated this region to define the structural basis of 1-alkanol binding site in dShaw2 K+ channel. L45 peptides derived from dShaw2 and hKv3.4 K+ channel, which, if part of the complete channel, demonstrate different sensitivity to 1-alcohols. Specifically, dShaw2 is alcohol sensitive and hKv3.4 is alcohol resistant. Structural analysis of L45 with NMR and CD suggested a direct correlation between alpha-helicity and the inhibition of dShaw2 channel by 1-butanol. We used CD and NMR to determine the structure of L45 peptides in micelles and vesicles. We measured spin-lattice relaxation time (T1) and determined the location and surface accessibility of L45 in micelles. These experiments confirm that L45 of dShaw2 adopts an α-helical conformation, partially buried in the membrane and parallel to the surface. The binding and accumulation of rev proteins to an internal loop of RRE (rev responsive element) of unspliced mRNA precursors is a key step of propagation of human immunodeficiency (HIV) virus. Molecules that interfere with this process can be expected to show anti-HIV activity. Our work is based on an assumption that zinc fingers could compete with rev proteins, therefore impeding the life cycle of HIV and stopping its infection. We studied the influence of different cations, anions, and the concentration of salts and osmolytes on the binding affinity with Polyacrylamide Gel Electrophoresis (PAGE) and Isothermal Titration Calorimetry (ITC). We conclude that the types of anions and/or cations and their concentrations affect the enthalpy and entropy of the binding interacitons. Using a gel assay, we confirm that there are three products in RNA-Protein reaction, and both EDTA and salts (and their concentrations) in the gel or samples interfere with RNA-protein complex mobility.

Plantas de café Arábicas com 4,5 anos de idade foram comparadas de agosto a dezembro crescendo a pleno sol e sob condições de sombreamento natural. As plantas apresentaram diferenças significativas em termos de crescimento e aspectos fisiológicos e bioquímicos O fornecimento de nitrogênio no mês de outubro proporcionou um aumento significativo nos teores de aminoácidos totais e na atividade da redutase do nitrato. As folhas de plantas crescendo em condições de sombreamento natural apresentaram maior peso fresco a que pleno sol. O teor de proteínas totais decresceu abruptamente a partir do mês 10 em todas as plantas analisadas, época em coincidiu com o inicio do enchimento dos grãos. O aumento nos teores de aminoácidos totais também foi acompanhado por um aumento nos dias de chuva, na precipitação e também na radiação global. Não houve efeito significativo com relação á localização da folhas (parte superior x parte inferior) nos aspectos analisados. O fornecimento de nitrogênio no mês 10 não alterou a concentração de clorofila medida pelo clorofilômetro SPAD, e a clorofila total extraída não se alterou significativamente até o mês 11 exceto nas plantas de sol as folhas do parte superior tiveram uma maior síntese no mês 9. No mês 12 houve um aumento nas taxas de clorofila em todos os tratamentos. Os teores de N foliar tiveram correlação positiva com as leituras de clorofila analisadas pelo SPAD, ate o mês 11. Tivemos também no período de analise, um aumento significativo na concentração de carotenóides a partir do mês 8, evidenciando seu efeito foto protetor uma vez que houve também um aumento na radiação global no período ao redor de 50%. As folhas em condições de sombreamento natural apresentaram maior peso seco que as de pleno sol. O teor de proteínas totais decresceu abruptamente a partir do mês 11 em todas as plantas, época que coincidiu com o início da produção/enchimento dos grãos. Houve um aumento nos teores de amido após o mês 10 (exceto nas folhas da parte inferior das plantas de sol), vindo a diminuir em função do enchimento dos grãos. Também se observou uma síntese acentuada de açúcares redutores no início, decrescendo em função do início da formação dos grãos. Com relação aos nutrientes nas folhas analisadas, o manganês teve um comportamento significativamente diferente em relação aos demais, onde nas folhas a pleno sol a concentração do elemento foi de 6 a 8 vezes á das folhas sombreadas.
Plants of Arabian coffee with 4,5 years of age, had been compared from August to December growing under full sun and under natural shade conditions. The plants presented significant differences in growth terms and physiological and biochemical's aspects. The nitrogen supply in the October month provided a significant increase on levels of amino acid totals and the activity of nitrate redutase. The leaves of plants growing under natural shade presented greater fresh weight than full sun. The total protein levels abruptly decreased from the month 10 in all analyzed plants, time in coincidence with the beginning of filling up the grains. The increase on levels of total amino acids also was followed by an increase in the days of rain, the precipitation and also the global radiation. Didn't have significant effect with relation of localization of leaves (upper part X inferior part) within analyzed aspects. The nitrogen supply in month 10 did not modify the concentration of chlorophyll measured by chlorophyll meter SPAD, and chlorophyll total extracted did not change significantly until month 11 except in the sunny plants leaves of upper part that had a bigger synthesis in month 9. In month 12 had an increase on the levels of chlorophyll in all the treatments. The levels of foliar N had had positive correlation with the readings of chlorophyll analyzed by the SPAD, until month 11. We had also in the period of analyzes, a significant increase on the concentration of carotenoids from month 8, evidencing its photo protective effect. During this time also we had an increase in the global radiation in the period around 50%. The leaves in conditions of natural shade had presented greater dry weight that of full sun. The total protein levels abruptly decreased from the month 11 in all plants, time coincident with the beginning of the production/filling up of the grains. The Month 10 also had an increase in starch levels (except in leaves of third inferior of the sunny plants), decreasing in function of the filling up of the grains. Also an increased synthesis of reducing sugars was observed at the beginning and a decrease during the beginning of the formation of the grains. With regard to the nutrients in analyzed leaves, the manganese had a behavior significantly different in relation to others, where in leaves at the full sun the concentration of the element was 6 to 8 times higher than shaded leaves.

Prion protein (PrP) is best known for its involvement in prion diseases. A normal, dynamic isoform of prion protein (PrP^C) transforms into a pathogenic, compact isoform (PrP^Sc) during prion disease pathogenesis. The PrP^Sc, acting as a template upon which PrP^C molecules are refolded into a likeness of itself, accumulates in the brain neurones and causes disease. It is the only known component of prions, proteinaceous infectious particles. Both prion protein isoforms have the same primary amino acid structure and are encoded by the same prion protein gene (PRNP). PRNP determines susceptibility/disposition to prion diseases and their phenotypes. ¶ ... ¶ Depth of comparative genomic analysis, strategy to understand biological function, depends on the number of species in comparison and their relative evolutionary distance. To understand better evolution and function of mammalian PRNP, I isolated and characterized the PRNP gene from Australian model marsupial tammar wallaby (Macropus eugenii). Marsupials are mammals separated from their eutherian relatives by roughly 180 million years. Comparison of the tammar wallaby and Brazilian opossum PrP with other vertebrate PrPs indicated patterns of evolution of the PrP regions. Whereas the repeat region is conserved within lineages but differs between lineages, the hydrophobic region is invariably conserved in all the PrPs. Conservation of PrP between marsupials and eutherians suggests that marsupial PrP could have the same pathogenic potential as eutherian PrPs. Using the marsupial PRNP gene in comparison with the PRNP genes from eutherian species in which prion diseases occur naturally (human, bovine, ovine) or experimentally (mouse), I defined gene regions that are conserved mammalian-wide and showed the utility of the marsupial genomic sequence for cross-species comparisons. These regions are potential regulatory elements that could govern gene expression and posttranscriptional control of mRNA activity. These findings shed new light on the normal function of mammalian PRNP supporting best the signal-transduction hypothesis. The normal function of PRNP may be triggering of signalling cascades which contribute to cell-cell interactions and may act anti-apoptotically. Yet, in the heterogenous set of cells expressing PrP^C these pathways will contribute to a number of cell-specific phenotypes, such as the synaptic plasticity and activation of lymphoid cells

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Shadoo (Sho) is a member of the prion protein (PrP) family, found mainly in the brain. PrP is well-known as the central agent involved in the prion diseases - a form of fatal neurodegenerative disease. Despite decades of intense research, the natural functions of PrP have not been clearly elucidated. The reasons for this are complex, but it is likely that there is a level of redundancy in the functions performed by PrP. In particular, it has been suggested that Sho may have overlapping functions with PrP and that both proteins can compensate for one another. Therefore, investigating the natural function/s of Sho may provide some insight into the roles that the non-pathogenic form of PrP may play in vivo. Although the natural function of Sho is currently unknown, recent studies point to it having a role in neural tube development and neuroprotection. Comparative genomics is a powerful technique for providing insight into functionally important protein domains. Comparing Sho protein sequences over the course of evolution highlighted a strongly conserved sequence at the beginning of the N-terminus. Literature-based data mining led me to hypothesize that it may constitute an RGG box, a known RNA binding motif. This hypothesis provides an interesting link to PrP, which can bind RNA, although an RNA binding function has not yet been identified. However, the RNA binding region of PrP, also located at the beginning of the N-terminal region, does not have the characteristics of an RGG box. This thesis reports the results of a series of studies designed to test the hypothesis that Sho has an RGG box and establish the plausibility that Sho may play a functional role as an RNA-binding protein. The RGG box of Sho has strong sequence similarity to the RGG box of the Fragile X Mental Retardation Protein (FMRP), which is known to bind a range of mRNAs and play an important role in neural plasticity. Comparison of the RGG boxes of Sho and FMRP reveals that, like many RNA binding domains, the RGG boxes of Sho and FMRP lie within disordered protein domains. This work examines the nature of these flexible protein domains using molecular dynamics (MD) simulations. The RGG box of FMRP has an affinity for G-quadruplex RNA, which is also the form of RNA that binds most strongly to PrP. Here, MD simulations and biophysical experiments are used to investigate whether Sho, too, binds G-quadruplex RNA. Binding of 5 different RNA transcripts to the RGG box regions of Sho and FMRP and the N-terminus RNA binding domain of PrP was explored through circular dichroism, fluorescence and surface plasmon resonance experiments. The results of both MD simulations and biophysical experiments show that a peptide derived from the RGG box region of Sho is capable of binding G-quadruplex RNA with physiologically relevant affinity. The Sho RGG box peptide binds to certain RNA transcripts with similar affinity to peptides comprising the FMRP RGG box and the RNA-binding region of PrP. However, some differences in RNA-binding affinities across all three peptides also indicate their ability to discriminate between different RNA targets. Overall, the findings reported here suggest that Sho is likely to function, in some capacity, as an RNA-binding protein. As Sho and PrP are capable of binding G-quadruplex RNA with similar affinities, it is possible that they share an RNA binding function. Given the primary location of Sho and PrP on the outer cell membrane it seems most plausible that they bind extracellular RNA in a signaling context. This finding is particularly interesting as recent research indicates that RNA may be a cofactor in the conversion of PrP to its disease-producing isoform. There is also growing interest in the role of extracellular RNA as a target for cell surface receptors. Future studies to identify likely RNA binding partners for Sho provide a promising avenue for elucidating the natural function of this protein.

碩士
國立中山大學
海洋生物研究所
100
Xenograpsus testudinatus is a dominant species in shallow hydrothermal vents off Kueishan Island, Taiwan. But we know little about crab populations in vent areas. In this study, stable isotope and one dimension protein expression profiles were studied to evaluate the differences of X. testudinatus from different habitats, i.e. white and yellow vents. Results indicated food sources between the two habitats were different in 2009, but similar in 2010. The ranges of δ15N value of X. testudinatus were broad which might result from abundant food in white vent. Furthermore, zooplankton might not be the major food source of X. testudinatus. Significant difference in protein expression profiles between habitats indicate that they adapt to different habitats in small scales with migration between habitats. And, crabs may have other habitats outside vent areas. After culture in laboratory for 12 hours, crab protein expression profiles were different from their original ones, indicating their acclimation to a new environment is relative fast.