Le déterminisme de la formation de la grappe reste parmi les phénomènes les moins bien précisés de la physiologie de la Vigne. La question a été abordée de plusieurs façons mais les éléments de réponse obtenus ne permettent pas encore de se faire une idée synthétique des mécanismes fondamentaux qui produisent l’apparition des primordia inflorescentiels (PI) en année N-1 et qui pourraient régir jusqu’à 65 à 70% du rendement final en année N. Notre étude s’est donc focalisée sur (i) la caractérisation du processus de formation des PI dans les bourgeons latents de vigne (Vitis vinifera L. Merlot) et la redéfinition de la constitution du rendement, (ii) la compréhension des mécanismes d’initiation et de différenciation des PI et (iii) l’évaluation des impacts de différents facteurs (ABA exogène, lumière et effeuillage) sur ces mécanismes.Cette étude intégrale combinant des approches multi-échelles et multi-disciplinaires a permis dans un premier temps de mettre en évidence le déterminisme de la phase d’induction inflorescentielle dans la construction du rendement en identifiant trois phases clés du processus de formation des PI et en y intégrant une nouvelle notion qu’est le « degré de ramification des PI » ; puis dans un second temps, de proposer certains modes de régulation des mécanismes d’initiation et de différenciation des PI en identifiant trois types de marqueurs précoces de la fertilité potentielle : des marqueurs moléculaires (VvCLE25-1, VvTFL1a et VvMFT1), biochimiques (équilibre CKs/AIA, D-glucose et amidon), et environnementaux (ensoleillement et température journaliers cumulés). L’ensemble des résultats pourra contribuer au développement d’un outil prédictif précoce de la « fertilité potentielle » (et donc du rendement probable maximum) afin de mieux guider et gérer la taille et les pratiques culturales. / The grape cluster formation determinism remains the one of the undefined phenomena in grapevine physiology process. The topic has been tackled in multiple ways. However, based on all the known information, we still are not allowed to get a synthetic idea of the fundamental mechanisms that produce the appearance of inflorescential primordia in year N-1 and which could govern up to 65 to 70 % of final yield in year N. Our study focuses thus on (i) the characterization of IP formation process in grapevine latent buds (Vitis vinifera L. Merlot) and the redefinition of yield constitution, (ii) understanding IP initiation and differentiation mechanisms, and (iii) evaluating the impacts of different factors (exogenous ABA, light and leaf removal) on these mechanisms.This comprehensive study, which combines multi-scale and multi-disciplinary approaches, firstly allowed us to highlight the determinism of inflorescence induction phase in yield construction by identifying three key phases in IP formation process and by incorporating a new notion which is "IP branching degree". Secondly, it proposed certain modes of regulation of IP initiation and differentiation mechanisms by identifying three types of early markers of bud fruitfulness: molecular markers (VvCLE25-1, VvTFL1a and VvMFT1), biochemical markers (CKs/IAA balance, D-glucose and starch), and environmental markers (cumulative daily sunshine and temperature).The all-inclusive results contribute to the development of an early predictive tool of "bud fruitfulness" (and thereby the probable maximum yield) to better guide and manage the grapevine training and other cultural practices.

Doenças envolvendo órgãos endodermicamente derivados afetam milhares de pessoas no mundo. O sucesso da terapia celular para o tratamento de doenças de órgãos oriundos da mesoderme e ectoderme gera ótimas perspectivas para o uso do mesmo em tratamento de doenças de órgãos de origem endodérmica (tireóide, pulmões, fígado, vesícula biliar e pâncreas). Particularmente, no fígado, ainda que sejam atribuídas propriedades regenerativas em muitas lesões o mecanismo de reparação é insuficiente, tornando o transplante hepático à única opção definitiva. Dentre as células-tronco embrionárias, as células de linhagens hepáticas fetais podem estabelecer-se como fonte importante para a terapia celular em indivíduos com doenças hepáticas, pois possuem um alto índice de diferenciação de hepatócitos e células do ducto biliar in vitro. Evidências apontam a progranulina como um fator de crescimento de grande habilidade para a indução de proliferação celular, uma vez que está envolvida no desenvolvimento embrionário e neonatal. Sendo assim, neste trabalho foram utilizados embriões de ratos Fischer 344 com idades gestacionais 12,5; 13,5; 14,5; 15,5; 16;5 para caracterizar o papel do novo fator de crescimento progranulina na hepatogênese. Foram realizadas análises histológicas, de microscopia eletrônica de transmissão e imuno-histoquímicas nos embriões. Houve expressão de progranulina e Oct-4 (marcador de célula tronco indiferenciada) principalmente nas idades gestacionais de 13,5 a 16,6 dias e 12,5 a 16,5 dias para PCNA (marcador de proliferação celular). Dessa forma acredita-se que, a progranulina atua nos processos de proliferação celular e diferenciação das células-tronco do broto hepático, podendo ser usada como um fator de diferenciação em culturas in vitro visando à diferenciação de células-tronco do broto hepático em hepatócitos funcionais para a terapia celular. / Diseases involving endodermal-derivated organs affect thousands of people all over the world. The cell therapy success for treating diseases of organs originated from mesoderm and ectoderm generates excellent perspectives for its use in treating diseases of organs of endodermal origin (thyroid, lungs, liver, gallbladder and pancreas). Particularly in the liver, although regenerative proprieties are attributed in many lesions, the repairing mechanism is insufficient, making the hepatic transplant the only definitive option. Among the embryo stem cells, the fetal hepatic lineage cells may establish themselves as important sources for cell therapy in individuals with hepatic diseases, since they have high ability in hepatocytes and billiar duct cells differentiation in vitro. However, for the use of hepatic lineage embryo stem cells as a bi-potential source of differentiation, it is necessary the establishment of efficient proliferation methods in this type of cells. Evidences point to progranuline as a growth factor with high capability for the induction of cell proliferation, since it is involved in the embryo and neo-natal development. Thus, this study used embryos of Fischer 344 rats with gestational ages 12.5, 13.5, 14.5, 15.5 and 16.5 to characterize the role of the new growth factor progranuline in hepatogenesis. We conducted histological, transmission electron microscopy and immunohistochemical in embryos. There was expression of progranuline and Oct-4 (undifferentiated stem cell marker), especially at gestational ages 13.5 to 16.5 days and 12.5 to 16.5 days for PCNA (proliferation marker). So it is believed that progranuline acts on cell proliferation and differentiation of stem cells from the liver bud, which can be used as a differentiating factor in order to cultures in vitro differentiation of stem cells from the liver bud into functional hepatocytes for cell therapy.

As células derivadas de fígado embrionário tanto de animais quanto de humanos tem sido cada vez mais estudas devido ao seu potencial antiinflamatório, imunomodulador e regenerativo, demonstrado as mesmas bipotencial de diferenciação em hepatócitos e colangiocitos. Na presente pesquisa utilizou-se células derivadas de fígados embrionários de ratos com 14,5 dias de gestação. As células apresentaram marcadores de células progenitoras hepáticas, bem como marcadores de células hepáticas e biliares diferenciadas confirmando, seu bipotencial. A terapia celular utilizando as células supracitadas, reduziu significativamente a progressão da fibrose hepática, diminuindo a inflamação e ainda estimulando a regeneração hepática de ratos submetidos à cirrose por ligadura do ducto biliar. As análises realizadas mediante avaliação quantitativa pela técnica de morfometria, demonstraram redução da deposição de fibras de colágeno, bem como menor proliferação de ductos biliares nos animais tratados. Os resultados foram ainda complementados por analise semiquantitativa, a qual avaliou a intensidade da necroinflamação dos tecidos hepáticos analisados, apontando menor escore de inflamação dos animais tratados. As células poderão ter efeito benéfico para o tratamento de doenças hepáticas crônicas, que estimulam a formação de fibrose. A cirrose é o estágio final comum à doenças hepáticas crônicas por causadas por fatores de diversas etiologias. Esta ocupa a decima quarta causa mundial de mortalidade em humanos, sendo que o único tratamento definitivo atualmente é transplante do órgão. Entretanto o número de transplantes está longe de suprir a demanda atual, visto que há um déficit de doadores do órgão. Terapias que possam oferecer uma alternativa de tratamento confiável, segura e acessível são bastante oportunas. Nossos resultados sugerem que as células utilizadas neste trabalho podem modular a fibrogênese, e consequentemente retardar o estabelecimento da cirrose em doenças hepáticas crônicas. / Studies on human and animal embryonic liver stem cells have been growing due to its anti-inflammatory, immunomodulatory and regenerative potential. These cells show also a bipotential do differentiate into hepatocytes and cholangiocytes. In the present study, it was used rodent embryonic liver with 14.5 of gestation. The cells presented hepatic progenitor, as well adult hepatic and biliary cells markers, confirming their bipotential. Previous studies with these cells in therapy decreased hepatic fibrosis progression in rat models submitted to cirrhosis by biliary duct ligation. Quantitative analysis was performed by morphometry showed decreased collagen fibers deposition and lower proliferation of biliary ducts in treated animals. Results were complemented with semiquantitative analysis with evaluation of necroinflammation of the analyzed hepatic tissues, in which a decreased inflammation score was observed. Cirrhosis is a common final stage for chronic hepatic diseases caused by different factors in several etiologies. It occupies the 14th world cause of mortality in human. However, the number of liver transplants is insufficient for current demand, caused by deficit in organs donors. Therapies that could offer an alternative for a reliable, safe and accessible treatment is opportune. Our results suggest that cells used in this study can modulate fibrogenesis and consequently delay the establishment of cirrhosis in chronic liver diseases.

1 Viruses are submicroscopic obligate parasites that depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. In the recent times, many new RNA viruses have evolved that possess the potential threat to plants and animals. One among them is Tospovirus (Family Bunyaviridae) which has severely affected the agricultural productivity in India. One of the Tospoviruses GBNV is a major challenge of crop production in south India. Tospoviruses shares several features such as morphology, genome structure and organization with members of other genera in the family Bunyaviridae. Virus particles are 80–120 nm in diameter. The genome includes three RNAs referred to as large (L), medium (M) and small (S). The L RNA is in negative-sense while the M and S RNAs are ambisense. The L RNA codes for the RNA-dependent RNA polymerase (RdRp), and the M RNA for the precursor of two glycoproteins (GN and GC) and a non-structural protein (NSm). The S RNA codes for the N protein and another non-structural protein (NSs). Tospovirus infection is an emerging threat for agricultural productivity in India. Therefore, biochemical and molecular characterization of these viruses is essential for developing various strategies for control of these diseases. 2 Present thesis deals with biochemical characterization of nonstructural protein, NSs of GBNV. 3 A review of literature on Tospovirus genome organization, replication, transcription, translation and assembly is presented in Chapter I. This chapter also includes the recent work on all the proteins encoded by the tospoviruses. 4 The objectives of the present study are as follows; a. Cloning, expression, purification and biophysical characterizations of rNSs. b. Analysis of its NTPase/dATPase activity c. Demonstration of nucleic acid 5’ phosphatase activity d. Characterization of nucleic acid unwinding activity of rNSs 5 The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in bacteria, protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology are described in chapter II 6 NSs of TSWV was shown to be suppressor of gene silencing (PTGS) in 2002. Since then there has been no further work on this protein. Till date neither in vitro nor in vivo study of NSs of any tospovirus has been carried out in detail. To gain insight into the biochemical function of rNSs, the NSS gene was cloned, overexpressed in E.coli and purified. The NSS gene, was cloned into pRSET-C vector. 7. Chapter 3 deals with cloning, overexpression, purification and biophysical characterization of GBNV NSs in terms of secondary structure analysis as well as its interaction with siRNA and ssRNA. The results provide the evidence that rNSs was successfully expressed in E.coli and purified (Fig. 3.1). Molecular mass of purified rNSs was confirmed by MALDI TOF, which gave the molecular mass of expected size 51.5 kDa (Fig. 3.2) Circular dichroism study revealed that rNSs has negative ellipticity peak at 215 and 223 nm typical of a globular protein. The protein had an emission maximum at 340 nm (Fig 3.3 B) when exited at 280 nm, which reflects that rNSs is well folded. Thermal melting study (Fig 3.3 C) showed rNSs had a reasonably high Tm (65°C). So overall, spectral study suggested that purified rNSs was soluble, well folded and thermally stable and could be used for further biochemical assay. The oligomeric status of the protein was determined by size exclusion chromatography to be trimeric (156 kDa, Fig 3.5). Purified rNSs was used to raise the polyclonal antibodies in rabbit. The antiserum could detect rNSs specific band only in IPTG induced sample not in uninduced sample (Fig 3.6). 50% binding was observed at 100 ng/ml of antigen showing that these antibodies were of high affinity (Fig 3.7 B). Further, the 50% binding was observed at 1:34000 dilution of the antiserum, which suggests that high titer antibodies against rNSs were obtained (Fig 3.7 A). 8 Further, the RNA binding property of rNSs was examined. Synthetic 21 bp siRNA and in vitro transcribed 100 nt ssRNA was used to analyze the RNA binding property of rNSs. Indeed rNSs was able to bind with 100 nt ssRNA (Fig 3.8 A) or 21 nt siRNA in a protein concentration dependent manner (Fig 3.8 B). The binding however did not require presence of divalent cation such as Mg 2+ (Fig 3.8 C). In order to understand the biological function of rNSs, its interaction with the structural protein, NP by ELISA was investigated. rNSs could interact with the NP protein (Fig 3.9) . Further 15 amino deletions from C terminus of NP did not affect its interaction with rNSs protein (Fig 3.9), which suggest that the C terminal 15 amino acid residues of NP are not essential for interaction with rNSs in vitro. 9. Sequence analysis of GBNV NSs revealed the presence of Walker motifs A (GxxxxGKT) and B (DExx) in its primary structure (Fig 4.2). The proteins that possess the Walker motifs A and B exhibit ATPase activity. Therefore, the purified rNSs was tested for its ability to hydrolyze ATP in the absence and presence of poly(A) (chapter IV). rNSs could hydrolyze [γ-32P] ATP in a concentration-dependent manner (Fig. 4.3 A). Further, ATPase activity was stimulated in presence of poly(A) (Fig. 4.3 B). Quantitative analysis of reaction product suggested that the reaction was linear in the presence of poly(A) upto 1.6 µg of rNSs (Fig. 4.3 C). 10. The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase, a positive control used in the assay. In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, purified in same way as rNSs, did not show the release of phosphate, suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate could be seen when immunodepleted rNSs was used suggesting that the activity was inherent to the protein and was not due to bacterial contamination (Fig 4.3 lane 7). Time course analysis of ATPase activity revealed that the reaction is linear up to 25 mins (Fig 4.4). Further, pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig 4.5 A) and the temperature optimum was at 25 °C(Fig 4.5 B). Most of the known viral ATPases require the divalent cation for their activity. The rNSs exhibited the optimum ATPase activity between 2-2.5 mM of MgCl2. The reaction was inhibited by increasing concentration of EDTA demonstrating the requirement of Mg2+ for ATP hydrolysis (Fig. 4.7). Further, the ATPase activity of rNSs was inhibited by increasing concentrations of non-hydrolyzable analog of ATP (Fig. 4.8) and was not inhibited by AMP (Fig 4.9) suggesting that rNSs is not a nucleotidyl phosphatase and is a true ATPase. Limited proteolysis of rNSs suggested that core domain was 23 kDa in size and could catalyze ATP hydrolysis (Fig. 21 and 4.22). 11. Interestingly rNSs not only cleaved ATP rather it could hydrolyze all rNTPs as well as dATP (Fig 4.10). Kinetic parameters were determined for its enzymatic activity. Comparison of the kinetic constants of rNSs NTPase activity revealed little variation, suggesting that the rNSs has a broad substrate specificity (Fig 4.10- 4.15 and table 4.1). 12. To assess the role of amino acids in Walker motif A and B (Fig. 4.16) site specific mutants K189A and D159A were generated ( Fig 4.17) confirmed by sequencing, overexpressed in E.coli and purified (Fig. 4.18). Point mutation in Walker motif B (D159A) reduced the ATPase activity (Fig 4.19) where as point mutation in Walker motif A (K189A abolishes the activity (Fig 4.19). 13. Chapter V deals with the nucleic acid 5’ phosphatase activity of rNSs. Experimental evidence presented in this chapter clearly shows that rNSs can cleave the single phosphate from the ssDNA, ssRNA, dsRNA and dsDNA. Nucleic acid 5’ phosphatase activity of rNSs was inhibited by AMP and ATP (Fig 5.2 and Fig 5.3). Interestingly the K189A mutant rNSs was as active as wild type rNSs where as D159A mutant showed slightly reduced activity (Fig 5.7 C). 14. As mentioned earlier, rNSs was shown to possesses the RNA stimulated NTPase/dATPase activity, a hallmark of all known helicases. Therefore, its nucleic acid unwinding activity was examined using dsDNA and dsRNA as a substrate. rNSs was able to unwind the dsDNA as well as dsRNA in a ATP dependent manner (chapter VI, Fig. 6.1 and 6.5 respectively). ATP and Mg2+ are essential cofactors for the unwinding activity (Fig. 6.1). While the unwinding activity could be observed with ATP and to some extent with dATP, all other NTPs and dNTPs failed to support the helicase function of rNSs (Fig 6.2) Further experimental evidence suggested that rNSs is a bidirectional helicase (Fig. 6.3). D159A mutation in Walker motif B resulted in reduced helicase activity where as K189A mutation in walker Motif A completely abolished the DNA as well as RNA helicase activity of rNSs (Fig. 6.6 and Fig 6.7 respectively). Therefore, mutational analysis clearly suggests that helicase activity is an intrinsic property of rNSs. 15. In conclusion rNSs of GBNV is multifunctional enzyme. This is the first report on the demonstration that rNSs is an non canonical ATP dependent helicase in the Bunyaviridae family. In addition to being a suppressor of PTGS, NSs may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. This new research finding on NSs might pave way for further studies on its role in viral replication and transcription.

Dormancy is one of the most important adaptive mechanisms developed by perennial plants, in order to survive the low temperatures of autumn and winter in temperate climates. The study of the genes regulated during dormancy release is crucial to understand the process, with the final objective of the development of new varieties with a better adaptation to certain environments; in particular in the Mediterranean area. The general aim of this work is to understand the molecular and physiological mechanisms underlying the maintenance and release of seasonal dormancy in peach. The first part of this work is focused on the identification of peach genes related to dormancy release by suppression subtractive hybridization (SSH) and microarray hybridization. A significant number of genes identified in this work were homologous to ABA and drought related genes from other species. Our data contribute to highlight a prominent role of ABA in dormancy processes and also uncover elements of the ABA and drought regulatory response in peach, as an ABA-INSENSITIVE5 (ABI5) binding protein (AFP)-like, a dehydration-responsive element (DRE)-binding protein (DREB2C)-like, a calcium-binding annexin, and several genes regulated by stress signalling pathways. Other identified genes were also evaluated to assess the chilling requirement of cultivars by analysis of expression showing a very good correlation between the expression pattern of DAM5, together with other transcripts (BD396, DB247, SB280 and PpB63) and the chilling requirements values of five different varieties ('Big Top', 'Catherina', 'Fergold', 'Maruja' and 'Springlady') measured following Utah and Dynamic models. Furthermore, a study of chromatin modifications associated to dormancy release in the DAM6 gene is presented. A ChIP analysis of DAM6 promoter and structural gene revealed chromatin modification events similar to those observed in vernalization of Arabidopsis and cereals. / Leida, CA. (2012). Molecular aspects of dormancy in peach (Prunus persica [L.] Batsch.) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/15864 / Palancia

In loquat, apex of a current shoot changes from vegetative to reproductive stage during summer, i.e. under high temperature conditions. Indeed, just before floral bud differentiation, a decline in the growth rate due to high temperature takes place. The aim of this work is to study the role of this 'summer rest period' on the apex transition from vegetative to reproductive stage. For this purpose 1) sprouting of secondary shoots was promoted at different times, removing the main shoot, before, during and after floral bud differentiation occurred and 2) groups of trees were shifted to a greenhouse under average maximum temperature not exceeding 25 ° C during different periods from June to October. Floral bud differentiation was evaluated. LEAFY (LFY), APETALA (AP1), TERMINAL FLOWERING 1 (TFL1) and FLOWERING LOCUS T (FT1) expression and hormonal content in abscisic acid (ABA), gibberellins (GAs), indoleacetic acid (IAA) and cytokinins (CKs) were analyzed in bud collected during the summer. Results suggest that the date of shoot apex removal determining floral bud differentiation of new shoots, so that the percentage of the new reproductive shoots reduced with the delaying of apex removal. On the other hand, maximum average temperature not exceeding 25 ° C prevented floral bud differentiation. Buds of the trees under indoors conditons displayed lower expression of identity floral genes EjLFY and EjAP1 than buds of trees grown in field. On the contrary, the floral repressor EjTFL1 and EjFT1 gene expressed higher in buds of the trees grown indoors. Time-course of ABA decreased in buds of trees grown in field during studied period while in buds of trees under greenhouse conditions displayed a growing trend. Time-course of GAs, IAA and CKs concentrations did not show remarkable differences between buds of trees growing under field and indoors conditions. Accordingly, 1) secondary shoots emerged from mid- August are unfitness to flower and 2) maximum average tempertature 25±1 °C during the summer prevents floral bud differentiation, enhances ABA biosynthesis, reduces EjLFY and EjAP1 expression and enhance EjTFL1 expression in the apex. / El níspero japonés diferencia sus yemas durante el verano, después de un periodo de ralentización del crecimiento vegetativo ligado a las altas temperaturas que se conoce como reposo estival. El objetivo de esta tesis fue estudiar la influencia de la parada estival en la diferenciación floral de esta especie. Para ello se diseñó un experimento en el que se forzó la brotación de brotes anticipados eliminado el ápice principal en diferentes fechas entre julio y septiembre, antes, durante y después de la parada estival. Paralelamente se diseñó otro experimento en el que se cambiaron las condiciones climáticas a grupos de árboles manteniéndolos en un invernadero a una temperatura máxima media de 25 °C durante diferentes periodos de diversa duración. Se evaluó la diferenciación floral y se analizó la expresión de los genes relacionados con la floración LEAFY (LFY), APETALA (AP1), TERMINAL FLOWERING 1 (TFL1) and FLOWERING LOCUS T (FT1) y el contenido hormonal en ácido abscisico (ABA), giberelinas (GAs), ácido indolácetico (AIA) y citoquininas (CKs) en yemas terminales muestreadas a lo largo del verano. Los resultados indican que la fecha de brotación modifica la diferenciación floral de los brotes anticipados siendo el porcentaje de brotes reproductivos inversamente proporcional a la fecha de eliminación del meristemo. Del mismo modo unas condiciones de temperatura máxima no superior a 25 °C impidieron la diferenciación floral. Las yemas de los árboles que estuvieron bajo dichas condiciones mantuvieron unos niveles de expresión de los genes de identidad floral, EjLFY y EjAP1, mucho menor que la de los árboles en condiciones de campo. Por el contrario, la expresión del represor EjTFL1 y del gen EjFT1 fue mayor en los árboles en invernadero. Por otro lado, el contenido endógeno de ABA descendió en los árboles situados en el campo durante el periodo de estudio mientras que en los árboles situados en el invernadero tuvo una evolución ascendente. Las concentraciones de GAs, AIA y CKs no mostraron prácticamente diferencias entre los ápices de los árboles mantenidos en campo y en invernadero. De acuerdo con ello, 1) los brotes anticipados surgidos a partir de mitad de agosto son incapaces de florecer y 2) la ausencia de altas temperaturas del verano promueve la acumulación de ABA, aumenta la expresión del gen represor (EjTFL1) y reduce la expresión de los genes de identidad floral (EjLFY y EjAP1) en yemas de níspero impidiendo su diferenciación floral. / El nispro japonés diferència les seus gemmes durant l'estiu, després d'un període d'alentiment del creixement vegetatiu lligat a les altes temperatures que es coneix com repòs estival. L'objectiu d'aquesta Tesi va ser estudiar la influència de la parada estival en la diferenciació floral d'aquesta espècie. Per a això es va dissenyar un experiment en què es va forçar la aparició dels brots anticipats eliminat l'àpex principal en diferents dates entre juliol i setembre, abans, durant i després de l'aturada estival. Paral·lelament es va dissenyar un altre experiment en què es van canviar les condicions climàtiques a grups d'arbres mantenint-los en un hivernacle a una temperatura màxima mitjana de 25 °C durant diferents períodes de diversa durada. Es va avaluar la diferenciació floral i es va analitzar l'expressió dels gens relacionats amb la floració LEAFY (LFY), APETALA (AP1), TERMINAL FLOWERING 1 (TFL1) and FLOWERING LOCUS T (FT1) i el contingut hormonal en àcid abscísic (ABA) , gibberel·lines (GAs), àcid indolacètic (AIA) i citoquinines (CKs) en gemmes terminals mostrejades al llarg de l'estiu. Els resultats indiquen que la data de brotació modifica la diferenciació floral dels brots anticipats i el percentatge de brots reproductius es inversament proporcional a la data d'eliminació del meristema. De la mateixa manera unes condicions de temperatura màxima no superior a 25 ° C varen impedir la diferenciació floral. Les gemmes dels arbres que van estar sota aquestes condicions van mantenir uns nivells d'expressió dels gens d'identitat floral, EjLFY i EjAP1, molt menor que la dels arbres en condicions de camp. Per contra, l'expressió del repressor EjTFL1 i del gen EjFT1 va ser més gran en els arbres en hivernacle. D'altra banda, el contingut endogen d'ABA va baixar en els arbres situats al camp durant el període d'estudi mentre que en els arbres situats a l'hivernacle va tenir una evolució ascendent. Les concentracions de GAs, AIA i CKS no van mostrar pràcticament diferències entre els àpexs dels arbres mantinguts en camp i en hivernacle. D'acord amb això, 1) els brots anticipats sorgits a partir de meitat d'agost són incapaços de florir i 2) l'absència d'altes temperatures de l'estiu promou l'acumulació d'ABA, augmenta l'expressió del gen repressor (EjTFL1) i redueix l'expressió dels gens d'identitat floral (EjLFY i EjAP1) en gemmes de nispro del Japó impedint la seva diferenciació floral. / García Lorca, AL. (2017). Temperature regulating floral bud differentiation in loquat (Eriobotrya japonica Lindl.). Hormonal and genetic aspects [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79873 / TESIS

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22879号 / 医博第4673号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 柳田 素子, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Field and laboratory experiments were conducted to elucidate various factors influencing the temperature-dependent larval eclosion of winter moth, Operophtera brumata L, in New England. We found no difference in duration of the embryonic stage of eggs reared from larvae collected in Massachusetts (MA) and on Vancouver Island, British Columbia (BC), where winter temperatures are rarely below freezing. The number of growing degree days (GDD) required for larval eclosion declined with the number of days chilled in the laboratory and number of days below freezing in the field, confirming the findings of previous studies. Thus, eggs hatched with fewer GDD, when the spring came later than usual. Date of oviposition had no effect on date of hatch. Eggs laid by naturally occurring (feral) females hatched sooner with lower GDD than eggs from laboratory-reared females from MA and BC held on the same trees over the winter. South-facing eggs on the stems of trees hatched on average 1.6 days sooner than north-facing eggs. Growing degree days calculated from bi-hourly measures of temperature were 15% greater than GDD estimates based on the average of daily maximum and minimum temperatures, as used by many GDD estimates made for online sources. Over two years, the mean GDD in ⁰C for hatch of feral eggs based on bihourly temperature measurements, a 1 Jan start date and a 3.9⁰C developmental threshold was 176.53 ± 6.35SE

<p>  </p> <p>Congenital diseases of the kidney are the leading cause of chronic kidney disease in pediatric patients. Tissue engineering models used to investigate these diseases are limited by an immature phenotype. Models cultured in an extracellular matrix (ECM), a network of proteins and glycosaminoglycans surrounding cells and providing structural support that mimic the matrix found in development will be likely more mature. However, developing kidney ECM composition and structural dynamics are unknown. To address this gap, we studied ECM composition using mass spectrometry and organization by visualizing the ECM in 3D.</p> <p>In this work, we used mass spectrometry to resolve ECM basement membrane and interstitial matrix dynamics between embryonic, perinatal, and adult kidneys. Surprisingly, we observed a transient upregulation of interstitial matrix structures that corresponded to dynamic 3D structures in the cortex (vertical fibers) and at the corticomedullary junction (medullary ray sheath fibers). Notably, in a model of abnormal <em>Foxd1</em>+ stromal cells, the vertical fibers were disorganized, and medullary ray sheath fibers were no longer associated with blood vessels, suggesting the dynamic 3D structures depended on stromal cell modulation.</p> <p>One of the effects of abnormal kidney development is decreased amniotic fluid, which limits embryonic movement and subsequent limb development. In additional studies, we looked at the implications of the lost motility in the muscular dysgenesis (<em>mdg</em>) mouse on the development of the myotendinous junction (MTJ). The MTJ links contractile muscle with tendon. We found the ECM protein COL22A1 was specific to the developing MTJ as early as embryonic day (E)13.5. The development of the MTJ from a linear structure to a cap-like structure with invaginations in adolescent mice depended on muscle contraction. Furthermore, we used a model to decouple the muscle-tendon-bone complex at an ectopic lateral triceps insertion (<em>Prrx1Cretg/+; Tbx3fl/fl</em>). We observed disorganized tendon and MTJ markers at the termination of the ectopic lateral triceps muscle but negligible cartilage markers. Together, this indicated MTJ maturation depended on motility but not on the enthesis.</p> <p>The information gleaned from our studies on how stromal cells affect dynamic 3D interstitial ECM structures and composition change during kidney development can be used as a template for 3D kidney culture systems. Combined with forelimb MTJ development, our results indicate the importance of the interstitial matrix in tissue morphogenesis.</p>

In this thesis the regulation of chitin synthases by proteolytic activation has been analyzed in yeast and insects. It was shown that the solubilized chitin synthase 2 of Manduca sexta (MsChs2) is an oligomeric complex of about 10 nm in diameter. In contrast to MsChs2 in membrane fractions, it can be activated by trypsin and chymotrypsin in the solubilized and purified state. In yeast, proteolytic activation of chitin synthases has been described almost 40 years ago. However, no protease has been identified stimulating chitin synthesis in vivo. Recently, Martinez-Rucobo et al. (2009) demonstrated, that the chitin synthase 2 (Chs2) of Saccharomyces cerevisiae is activated by a still unknown soluble endogenous protease. A global screening for protein-protein interactions indicated that the metalloprotease Ste24 interacts with chitin synthase 3 (Chs3). Ste24 is a membrane-integral CaaX protease residing in the endoplasmic reticulum (ER). In yeast, the only known substrate of Ste24 is the mating factor a (MFa) precursor. The interaction between Ste24 and Chs3 was verified by yeast two hybrid analysis and the interacting domains were mapped. Further investigations focused on the characterization of Ste24’s influence on chitin synthesis. Growth tests demonstrated that ste24D mutants are resistant to Calcofluor White (CFW). Mutant cells expressing a catalytically inactive version of Ste24 were also CFW resistant and showed a decrease in chitin levels. Overexpression of STE24 resulted in hypersensitivity to CFW and a slight increase in chitin levels. The CFW phenotype of ste24D cells could be rescued by its human and insect orthologues. Additionally, Chs3-GFP localized less frequently at the bud neck in ste24D cells. Although Chs3 binds to Ste24, it appears not to be a substrate of this protease. Instead Ste24 modulates the chitin synthesis by cleaving the CaaX motif of prenylated Chs4, a known activator of Chs3, since in cells expressing non-prenylated Chs4, deletion or overexpression of Ste24 had no influence on chitin synthesis. Moreover, the data suggests that Chs3 and Ste24 form a complex in the ER that facilitates proteolytic activation of Chs4, a known activator of Chs3 with a C-terminal CaaX motif, leading to a more efficient localization of Chs3 at the plasma membrane.