[EN] The general aim of this thesis is to study the dormancy process from a molecular point of view identifying mechanisms and targeting genes that control it. In order to do that we have focused on the study of three genes that are differentially expressed during reproductive bud development within the conceptual framework of the three major processes that converge spatially and temporally in a reproductive bud: dormancy, stress tolerance and flower development. The first gene is down-regulated in dormancy release flower buds and encodes a STRESS ASSOCIATED PROTEIN (PpSAP1) that contains Zn-finger domains A20 and AN1. SAP proteins have been related to stress tolerance response in both plants and animals and in fact, we have shown that drought stress induces its expression in buds, resembling other SAP genes in plants. Moreover, the constitutive expression of PpSAP1 in plum increases its tolerance to water stress by increasing water retention. Likewise, transgenic plum plants show leaf alterations related to reduced cell size concomitant with the down-regulation of genes involved in cell growth. All these studies suggest a dual role of PpSAP1 in stress tolerance response and cell growth during peach dormancy. The second gene is PpeS6PDH, coding for an enzyme with sorbitol-6-phosphate dehydrogenase activity. PpeS6PDH is differentially regulated during bud development, highly expressed in dormant buds consistently with sorbitol accumulation. Concomitantly with PpeS6PDH down-regulation in dormancy-released flower buds, chromatin around the translation start site of the gene shows changes in the methylation state of specific residues of histone H3 (H3K4 and H3K27). These data suggest the transcriptional regulation of PpeS6PDH expression by chromatin modification mechanisms. Moreover, abiotic stresses affect PpeS6PDH expression. Low temperature treatments induce gene expression in buds and leaves, whereas desiccation up-regulates PpeS6PDH in buds and represses the gene in leaves. These data suggest the participation of PpeS6PDH in tolerance against cold and water deficit stresses in buds. Finally, the third gene is PpeDAM6, one of the major regulators of bud dormancy in peach. PpeDAM6 is sharply down-regulated during bud development concomitantly with dormancy release events. This repression is in part due to the direct binding of PpeBPC1, a BASIC PENTACYSTEINE PROTEIN, to the GAGA motifs present in an intronic regulatory region of PpeDAM6 gene that becomes enriched in H3K27me3 chromatin modification after dormancy release. In addition, the ectopic expression of PpeDAM6 in Arabidopsis shows abnormal flower phenotypes resembling 35S::SVP plants. On the other hand, overexpression in plum causes stunted growth in the transgenic lines due to an altered hormonal homeostasis. The changes in hormone content are mediated by the modulation of genes involved in jasmonic acid, cytokinins and gibberellic acid metabolism and signalling pathways. These results suggest that PpeDAM6 works as a master growth repressor maintaining dormancy, stress tolerance response and flowering inhibition by mainly modulating hormone homeostasis. Therefore, this thesis provides a dynamic snapshot of different molecular mechanism that take place inside the bud. The studied genes have a crucial role regulating dormancy processes, stress tolerance response and flowering pathways and all of them are potential candidate genes for breeding new plants more adapted to the climate change. / [ES] El objetivo general de esta tesis es el estudio de la latencia desde un punto de vista molecular, identificando mecanismos y genes diana que la controlen. Para ello, nos hemos centrado en el estudio de tres genes que se expresan de manera diferencial durante el desarrollo de una yema reproductiva en melocotón, bajo el marco conceptual de los tres procesos que convergen espacialmente y temporalmente en una yema reproductiva: latencia, tolerancia a estrés y desarrollo floral. El primer gen que se estudió codificó para una STRESS ASSOCIATED PROTEIN (PpSAP1) con dos dominios tipo Zn-finger, A20 y AN1 que disminuye su expresión durante la latencia. Las proteínas tipo SAP se han relacionado con resistencias a distintos tipos de estrés tanto en plantas como en animales. De hecho, se ha visto que PpSAP1 aumentó su expresión en yemas de melocotón bajo condiciones de estrés por sequía, de forma similar a como lo hacen otras SAP en distintas plantas. Además, la expresión ectópica de PpSAP1 en ciruelos transgénicos ha permitido aumentar la tolerancia a estrés hídrico en estas líneas al incrementar la cantidad de agua retenida. Asimismo, estas plantas transgénicas también mostraron alteraciones en el tamaño de las hojas, provocadas principalmente por una menor área celular de las células que formaban parte de ellas y relacionadas con una represión de distintos genes implicados en crecimiento celular. Todo ello sugiere que PpSAP1 probablemente tenga una doble función relacionada tanto con resistencia a estrés como con crecimiento celular durante la latencia de melocotonero. El segundo gen de estudio fue PpeS6PDH, el cual codifica para una enzima con actividad sorbitol-6-fosfato deshidrogenasa. PpeS6PDH está diferencialmente regulado durante el desarrollo de la yema, aumentando su expresión en yemas latentes de manera consistente a la acumulación de sorbitol. Simultáneamente a la disminución de PpeS6pDH en las yemas no latentes, alrededor del sitio de inicio de la traducción del gen se mostraron cambios a nivel de cromatina en el estado de metilación de los residuos específicos de la histona H3 (H3K4 y H3K27). Estos datos apuntan a la existencia de una regulación transcripcional de PpeS6PDH a nivel de modificaciones de la cromatina. Además, también se ha visto que distintos tipos de estrés abiótico afectan a la expresión de PpeS6PDH. Tratamientos con bajas temperaturas inducieron su expresión tanto en yemas como en hojas, mientras que la desecación aumentó la expresión en yemas pero no en hojas. Estos estudios sugieren que la función de PpeS6PDH durante la latencia de melocotonero es dar tolerancia a estrés por frío y sequía. Finalmente, el tercer gen de estudio fue PpeDAM6, uno de los mayores reguladores de la latencia en yemas de melocotonero. PpeDAM6 está fuertemente reprimido durante el desarrollo de la yema con una relación directa con los eventos de salida de latencia. Esta represión se debe en parte a la unión directa de PpeBPC1, una BASIC PENTACYSTEINE PROTEIN, a dos motivos GAGA presentes en la región intrónica reguladora de PpeDAM6. Justamente esta región se encuentra modificada a nivel de cromatina con un enriquecimiento en H3K27me3 después de la salida de latencia. Además, la expresión ectópica de PpeDAM6 en Arabidopsis mostró fenotipos de floración anormal parecidos a los producidos en plantas 35S::SVP. Por otro lado, la sobreexpresión en ciruelos provocó retrasos en el crecimiento de las líneas transgénicas, debido a una alteración en los niveles hormonales. Así mismo, se determinó que estos cambios en la homeostasis hormonal estaban producidos por la regulación diferencial de genes involucrados en las rutas del ácido jasmónico, las citoquininas y del ácido giberélico en las plantas transgénicas. Estos resultados sugieren que PpeDAM6 actúa como un represor máster del crecimiento, manteniendo la latencia, la respuesta de tolerancia a estrés y la inhibición floral a través de la regulación del equilibrio hormonal. Con todo ello, esta tesis proporciona una instantánea dinámica de los diferentes mecanismos moleculares que tienen lugar dentro de la yema. Los genes estudiados tienen una función crucial regulando tanto el proceso de latencia como la respuesta de tolerancia a estrés y las rutas de floración, y todos ellos son potenciales candidatos para mejorar nuevas plantas más adaptadas al cambio climático. / [CA] L'objectiu general d'aquesta tesi és l'estudi de la latència des d'un punt de vista molecular, identificant mecanismes i gens diana que la controlen. Per això, ens hem centrat en l'estudi de tres gens que s'expressen d'una manera diferencial durant el desenvolupament d'una gemma reproductiva en el préssec, sota el marc conceptual dels tres processos que convergeixen espacialment i temporalment en una gemma reproductiva: latència, tolerància a estrés i desenvolupament floral. El primer gen d'estudi codifica per a una STRESS ASSOCIATED PROTEIN (PpSAP1) amb dos dominis tipus Zn-finger, A20 i AN1, i disminueix la seua expressió durant la latència. Les proteïnes tipus SAP s'han relacionat amb resistències a diferents tipus d'estrés tant en plantes com en animals. De fet, s'ha vist que PpSAP1 va augmentar la seua expressió en gemmes de préssec sota condiciones d'estrés per sequia, de manera similar a com ho fan altres SAPs en diferents plantes. A més, l'expressió ectòpica de PpSAP1 en pruneres transgèniques ha permés augmentar la tolerància a estrés en aquestes línies en incrementar la quantitat d'aigua retinguda. Així mateix, aquestes plantes trnasgèniques també mostraren alteracions en la mida de les fulles, causades principalmente per una menor àrea cel¿lular de les cèl¿lules que formen part d'elles i relacionades amb una repressió de diferents gens implicats en el creixement cel¿lular. Tot aço, suggereix que PpSAP1 probablement tinga una doble funció relacionada tant amb resistència a estrés com amb creixement cel¿lular durant la latència del préssec. El segon gen d'estudi va ser una PpeS6PDH, la qual codificava per a un enzim amb activitat sorbitol-6-fosfato dehidrogenasa. PpeS6PDH està diferencialment regulada durant el desenvolupament de la gemma, augmentant la seua expressió en gemmes latents de manera consistent a l'acumulació de sorbitol. Simultàniament a la disminució de PpeS6PDH en les gemmes no latents, al voltant del lloc d'iniciació de la traducció del gen es van mostrar canvis a nivell de cromatina en l'estat de metilació dels residus específics de la històna H3 (H3K4 i H3K27). Aquestes dades assenyalen l'existència d'una regulació transcripcional de PpeS6PDH a nivell de modificacions de la cromatina. A més, també s'ha vist que diferents tipus d'estrés abiòtic afecten a l'expressió de PpeS6PDH. Tractaments amb baixes temperatures van induir la seua expressió tant en gemmes com en fulles, mentres que la desecació va augmentar l'expressió en gemmes però no en fulles. Aquests estudis suggereixen que la funció de PpeS6PDH durant la latència del préssec és donar tolerància a estrés per fred i sequia. Finalment, el tercer gen d'estudi va ser PpeDAM6, un dels majors reguladors de la latència en gemmes de préssec. PpeDAM6 està fortament représ durant el desenvolupament de la gemma amb una relació directa amb els events d'eixida de la latència. Aquesta repressió és deguda en part a la unió directa de PpeBPC1, una BASIC PENTACUSTEINE PROTEIN, a dos motius GAGA presents en la regió intrònica reguladora de PpeDAM6. Justament aquesta regió es troba modificada a nivell de cromatina amb un enriquiment en H3K27me3 després de l'eixida de latència. A més, l'expressió ectòpica de PpeDAM6 en Arabidopsis va mostrar fenotips de floració anormal semblants als produïts en plantes 35S::SVP. Per un altra banda, la sobreexpressió en pruneres va provocar retards en el creixement de les línies transgèniques a causa d'una alteració en els nivells hormonals. Aixi mateix, es va determinar que aquests canvis en l'homeostasi hormonal estaven produïts per la regulació diferencial de gens involucrats en les rutes d'àcid jasmònic, citoquinines i àcid gibberèl·lic en les plantes transgèniques. Aquests resultats suggereixen que PpeDAM6 actua com un repressor master del creixement, mantenint la latència, la resposta de tolerància a estrés i la inhibició floral a través de la regulació de l’equilibri hormonal. Com a conclusió, aquesta tesi proporciona una instantània dinàmica dels diferents mecanismes moleculars que tenen lloc dins de la gemma. Els gens estudiats tenen una funció fonamental, regulant tant el mateix procés de la latència com la resposta de tolerància a estrés i les rutes de floració i tots ells són potencials candidats per a millorar noves plantes més adaptades al canvi climàtic. / Lloret Compañ, A. (2020). A dynamic snapshot of bud dormancy in peach [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153795 / TESIS

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20078号 / 医博第4171号 / 新制||医||1018(附属図書館) / 33194 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

I Sverige är bostadsmarknaden ett centralt och återkommande samtalsämne i såväl folkmun som media. Hetsiga budgivningar och en oreglerad lagstiftning har medfört att parterna ifrågasatt huruvida processen varit korrekt, och vidare skapat misstro mot hela fastighetsmäklarbranschen. Avsaknaden av strikt lagreglering, mer specifikt bindande bud, kan ha en bidragande effekt till att bostadsmarknaden ser hetare ut än vad den är. Detta då spekulanter kan delta i flertalet budgivningar samtidigt utan intentionen att faktiskt genomföra ett köp. En tänkbar förändring för ett mer hållbart klimat på bostadsmarknaden är därför att införa bindande bud. En utredning kring huruvida detta är applicerbart har legat som grund för denna uppsats. För att undersöka detta har en komparation gjorts mellan det svenska systemet och våra grannländer Norge och Danmark som har andra utgångspunkter där budgivningsprocesserna lyder under betydligt striktare lagrum. En kvalitativ metod har använts där primärdata har insamlats genom intervjuer med representanter från såväl tillsynsmyndigheter, branschorganisationer samt praktiker aktiva i branschen. Detta tillsammans med sekundärdata från bland annat mediala artiklar har påvisat en rådande meningsskiljaktighet huruvida det finns en önskan om att införa bindande bud i Sverige. Ett sammanslaget resultat visar på att ett införande av en modell likt den norska skulle ha en svalkande och hälsosam effekt på bostadsmarknaden. Författarna är dock oeniga om inom vilken tidsram ett införande skulle ske då justeringen av lagen skulle vara tidskrävande. Önskvärt skulle därför, som ett första steg, vara att i närtid införa obligatorisk Id-kontroll av samtliga budgivare. Detta för att skapa ett seriösare och säkrare klimat för såväl köpare som säljare. / Throughout the years, the Swedish housing market has been a central topic for discussions. Over inflated biddings and a lack of regulating law regarding the bidding process has resulted in questions regarding the process correctness and has created a distrust towards the Swedish real estate brokers. The lack of regulations which makes it possible for a bidder to participate in more than one bidding at the same time without any intention of actually buying, might have a contributing effect to making the real estate market look hotter than it in reality is. One solution to the problem could be to implement binding bids. Whether or not that is a possibility is the foundation for this thesis. A comparison between the Swedish, Norwegian and Danish law has been made to examine this. A qualitative method has been used where the primary data has been collected through interviews with respondents from the regulators, trade organizations and active practitioners. Together with secondary data including articles published in media has shown a divided opinion regarding the problem. A conclusion has been drawn that an implementation of the Norwegian model will have a dampening and sound effect on the Swedish real estate market. The authors of this thesis are in a disagreement on the time frame of the implementation. Due to the long process of changing the law a first desirable step would be the implementation of ID-control when placing a bid. This could contribute to a more serious and secure climate for both sellers and buyers.

No description available.

The housing market in Sweden has been a central debate topic in media during the past few years. The increase of prices in the big cities has caused hectic auctions that leaves the bidders to question whether the process really passed as it should. The bidding process has today no regulation by law and it is therefore up to the seller to decide how he wants it to proceed. This brings consequences in form of unhappy customers that does not know what to expect of this process.One solution, that has been discussed in media already, is to make the bidding process more safe by making the bids legally binding. This thesis is written in order to find out how this kind of regulation would change the swedish housing market. To do so we will investigate the system in Norway to see how they handle the binding bids.The investigation is based on interviews with industry professionals that has knowledge of the subject. The interviews showed that there is split opinions about whether binding bids in the housing market would be a good regulation since both advantages and disadvantages were discussed. The regulation of the law is comprehensive and it would take many years to process before it would be applied. Thus the result of the investigation is that both the real estate industry as well as the customers will need time to adapt to the new law, since it is a major change. On the other hand, binding bids would contribute to a safer bidding process in the sense that the risk of false bids probably would be eliminated. That in combination with a slower and more serious bidding process makes the regulation reasonable in the long run. / The housing market in Sweden has been a central debate topic in media during the past few years. The increase of prices in the big cities has caused hectic auctions that leaves the bidders to question whether the process really passed as it should. The bidding process has today no regulation by law and it is therefore up to the seller to decide how he wants it to proceed. This brings consequences in form of unhappy customers that does not know what to expect of this process.One solution, that has been discussed in media already, is to make the bidding process more safe by making the bids legally binding. This thesis is written in order to find out how this kind of regulation would change the swedish housing market. To do so we will investigate the system in Norway to see how they handle the binding bids.The investigation is based on interviews with industry professionals that has knowledge of the subject. The interviews showed that there is split opinions about whether binding bids in the housing market would be a good regulation since both advantages and disadvantages were discussed. The regulation of the law is comprehensive and it would take many years to process before it would be applied. Thus the result of the investigation is that both the real estate industry as well as the customers will need time to adapt to the new law, since it is a major change. On the other hand, binding bids would contribute to a safer bidding process in the sense that the risk of false bids probably would be eliminated. That in combination with a slower and more serious bidding process makes the regulation reasonable in the long run.

<p>Repertoar:</p><p>Budpology- Jonatan Erlingsson</p><p>Strictly Confidential- Bud Powell</p><p>Tempus Fugit- Bud Powell</p><p>The Fruit- Bud Powell</p><p>Celia- Bud Powell</p><p>I´ll Keep Loving You- Bud Powell</p><p>I´ll Remember April- Gene de Paul</p><p></p><p>Medverkande Musiker:</p><p>piano, arrangemang, komposition - Jonatan Erlingsson</p><p>kontrabas - August Eriksson</p><p>trummor - Henrik Jäderberg</p><p>saxofon - Amanda Sedgwick</p><p>violin 1 - Tor Alvin Wika</p><p>violin 2 - Natasja Dluzewska</p><p>viola - Stina Ahlgren</p><p>cello - Åse-Maria Bygland Larsen</p>

This thesis introduces public relations scholars and practitioners to the benefits of using an invitational rhetoric approach to the corporate voice instead of relying on traditional rhetorical approaches that are grounded in persuasive, authoritative intent. Because of societal changes inspired by changing gender norms, the traditional masculine corporate voice may no longer be the most effective corporate communication style. Traditional approaches to corporate communication are being challenged by more inclusive approaches that emphasize meaning co-creation with the public over purely persuasive approaches. This thesis conducts a case study of the Bud Light brand's use of Twitter. The purpose of this thesis is to determine if the brand uses an invitational rhetoric approach, and, if so, explore how Bud Light uses this approach. The thesis also analyzes the implications of this approach for corporate public relations practice. I argue an invitational rhetoric approach to corporate public relations may encourage more equitable communication between organizations and publics rather than privilege one authoritative, persuasive corporate voice over other voices involved in the conversation. / Master of Arts / This thesis introduces public relations scholars and practitioners to the benefits of using a more inclusive approach to their corporate voice on social media through an approach called invitational rhetoric. Invitational rhetoric is defined by Foss and Griffin (1995) as an "invitation to understanding as a means to create a relationship rooted in equality, immanent value, and self-determination" (p. 5). Because of societal changes inspired by changing gender norms, the traditional masculine corporate voice that is grounded in persuasive, authoritative intent may no longer be the most effective corporate communication style. Traditional approaches to corporate communication are being challenged by more inclusive approaches that emphasize interaction and understanding with the public over purely persuasive approaches. This thesis conducts a case study of the Bud Light brand's use of Twitter. The purpose of this thesis is to determine if the brand makes use of an invitational rhetoric approach, and, if so, explore how Bud Light uses invitational rhetoric. The thesis also analyzes the implications of an invitational rhetoric approach for corporate public relations practice. I argue an invitational rhetoric approach to corporate public relations may encourage more equitable communication between organizations and publics rather than privilege one authoritative, persuasive corporate voice over other voices involved in the conversation.

No description available.

Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this study are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.

Chapter 3 Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this chapter, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. Further NSm was shown to be phosphorylated by N. benthamiana and tomato crude sap as observed in other movement proteins. Chapter 4 This chapter deals with localization of NSm to PD and identification of domain involved in localization. For this purpose NSm and its mutants were cloned in pEAQ:GFP vector and transiently expressed in N. benthamiana by infiltration of transformed Agrobacteria. The GFP tagged NSm was visualized by confocal microscopy. The results demonstrated that NSm forms punctate structures and localizes to PD as confirmed by colocalization of mCherry: PDLP1a, a PD marker which resides in PD, with GFP:NSm. To find out the domain involved in PD localization, sequential deletion mutants were made. It was found that C-terminal domain is involved in PD localization. On the other hand, N-terminal unfolded region was dispensable for PD localization. This is the first report of a coiled coil domain shown to be involved in PD localization. It has also been demonstrated that GBNV NSm interacts with NP. Further, membrane floatation assay carried in presence of NP suggested that interaction of NSm and NP affected membrane association of NSm. These results were further confirmed by localization studies of NSm in presence of NP. It was found that there was considerable relocalization of both NSm and NP. NSm was observed to be present in cytoplasm as well as on the membrane. At the same time, NP was observed on membrane apart from being present in the cytoplasm. When N-terminal 50 amino acids (unfolded) region of NSm was deleted and colocalization studies were carried out, it was found that NSm and NP do not colocalize, suggesting that NSm interacts with NP via the unfolded region and helps in the relocalization of NP to the membrane. Chapter 5 This chapter deals with the pathway of targeting NSm to PD. To decipher the pathway, followed by NSm, an inhibitor of endomembrane or vesicle mediated transport, Brefeldin A (BFA) was used. When GFP-NSm was expressed it was observed to form punctate structure at PD as before. Upon treatment with BFA, green islands were observed in the cytoplasm suggesting that ER was involved in targeting NSm to PD. Similarly, LatB, inhibitor of actin mediated targeting of protein to membrane, also abrogated the localization of NSm to PD. In order to further understand the role of ER in targeting NSm to PD, an ER marker, ER-GFP (GFP fused to HDEL peptide that directs it to ER) was coexpressed with GBNV NSm fused to mCherry. It was observed that NSm colocalizes with ER-GFP as yellow puncta on PD. The puncta appeared as patches and the whole ER-network was converted to vesicles. This was further confirmed by coexpressing ER-GFP with NSm without any tag. The green fluorescent vesicles were observed preferentially near cell membrane. To delineate the region of NSm involved in vesicle formation, point mutants and deletion mutants of NSm were generated without the tag and coexpressed with ER-GFP. When N-terminal 203 amino acids were deleted, NSm was able to transform ER membranes to vesicles suggesting that these residues are dispensable for vesicle formation. Interestingly, the deletion of coiled coil domain leads to cytosolic location of NSm. Furthermore, the C-terminal coiled coil domain when expressed alone was capable of inducing vesicle formation. This is the first report of involvement of such a domain in ER membrane association and vesicle formation.