Dissertations / Theses on the topic 'Protest paradigm'

In a global media environment characterized by change and conflict, narratives are especially useful to understand how the media form and distribute shared understanding of how the world works and who the important actors are. As the borders between local and global politics are blurred in the digital media landscape, protesters are in increased rate turning their placards to global broadcasters’ cameras, especially when political movements such as the U.S.-based Black Lives Matter movement get international counterparts. The scholarship concerned with the framework through which the media report protests argue the protest paradigm offers useful variables for the study of protests, while problematizing the lack of research on global broadcasting media. Global broadcasters, International Relations scholars argue, need to be understood as resources of soft power that distribute strategic narratives, but they have yet to develop a methodology for how broadcasts can be empirically studied. With this research gap as a point of departure, the chosen case study is the unrest in Ferguson in August 2014. A quantitative mapping and a comparative narrative analysis focusing on the narrative structure were conducted on 16 days of news bulletins from Al Jazeera English, BBC World News and RT. The results show several differences in the reports, the first concerns the amount of attention that was given to Ferguson by each broadcaster, where RT gave almost twice the amount of attention as the other two broadcasters. Further differences were found in the sources each broadcaster used and how they used violence as an entry-point to what their narratives where about, which in the case of AJE was the effects violence has on a society; BBCW’s narrative was of a political issue of high importance that concerns people of color; whereas RT’s narrative was about the militarization of the U.S. police force. The results imply the global broadcasters offer distinctive narratives, which through different storytelling techniques convey different attitudes and morals.

Since Greta Thunberg started her environmental school strike in August 2018 she has become a world famous activist and has been portrayed in news media everywhere. The aim of this bachelor study has been to examine how Greta Thunberg has been portrayed in the Swedish news media through examining two daily press newspapers and two evening press newspapers. This resulted in analyzing articles in Aftonbladet, Expressen, Dagens Nyheter and Svenska Dagbladet. By using a qualitative and a quantitative content analysis we investigated three different events connected to Greta Thunberg through published articles. During 2018 the event that started her journey, which is the school strike, was selected as one of these happenings. During 2019 we chose her UN- speech in New York, and for the last event her speech at the World Economic Forum's Annual Meeting in Davos was chosen. By choosing these specific events we could build a perception of what the media's portrayal of Greta Thunberg has been like during these three years. Therefore this study also aimed to answer the following research questions: - How has Greta Thunberg been portrayed in the daily press and the evening press? - Has the portrayal of Greta Thunberg changed over time? In this study we used the Framing Theory and also the protest paradigms as guidelines and we also applied von Zabern & Tulloch (2020) and Bergmann & Ossewaardes (2020) different frames. This study then led to an identification of a new framing of Greta Thunberg which we named “Speaking without speaking”, mostly consisting of what politicians and celebrities are saying about Greta Thunberg in the press. We also found that by examining these specific events that Greta Thunberg has been portrayed in different ways. Therefore we could also conclude that the framings and portrayals of Greta Thunberg changed throughout time. This means that she went from being portrayed as a young girl skipping school for the climate, to a world famous environmental activist and finally becoming a trustworthy leader. Therefore our study shows that Greta Thunberg was being undermined due to her young age in the ibeginning, but as her status grew the media changed their portrayal of her into the role model that she has become today.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2002.<br>Includes bibliographical references (leaves 193-218).<br>Specific interactions between heparan sulfate glycosaminoglycans (HSGAGs) and proteins are central to a wide range of biological processes such as anticoagulation, angiogenesis and growth factor activation. The specificity involved in the HSGAG-protein interactions stems from the structural heterogeneity of HSGAGs, which are highly acidic biopolymers associated on the cell surface and in the extracellular matrix. It is believed that structural specificity in the HSGAG-protein interactions determines the biological functions mediated by HSGAG-binding proteins such as basic fibroblast growth factor (FGF2). A number of models have been proposed to account for the mode of FGF-FGFR interactions and the role of HSGAGs in modulating FGF2 signaling. It was hypothesized that one role played by HSGAGs was to stabilize FGF2 oligomers in a "side-by-side" or cis fashion for presentation to fibroblast growth factor receptor (FGFR). In this thesis research, we systematically examined different proposed modes of FGF2 dimerization and showed that extensive oligomerization of a FGF2 mutant protein could be achieved by oxidatively crosslinking. Heparin, a highly sulfated form of HSGAGs, was demonstrated to increase the extent of oligomerization. Therefore, the results reported here were consistent with the hypothesis that HSGAGs promoted FGF2 oligomerization in a "side-by-side" mode. The functional significance of a FGF2 dimer was tested using a genetically engineered dimeric FGF2 (dFGF2). Biochemical and biophysical properties of dFGF2, such as protein folding, heparin affinity and receptor<br>(cont.) binding, were assayed. dFGF2 was found to exhibit higher activities in stimulating cell proliferation and cell survival in vitro compared with the monomeric wildtype. An in vivo rat cornea pocket model further corroborated the in vitro findings. The functional role of HSGAGs derived from the cell surface was studied here. It was found that distinct HSGAG fragments released by heparinase treatment were capable of modulating FGF2-stimulated cell proliferation depending on the expression of FGFR isoforms. This founding is consistent with the proposal that structural specificity of distinct HSGAG fragments dictated the interaction of HSGAGs with FGF and FGFR. The role of heparinase-generated HSGAG fragments in inhibiting cell proliferation was investigated. B16 melanoma cells treated with heparinase III were found to exhibit biochemical and morphological hallmarks of apoptosis. Conditioned medium derived from heparinase-treated cells was shown to be effective in suppressing cell growth. Gene array experiments and caspase activity assays further suggested that apoptotic cell death was mediated through a caspase 8-, death receptor-dependent pathway. Thus, the present study lends further credence to the proposal that cell surface HSGAGs plays a critical role in orchestrating cell phenotype. This thesis work provides the framework for understanding the molecular mechanism of growth factor activation and the structure-function relationship of HSGAG-mediated cell signaling. Results from this study may potentially be useful for therapeutic protein engineering and carbohydrate-based drug discovery.<br>by Chi-Pong Kwan.<br>Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2003.<br>Includes bibliographical references (p. 211-223).<br>Glycosaminoglycans (GAGs) are a family of complex carbohydrates whose known biological roles have dramatically increased over the recent years. It is now becoming increasingly evident that sequence specific GAG-protein interactions play critical roles in cell growth, development, angiogenesis, cancer, anticoagulation and microbial pathogenesis. Therefore it is important to understand the specificity of glycan-protein interactions and how these specific interactions influence their structure-function relationships. This thesis addresses many challenges in this emerging area of glycomics, by taking an integrated approach that couples biophysical and biochemical methods with a bioinformatics framework to represent and process sequence information content in GAGs. With this motivation the thesis is divided into 4 components 1. Using heparin/heparan sulfate GAGs (HSGAGs) - fibroblast growth factor (FGF) as a model GAG-protein system, the first part focuses on determining the structural basis of FGF oligomerization, sequence specific FGF-HSGAG interactions and FGF-receptor (FGFR) interactions which collectively influence the specificity of HSGAG mediated FGF signaling. 2. The second part focuses on developing enzymatic tools for analysis of GAGs using chondroitinase B, HSGAG 2-0 sulfatase and 3-0 sulfotransferase as model enzymes. For each of these enzymes a theoretical model for the enzyme-substrate structural complex is developed and it is coupled with the site directed mutagenesis and biochemical studies to determine its catalytic mechanism and substrate specificity. 3. To deal with the heterogeneity and high information density of GAG sequences, an informatics based approach to decode GAG sequence information has been developed.<br>(cont.) A new property encoded nomenclature (PEN) computational framework has been formulated to encode and process information content in GAG sequences. The numerical nature of the PEN code facilitated the incorporation of diverse data sets from different analytical methods including mass spectrometry, electrophoresis and NMR as constraints to accurately determine the sequence of GAGs. Two practical methodologies for sequencing GAGs have been developed based on this approach. 4. The last part outlines the development of a powerful relational database that is capable of bridging sequence, structure and function information in glycomics. Thus this database and its associated computational tools to search and mine the data is an important resource for advancing glycomics.<br>by Rahul Raman.<br>Ph.D.

Modeling protean objects, i.e. objects adapting their structure and behavior dynamically with respect to a changeable environment, is often challenging in traditional object-oriented languages. According to the author, the root cause of this problem lies in the class-based conceptual framework embedded in the foundation of the object-oriented para-digm. The proposed paradigm Object Morphology (OM) is greatly influenced by prototype theory developed in the field of cognitive psychology. OM abandons the notion of class and suggests, instead, that the abstractions of protean objects should be established through the construction of morph models describing the possible forms of those objects. This the-sis defines the theoretical foundations of OM, which is further used to specify the elements of prototypical object-oriented analysis. An important part of this work is also a proof-of-concept implementation of an OM framework in Scala.

Made available in DSpace on 2016-04-25T20:21:29Z (GMT). No. of bitstreams: 1 Daniel Fassa Evangelista.pdf: 1550558 bytes, checksum: df1a30da1b1fc2dc04f1858b81b08d0a (MD5) Previous issue date: 2015-08-28<br>This dissertation is on the June 2013 demonstrations in Brazil, focusing on the city of São Paulo, where the protests convened by the Free Pass Movement (Movimento Passe Livre MPL) against the increase of public transport fare triggered an escalation of mass demonstrations that have taken more than one million people, mostly young, to the streets of 25 states capitals and hundreds of other cities across the country. Our main objectives were: to make a historical record of the demonstrations in the city of São Paulo; to interpret the demonstrations in the light of the social movements theories, paying particular attention to the role of online social networks; and to analyze in-depth interviews we conducted with six members of the National Youth Council from São Paulo (capital and/or metropolitan area) because they act in an institutional channel of interface between the state and the civil society and are members of organizations that are conducted by young people or have them as their target public. From the dialogue between the paradigm of resource mobilization and the paradigm of the new social movements, we conclude that, more than the necessary consequence of the dissatisfactions with the chronic problems of the country, the demonstrations of June must be understood as the result of the combination of strategic and cultural factors put into play by a multiplicity of actors interacting in civil society<br>A presente dissertação tem como tema as manifestações de junho de 2013, com foco na cidade de São Paulo. Foi a partir da capital paulista que protestos convocados pelo Movimento Passe Livre (MPL) contra o aumento da tarifa do transporte público desencadearam uma escalada de manifestações que levaram mais de um milhão de pessoas, sobretudo jovens, às ruas de 25 capitais e centenas de cidades de todo o país. Nossos principais objetivos foram: fazer um registro histórico das manifestações de junho na cidade de São Paulo; interpretar os acontecimentos à luz de uma revisão bibliográfica das teorias dos movimentos sociais, com particular atenção ao papel das redes sociais digitais; e analisar as entrevistas em profundidade que realizamos com seis integrantes do Conselho Nacional de Juventude originários de São Paulo (capital e/ou região metropolitana), pois atuam em um canal institucional de interface entre Estado e sociedade civil e são membros de organizações que são conduzidas por jovens ou os tem como público alvo. A partir do diálogo entre o paradigma da mobilização de recursos e o paradigma dos novos movimentos sociais, concluímos que mais que uma consequência necessária das insatisfações com os problemas crônicos do país, as manifestações de junho devem ser compreendidas como resultado de uma conjunção de fatores estratégicos e culturais colocados em jogo por uma multiplicidade de atores que interagem na sociedade civil

La régulation de la synthèse des protéines est une étape clé de la régulation de l'expression des gènes dans de nombreux processus cellulaires, permettant à la cellule de s'adapter rapidement au changement d’environnement en particulier en réponse à des stimuli externes et au stress. La majeure partie de la régulation de la traduction se produit à l'étape d'initiation, lorsque les ribosomes sont recrutés sur les ARNm, en perturbant eIF4F, le complexe fixé à l’extrémité 5' de l’ARNm via eIF4E, ou en réduisant la disponibilité du complexe ternaire lié au facteur d’initiation eIF2 (eIF2-GTP-Met-tARNMet). Au cours de ma thèse, j’ai montré comment ces étapes universelles sont régulées pour moduler spécifiquement les taux de traduction de différents ARNm.Nous avons montré qu'Angel1, une protéine interagissant avec eIF4E, est spécifiquement localisée dans le compartiment périnucléaire où elle régule la traduction d’ARNm spécifiques. Nous avons aussi décrit un nouvel opéron ARN caractérisé par la liaison spécifique de Hek2, une protéine de type hnRNP K de levure, à un sous-ensemble d'ARNm codant pour les pores nucléaires et régulant leur traduction. De plus, nous avons montré que la liaison de Hek2 à l'ARNm est empêchée par SUMOylation, une modification post-traductionnelle qui est contrecarrée par Ulp1, une SUMO protéase. Enfin, nous avons observé que la perturbation de l'intégrité des pores nucléaires suite à des mutations ou à un stress induisait l'accumulation de la forme SUMOylée de Hek2. Hek2-SUMO est incapable de se lier aux ARNm, dont la traduction se trouve ainsi augmentée dans un processus de rétroaction.Dans la dernière partie de ma thèse, nous avons réalisé la toute première étude de traductome d'une lignée de cellules β pancréatiques humaines en réponse à une stimulation par le glucose. Nous avons observé que le glucose stimule la traduction d'un ensemble défini d'ARNm pour lesquels nous avons identifié des caractéristiques spécifiques. Ces avancées sont importantes pour mieux comprendre la régulation de l’expression des gènes par le glucose.L’ensemble de nos résultats nous ont permis d’établir de nouveaux paradigmes de régulation traductionnelle<br>Regulation of protein synthesis is a key regulatory step of gene expression of many cellular processes allowing the cell to quickly adapt to the changing environment including external stimuli and stresses. Most of the translational regulation occurs at the the initiation step when ribosomes are recruited to the mRNAs, by disrupting eIF4F, the complex bound to the 5’ mRNA extremity through eIF4E, or by reducing the availability of the eIF2 ternary complex (eIF2-GTP-Met-tRNAMet). During my thesis I showed how these universal steps are regulated to specifically modulate the translation rates of different mRNAs.We showed that Angel1, an eIF4E interacting protein, is specifically localized to the perinuclear compartment where it regulates mRNA translation of specific mRNAs.We also described a novel RNA operon characterized by the specific binding of Hek2, a yeast hnRNP K-like protein, to a subset of nuclear-pore-encoding mRNAs regulating their translation. Moreover, we showed that Hek2 binding to the mRNA is impeded by the SUMOylation, a post-translational modification which is counteracted by Ulp1, a SUMO-protease. Finally, we reported that perturbation of the nuclear pore integrity by either mutations or stress, induced the accumulation of the SUMOylated form of Hek2. Hek2-SUMO is unable to bind to the mRNAs whose translation is thereby enhanced in a feedback process.In the last part of my thesis, we performed the first ever translatome study of a human pancreatic β cell line in response to glucose stimulation. We report that glucose stimulates translation of a defined set of mRNAs and identified their specific features providing important advances to better understand regulation of gene expression by glucose. Taken together our results allowed us to establish new paradigms of translational regulation

This dissertation examines the poetry of four Caribbean poets: Edward Brathwaite, Claire Harris, Olive Senior, and David Dabydeen. A presentation of the background issues which shape their voices of protest and prophecy, stemming from the colonization of the Caribbean region, governs the discussion. While the African ancestry of the poets Brathwaite, Harris, and Senior provides the cohesion of this critical analysis, Dabydeen, of East Indian ancestry, fits within the matrix of this analysis due to the thematic centering of his poetry on the issues of dislocation and dispossession surrounding the colonization of the Caribbean region. This analysis is organized into six chapters. Chapter One, the introduction, presents a historical overview ofthe Caribbean region and the scope of this dissertation. Chapters Two through Five are devoted to an analysis of selected works of each poet. Finally, Chapter Six synthesizes the powerful notes of protest and prophecy sounded by each of these poets in their quest for a home which empowers and embraces its people, a Paradise Regained.

Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition. Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations? Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle. Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone. Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.

La sécurité humaine est un concept qui a été formellement dégagé du rapport sur le développement humain du PNUD de 1994. Présentée par ce dernier comme une alternative au développement humain, la sécurité humaine a été institutionnalisée davantage comme une réponse aux préoccupations contemporaines en termes de sécurité globale et de bien être des individus, et comme un levier de garantie de la paix internationale.Dans la dynamique de la sécurité humaine, il sera observé une mutation du système normatif international par le renforcement de la place des droits de l’Homme et du droit international humanitaire dans l’ordre juridique. Aussi, la nécessité de la sécurité humaine va entrainer une nouvelle conception de l’objet de la sécurité collective. À cet effet, les Nations unies joueront un rôle déterminant en ce qu’elles vont développer de nouvelles compétences (en termes de garantie de la paix) et de nouvelles activités relatives à la sécurité humaine. Cet élan, d’une part, dynamisera un grand nombre d’acteurs internationaux qui s’investiront en faveur de la sécurité et de la protection des individus et, d’autre part, suscitera l’établissement de nouveaux mécanismes de paix et de sécurité internationales<br>Human security is a concept that was officially drawn out in the UNDP’s 1994 report on human development. In this report the concept was introduced as an alternative to human development and then became institutionalised as a response to contemporary preoccupations related to security matters. Human security therefore relates to threats to individuals, which are not only different to those that jeopardise State security but relate to the physical security and well-being of individuals.International peace and security factors were subsequently revised from a conceptual perspective, shifting from a State security-based focus to a focus on the global security of individuals.In the dynamics of human security, the international order’s normative system is shifting, especially owing to the growing importance of human rights and international humanitarian law in the legal order. Similarly, ensuring human security will redefine the objectives and activities of collective security. In this respect, the United Nations plays a crucial role. The Organisation will develop new competences in terms of peacekeeping and will carry out new operations for the benefit of human security. This renewed effort will reinforce many international stakeholders who will develop competences and establish organisations to contribute significantly to the security and protection of individuals and larger to the international peace

GATA1 and KLF1 are transcription factors that regulate genes which are important for the development of erythroid cells. The GATA1 transcriptional co-factor FOG1 has been shown to be essential in a wide range of GATA1 dependent cellular functions. Here we tried to understand the diverse mechanisms by which GATA1 and KLF1 recognize their binding sites, how the GATA1 recognition mechanisms are affected by complexation with either FOG1 or KLF1 and how the GATA1 recognition mechanisms affect the transcriptional regulation of the erythroid differentiation. We profiled the DNA binding specificities/affinities of a GATA1 fragment (mGATA1NC), that contains only the two DNA binding domains (N-terminal and C-terminal Zn finger), and the DNA binding specificities/affinities of a KLF1 fragment (mKLF1257-358), that contains the three DNA binding domains, using a novel methodology that combines EMSA with high throughput sequencing (EMSA-seq (Wong et al., 2011a)). We also profiled the DNA binding specificities of the C-terminal Zn finger of GATA1 alone (mGATA1C), the wt-mGATA1, the wt-mGATA1/wt-mFOG1 complex and the mGATA1NC/mKLF1257-358 complex. At first, we confirmed that the N-terminal Zn finger of GATA1 has a strong preference for the “GATC” motif, whereas the C-terminal Zn finger of GATA1 has a strong preference for the “GATA” motif. Next, we found that in the mGATA1NC, both DNA binding domains can bind simultaneously a wide range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. The wt-mGATA1 did not show the ability to bind in the same co-occurring motifs implying an effect of the non-DNA binding domains of the protein in the regulation of its DNA binding specificities. On the contrary, complexation of wt-mGATA1 with the wt-mFOG1 partially restored its ability to bind in a now limited range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. Similar observations were made for other pairs of GATA1 N-terminal and C-terminal Zn finger specific motifs. We then projected the GATA1 DNA binding specificities/affinities in vivo and we classified the GATA1 ChIP-seq peaks in low, medium or high affinity based on the number of the GATA1 motifs. We noticed that high affinity GATA1 ChIP-seq peaks tend to appear in regions with low nucleosome occupancy. We also noticed that GATA1 ChIP-seq peaks in the enhancer regions are usually high affinity whereas GATA1 ChIP-seq peaks in the proximal promoter regions are usually low affinity. Additionally, we observed that high affinity GATA1 ChIP-seq peaks are usually found in regions with increased levels of H3K4me2 and are associated with a higher decrease in the H3K4me3 levels on the TSS of the nearby genes. None of these GATA1 related in vivo observations were found for the KLF1 ChIP-seq positions. These findings significantly advance our understanding of the DNA binding properties of GATA1, KLF1 and their complexes and give an insight on the importance of the GATA1 DNA binding affinities in the regulation of the erythroid transcriptional program. Overall the work establishes an experimental and analytical framework to investigate how transcriptional co-factors can change the DNA binding specificities of specific transcription factors and how integration of the transcription factor DNA binding affinities with in vivo data can give novel insights into the transcriptional regulation.

Plant secondary products such as flavonoids have a variety of roles in plants including UV protection, antifeedant activity, pollinator attraction, stress response, flavor, and many more. These compounds also have effects on human physiology. Glucosylation is an important modification of many flavonoids and other plant secondary products. In grapefruit, glucosylation is important in the synthesis of the bitter compound naringin and several flavonoid glucosyltransferase (GT) enzymes have been characterized from young grapefruit leaf tissue. To study structure and function of flavonoid GTs, it is necessary to isolate cDNA’s that can be cloned and manipulated. In prior work, the plant secondary product glucosyltransferase (PSPG) box was used to identify putative GT clones. We report on results from experiments to test the hypothesis that PGT clones 4 and 11 are plant secondary product GTs, specifically flavonoid GTs. Previously, PGT 4 was cloned into a bacterial expression system, however all protein was localized into inclusion bodies and GT activity could not be tested. For this work, recombinant PGT 4 and PGT 11 were transformed into yeast and the proteins expressed and screened for glucosyltransferase activity with a variety of flavonoid substrates including flavanones, flavones, and flavonols.

Legionella pneumophila est une bactérie opportuniste qui émerge de l'environnement après multiplication dans des amibes et peut infecter accidentellement les macrophages alvéolaires humains, provoquant une pneumonie sévère, la légionellose. La capacité de L. pneumophila à survivre dans ses cellules hôtes est strictement dépendante du système de sécrétion de type 4 Dot/Icm, qui sécrète un large répertoire d'effecteurs dans le cytosol de l'hôte. Identifier la contribution individuelle de chaque protéine bactérienne sécrétée par le système Dot/Icm, dans le cycle infectieux de L. pneumophila reste un enjeu majeur pour comprendre les bases moléculaires de la virulence des légionelles. Mes travaux de thèse participent à cet objectif en caractérisant la voie cellulaire ciblée par la protéine kinase LegK2. Des tests d'interaction et de phosphorylation ont identifié le complexe nucléateur d'actine ARP2/3 comme cible de LegK2. Suite à l'adressage de LegK2 à la surface de la vacuole après sa translocation dans le cytosol de l'hôte, l'interaction LegK2-ARP2/3 inhibe la polymérisation d'actine sur le phagosome. Cette inhibition permet à Legionella de diminuer le trafic des endosomes tardifs et/ou des lysosomes vers le phagosome et favorise ainsi l'évasion du phagosome à la voie de dégradation endocytique. L'interaction LegK2-ARP2/3 met en évidence un mécanisme original de virulence dans lequel le remodelage local du cytosquelette d'actine de la cellule hôte permet à la bactérie de manipuler le trafic vésiculaire pour échapper aux défenses de l'hôte<br>Legionella pneumophila is an opportunistic bacterium that emerges from the environment after multiplication in protozoans and can accidentally infect human alveolar macrophages leading to a severe pneumonia, the legionellosis. The L. pneumophila ability to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Deciphering the individual contribution of each bacterial protein translocated by the Dot/Icm system in the L. pneumophila infectious cycle remains a major challenge to understand the molecular basis of Legionella virulence. My works contribute to this objective by characterizing the cellular pathway targeted by the protein kinase LegK2. Interaction and phosphorylation assays identified the actin nucleator ARP2/3 complex as the target of LegK2. Following the LegK2 addressing to the vacuole surface after its translocation into host cytosol, LegK2- ARP2/3 interplay inhibits the actin polymerization on the phagosome. This inhibition allows Legionella to decrease the late endosome/lysosome trafficking towards the phagosome and promotes the phagosome evasion from endocytic degradation pathway. LegK2-ARP2/3 interplay highlights an original mechanism of virulence wherein the local actin cytoskeleton remodeling of host cell allows bacteria to hijack the vesicles trafficking in order to escape host-cell defenses

Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.

This diploma thesis examines the image of Hong Kong's pro-democracy protests in 2019 in the Czech and Chinese media. The framing of these protests in selected media is compared using the method of quantitative content analysis, with the aim of revealing similarities and differences both between the individual Czech media in the context of the Czech public debate on China and between the Czech and Chinese media. The theoretical part of this thesis presents the concepts of media theory such as framing, agenda setting and social construction of reality and characterizes the specifics of given media systems. Furthermore, the work describes the previous findings of the protest paradigm, which are then employed in analytical part of this study. At the same time, the concept of soft power with Chinese characteristics and its specific manifestations in the efforts of the People's Republic of China to influence the Czech public debate on China is introduced. The methodological part then presents the research goal, research questions and hypotheses, defines the research sample and the research method used, including the characteristics of individual variables. The analytical part of the work presents the results of the research, which are then discussed in the framework of previous findings of the protest...

Aim: With particular regard to the traditional media and political participation in Sweden, Iintend to map how and in what way Greta Thunberg protests and the political engagement she expresses. Questions: • How is Greta Thunberg’s protesting framed and how can it be understood based on theprotest paradigm? • How can you understand Greta Thunberg´s political participation on the basis of ideasabout Civic Engagement? Method and material: This report is a qualitative study based on a textual analysis. For collectingthe empirical material, I have chosen to use texts from Swedish news articles. I used 27articles from 2 newspapers which is Aftonbladet and Expressen. Main results: The study shows that media puts greater focus on Greta Thunberg as a personinstead of the factual content and political agenda she stands for. This contributes to GretaThunberg being marginalized and delegitimized, since she is considered a symbol thatchallenges the political power structure. Greta Thunberg feels responsible for the climateissue and it is her duty to be able to contribute to a democratic society by getting involved.Greta Thunberg considers her participation in the media that she feels a moral obligation toengage with the climate.

A crise climática incorpora desafios sociais, ambientais e políticos inescapáveis que, continuando as atuais tendências de emissão de gases com efeito de estufa, impulsionam as sociedades atuais rumo ao colapso. O movimento social pelo clima, que incorpora e unifica o ativismo climático, procura responder a este desafio ao pressionar governos e organizações internacionais a tomar medidas decisivas de mitigação e adaptação às alterações climáticas. Esta pesquisa procura compreender, através do estudo de imprensa, de que forma estes ativistas têm sido representados para um público geral e de que forma são contextualizadas as suas ações. Para isso, foram analisados artigos publicados no jornal Público Online segundo indicadores identificados em outros estudos sobre ativismo e meios de comunicação, nomeadamente a presença do paradigma do protesto. Foram recolhidos 175 artigos, que foram analisados recorrendo à análise de conteúdo. A análise de dados revelou um paradigma distinto do que foi identificado em estudos internacionais, nomeadamente no respeita ao tipo de representações sociais dominantes e à presença do paradigma do protesto. O jornal em análise revelou um enquadramento do ativismo climático preferencial, no qual o ativista tem frequentemente oportunidade de auto-representação e as ações são contextualizadas segundo a sua motivação. A análise revela ainda que o paradigma do protesto não é prevalente no jornal analisado e que as representações sociais dominantes do ativista são maioritariamente favoráveis.<br>The climate crisis embodies inescapable social, environmental and political challenges that, if current trends in greenhouse gas emissions are to continue, current societies will be driven towards collapse. The climate movement, which embodies and unifies climate activism, seeks to respond to this challenge by putting pressure on governments and international organizations to take decisive action on climate change mitigation and adaptation. This research seeks to understand, through a press analysis, how these activists have been conveyed to a general public and how their actions are contextualized. For this purpose, articles published in Público Online newspaper were analysed according to indicators identified in previous studies on activism and media, namely the existence of the protest paradigm. 175 articles were gathered, which were analysed using content analysis. The data analysis revealed a different paradigm from what has been identified in international studies, namely in terms of the type of social representations that are prevalent and the presence of the protest paradigm. The newspaper under analysis revealed a preferred framing of climate activism, in which the activist often has the opportunity for self-representation and the actions are contextualized according to their motivation. The analysis also reveals that the protest paradigm is not prevalent in the newspaper and that the majority of the activists' social representations are favourable.

Whereas G-protein coupled receptors (GPCRs) have been long believed to signal through cyclic AMP exclusively at cell surface, our group has previously shown that GPCRs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent cAMP production. This phenomenon, which we originally described for the thyroid stimulating hormone receptor (TSHR) in thyroid cells, has been observed also for other GPCRs. However, the intracellular compartment(s) responsible for such persistent signaling and its consequences on downstream effectors were insufficiently characterized. The aim of this study was to follow by live-cell imaging the trafficking of internalized TSHRs and other involved signaling proteins as well as to understand the consequences of signaling by internalized TSHRs on the downstream activation of protein kinase A (PKA). cAMP and PKA activity was measured in real-time in living thyroid cells using FRET-based sensors Epac1-camp and AKAR2 respectively. The results suggest that TSH co-internalizes with its receptor and that the internalized TSH/TSHR complexes traffic retrogradely to the trans-Golgi network (TGN). This study also provides evidence that these internalized TSH/TSHR complexes meet an intracellular pool of Gs proteins in sorting endosomes and in TGN and activate it there, as visualized in real-time using a conformational biosensor nanobody, Nb37. Acute Brefeldin A-induced Golgi collapse hinders the retrograde trafficking of TSH/TSHR complexes, leading to reduced cAMP production and PKA signaling. BFA pretreatment was also able to attenuate CREB phosphorylation suggesting that an intact Golgi/TGN organisation is essential for an efficient cAMP/PKA signaling by internalized TSH/TSHR complexes. Taken together this data provides evidence that internalized TSH/TSHR complexes meet and activate Gs proteins in sorting endosomes and at the TGN, leading to a local activation of PKA and consequently increased CREB activation. These findings suggest unexpected functions for receptor internalization, with major pathophysiological and pharmacological implications<br>G-Protein-gekoppelte Rezeptoren sind nur in Eukaryonten vorhandeln und bilden die größte und diverseste Familie von Zellmembranrezeptoren. Sie reagieren auf eine vielfältige Gruppe von Stimuli die verschiedene Effektoren aktivieren und damit nachgelagerte Signalkaskaden auslösen, die letztlich entscheidend für die Zellphysiologie sind. Die Regelung der Ligand-vermittelten Signaltransduktion wird hauptsächlich durch die Desensibilisierung des GPCR mittels Dephosphorylierung (katalysiert durch GRK) und zusätzlich durch Internalisierung des GPCR gesteuert. Die Annahme, dass GPCRs für cAMP nur an der Zellmembran signalisieren und nicht mehr sobald sie in die Zelle internalisiert wurden, konnte durch wegweisende unabhängige Forschung an GPCRs im Besonderen an TSHR und PTHR geändert werden. So konnte gezeigt werden, dass sie für cAMP nicht nur an der Zellmembran signalisieren, sondern auch, wenn sie in intrazelluläre Zellkompartimente internalisiert wurde. Dieses Phänomen („sustained signaling“ hier „anhaltende Signalisierung“) wurde seitdem für andere GPCRs (z.B. 2-AR, V2R und LHR) beschrieben. Aber die Zellkompartimente wurden für nachhaltige intrazelluläre Signale nicht ausreichend charakterisiert. Das Ziel dieser Arbeit war es die Bewegung und die dynamische Natur der möglichen signalisierenden Kompartimente mittels „real-time TIRF“-Mikroskopie und die Signalisierung unter Verwendung von „real-time FRET“ in primären Maus Schilddrüsenzellen zu untersuchen. Die vorliegende Arbeit berichtet, dass TSH/TSHR Komplexe internalisieren und ein signifikanter Teil, welcher vom Retromer Komplex angeführt wird, gelangt über den retrograden (rückwärts gerichteten) Transport in das trans-Golgi-Netzwerk (TGN). Diese TSH/TSHR-Komplexe treffen nicht in den frühen Endosomen auf die Gs-Proteine, sondern in den „Sortierer Endosomen“ und in dem TGN. Ein direkter Beweis für Gs Protein Aktivierung und Signaltransduktion am TGN und in Sortierer Endosomen konnte mittels des nanobody Nb37, einem spezifischen Biosensor für das aktive Gs Protein, erbracht werden. Es konnte gezeigt werden, dass die Sequestrierung von Nb37 an diesen Kompartimenten ein szintillierendes Verhalten in Zeit und Raum zeigt. Die vorliegende Arbeit zeigt, dass die katalytische Untereinheit der PKA am Golgi/TGN angereichert ist. Die Behandlung mit Brefeldin A führt zum Verlust dieser PKA Lokalisation am Golgi. Die Beschädigung und Reorganisation des TGN durch Brefeldin A führt zu a) einer abgeschwächten cAMP Reaktion b) einer dreiphasigen PKA Reaktion charakterisiert durch eine schnelle erste Phase, eine langsame (deutlich abgeschwächte) zweite Phase und eine verzögerte dritte Phase und schließlich c) einer abgeschwächte CREB Phosphorylierung. Es gibt Anzeichen dafür, dass die Reorganisation des TGN Kompartimente betrifft, die verantwortlich für intrazelluläre cAMP- und PKA-Signalisierung sind. Zusammenfassend lässt sich sagen, dass das TGN eines der Kompartimente ist, das für die anhaltende TSHR-Signalisierung verantwortlich ist

BIOLOGICAL processes are governed through specific interactions of macromolecules. The three-dimensional structural information of the macromolecules is necessary to understand the basis of molecular recognition. A large number of protein structures have been determined at a high resolution using various experimental techniques such as X-ray crystallography, NMR, electron microscopy and made publicly available through the Protein Data Bank. In the recent years, comprehending function by studying a large number of related proteins is proving to be very fruitful for understanding their biological role and gaining mechanistic insights into molecular recognition. Availability of large-scale structural data has indeed made this task of predicting the protein function from three-dimensional structure, feasible. Structural bioinformatics, a branch of bioinformatics, has evolved into a separate discipline to rationalize and classify the information present in three-dimensional structures and derive meaningful biological insights. This has provided a better understanding of biological processes at a higher resolution in several cases. Most of the structural bioinformatics approaches so far, have focused on fold-level analysis of proteins and their relationship to sequences. It has long been recognized that sequence-fold or fold-function relationships are highly complex. Information on one aspect cannot be readily extrapolated to the other. To a significant extent, this can be overcome by understanding similarities in proteins by comparing their binding site structures. In this thesis, the primary focus is on analyzing the small-molecule ligand binding sites in protein structures, as most of the biological processes ranging from enzyme catalysis to complex signaling cascades are mediated through protein-ligand interactions. Moreover, given that the precise geometry and the chemical properties of the residues at the ligand binding sites dictate the molecular recognition capabilities, focusing on these sites at the structural level, is likely to yield more direct insights on protein function. The study of binding sites at the structural level poses several problems mainly because the residues at the site may be sequentially discontinuous but spatially proximal. Further, the order of the binding site residues in primary sequence, in most of cases has no significance for ligand binding. Compounding these difficulties are additional factors such as, non-uniform contribution to binding from different residues, and size-variations in binding sites even across closely related proteins. As a result, methods available to study ligand-binding sites in proteins, especially on a large-scale are limited, warranting exploration of new approaches. In the present work, new methods and tools have been developed to address some of these challenges in binding site analysis. First, a novel tool for site-based function annotation of protein structures, called PocketAnnotate was developed ( http://proline.biochem.iisc.ernet. in/pocketannotate/). PocketAnnotate, detects the putative binding sites from a given protein structure and compares them to known binding sites in PDB to derive functional annotation in terms of ligand association. Since the tool derives functional annotation at the level of binding sites, it has an advantage over other methods that solely utilize fold or sequence information. This becomes even more important for cases where there is no detectable homology with entries in existing databases, as Pocket Annotate does not depend on evolutionary based information for annotation. Second, a web-accessible tool for in silico almandine scanning mutations of binding site residues called ABS-Scan has been developed ( http://proline.biochem.iisc.ernet.in/abscan/). This tool helps in assessing the contribution of the individual residues of binding sites in the protein towards ligand recognition. All residues, one at a time, in a binding site are mutated systematically to an alanine and the ability of the corresponding mutant to bind a given ligand is analyzed. The contribution of each residue towards ligand binding is calculated through a G value derived by comparing the binding affinity to the wild-type protein-ligand complex. Third, a database called Protein-Ligand Interaction Clusters (PLIC) has been developed to identify and analyze the information of similarity across binding sites in PDB, which has been provided in the form of a web-accessible database ( http://proline.biochem.iisc.ernet/ PLIC). Protein-ligand interactions are primarily explored using three different computational approaches - (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of chemical probes, (ii) atomic contacts between protein and ligands (iii) binding energetics involved in interactions derived from scoring functions developed for docking. The information on variations in these features derived from different computational tools is also included in the database for enabling the characterization of the binding sites. As a case study to demonstrate the usefulness of these tools, they have been applied to decipher the complexity of S-adenosyl methionine interactions with the protein. Around 1,213 binding sites of SAM or SAM-like compounds could be extracted from the PLIC database. The SAM or SAM-like compounds were observed to interact with ∼18 different protein-fold types. The variations in different protein-ligand contacts across fold types were analyzed. The fold-specific interaction properties and contribution of individual residues towards SAM binding are identified. The tools developed and example analyses using them are described in Chapter 2. Chapter 3 describes a large-scale pocketome analysis from structural complexes in PDB, in an effort to characterize the known pocket space of protein-ligand interactions. Tools devel-opted as described in Chapter 2 are used for this. A set of 84,846 binding sites compiled from PDB, have been comprehensively analyzed with an objective of obtaining (a) classification of binding sites, (b) sequence-fold-site relationships among proteins, (c) a minimal set of physicochemical attributes sufficient to explain ligand recognition specificity and (d) site-type specific signatures in terms of physicochemical features. A new method to describe binding sites was developed in the form of BScIds such that the structural fold information is well captured. Binding sites and similarities among them were abstracted in the form of networks where each node represents a binding site and an edge between two nodes represents significant similarity between the sites at the structural level. Pocketome networks were constructed from the large-scale information on protein-ligand interactions in the PLIC database. The large pocketome network was then studied to derive relationships between protein folds and chemical entities they interact with. A classification of the binding pockets was achieved by analyzing the pocketome network using graph theoretical approaches combined with clustering methods. 10,858 clusters were identified from the network, each indicating a site-type. Thus, it can be said that there are about 10,858 site-types. Classification of ligand associations into specific site-types helps greatly in resolving the complex relationships by yielding specific site-type ligand associations. The observed classification was further probed to understand the basis of ligand recognition by representing the pockets through feature vectors. These features capture a wide range of physicochemical properties that can be used to derive site-type specific signatures and explore the pocket-space of protein-ligand interactions. A principal component analysis of these features reveals that binding site feature space is continuous in the entire PDB and minor changes in specific features can give rise to significant differences in ligand specificity, consequently defining their distinct functional roles. The weights were also derived for these features through the use of different information theoretic approaches to explain the multiple-specificity of protein-ligand interactions. Analysis of binding sites arising from contribution of residues from different protein fold-types revealed increasing diversity of physicochemical properties at the site, supporting the hypothesis that combination of folds could give rise to new binding sites. Given that a finer appreciation of the molecular mechanisms within the cell is possible only with the structural information, the next objective was to explore if a structural view of an entire proteome can be obtained and if a pocketome could be constructed and analyzed. With this in mind, the causative agent of tuberculosis - Mycobacterium tuberculosis (Mtb) was chosen. Mtb is also being studied in the laboratory from a systems biology perspective, which enabled exploration of how systems and the structural perspectives could be combined and applied for drug discovery. Chapters 4 to 6 describe this effort. The genome sequence of Mycobacterium tuberculosis (Mtb) H37Rv, indicates the presence of ∼4,000 protein coding genes, of which experimentally determined structures are available for ∼300 proteins. Further, advances in homology modeling methods have made it feasible to obtain structural models for many more proteins in the proteome. Chapter 4 describes the efforts for obtaining the Mtb structural proteome, through which the three-dimensional struc-tures were derived for ∼70% of the proteins in the genome. Functional annotation of each protein was derived based on fold-based functional assignments, binding-site comparisons and consequent ligand associations. PocketAnnotate, a site-based function annotation pipeline was utilized for this purpose and is described in Chapter 2. Besides these, the annotation covers detection of various sequence and sub-structural motifs and quaternary structure predictions based on the corresponding templates. The study provides a unique opportunity to obtain a global perspective of the fold distribution in the genome. The annotation indicates that cellular metabolism can be achieved with only 219 unique folds. New insights about the folds that predominate in the genome, as well as the fold-combinations that make up multi-domain proteins are also obtained. 1,728 binding pockets have been associated with ligands through binding site identification and sub-structure similarity analyses, yielding a list of ligands that can participate in various biochemical events in the mycobacterial cell. A web-accessible database MtbStructuralproteome has been developed to make the data and the analyses available to the community, ( http://proline.physics.iisc.ernet.in/Tbstructuralannotation). The resource, being one of the first to be based on structure-based functional annotations at a genome scale, is expected to be useful for better understanding of tuberculosis and for application in drug discovery. The reported annotation pipeline is fairly generic and can be applied to other genomes as well. Chapter 5 describes the characterization of the Mtb pocketome. For the structural models of the Mtb proteome described in chapter 4, a genome-scale binding site prediction exercise was carried out using three different computational methods and subsequently obtaining consensus predictions. The three methods were independent and were based on considering geometry, inter-molecular energies with probes and sequence conservations in evolutionarily related proteins respectively. In all, 13,858 consensus binding pockets were predicted in 2,877 proteins. The pocket space within Mtb was then explored through systematic all-pair comparisons of binding sites. The number of site-types within Mtb was found to be 6,584, as compared to the ∼400 structural folds and 1,831 unique sequence families. This reveals that the pocket space is larger than the sequence or fold-space, suggesting that variations at the site-level contribute significantly to functional repertoire of the organism. By comparing the pockets with the PDB sites enclosing known ligands, around 6906 binding sites were observed to exhibit significant similarity in the entire pockets to some or the other known binding site in PDB. 1,213 metabolites could be mapped onto 665 enzymes covering most of the metabolic pathways. The identified ligands serve as a predicted metabolome for unit abundances of the proteins. A list of proteins containing unique pockets is also identified. The binding pockets, similarities they share within Mtb and the ligands mapped onto them are all made available in a web-accessible database at http://proline.biochem.iisc.ernet.in/mtbpocketome/. The availability of structural information of the pocketome at a genome-scale opens up several opportunities in drug discovery. They can be directly applied for understanding mechanism of drug action, predicting adverse effects and pharmacodynamics of a drug. Moreover, it enables exploration of new ideas in drug discovery. Polypharmacology is a new concept that aims at modulating multiple drug targets through a single chemical entity. Currently, there are no established approaches to either select appropriate target sets or design polypharmacological drugs. In this study, a structural-proteomics approach is explored to first characterize the pocketome and then utilize it to identify similar binding sites. The knowledge of similarity relationships between the binding sites within the genome can be used in identifying possible polypharmacological drug targets. A pocket similarity based clustering of binding site residues resulted in identification of binding site sets, each having a theoretical potential to interact with a common ligand. A polypharmacological index was formulated to rank targets by incorporating a measure of drug ability and similarity to other pockets within the proteome. By comparing with known drug binding sites from databases such as the Drug Bank, the study has yielded a ready shortlist that includes sets of promising drug targets with polypharmacological possibilities and at the same time has identified possible drug candidates either directly for repurposing or at the least as significant lead clues that can be used to design new drug molecules against the entire group of proteins in each set. This analysis presents a rational approach to identify targets with polypharmacological potential, clues about lead compounds and a list of candidates for drug repurposing. This thesis demonstrates the feasibility of utilizing the structural bioinformatics approaches at a genome-scale. The tools developed for analyzing large-scale data on protein-ligand inter-actions could be applied to characterize the pocket-space of protein-ligand interactions. The network theory approaches applied in this work, make large-scale data tractable and enable binding-site typing. The binding site analysis at a genome-scale for Mtb is first of its kind and has provided novel insights into the pocket space. The binding site analysis performed on a genome-scale for Mtb provided an opportunity to rationalize the polypharmacological target selection and explore drugs for repurposing in TB. In the larger context, structural modelling of a proteome, mapping the small-molecule binding space in it and understanding the determinants of small-molecule recognition forms a major step in defining a proteome at higher resolution. This in turn will serve as a valuable input towards the emerging field of structural-systems biology, which seeks to understand the biological models at a systems level without compromising on the resolution of the study.

Dishevelled (Dvl) proteins are key transducers of Wnt signaling and are encoded by members of a multi-gene family in vertebrates. We report here divergent, tissue-specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression in the mouse. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, one of the outgroups to chordates, we find that the one Dvl gene is dynamically expressed in a tissue-specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation. Most strikingly, we report a novel role for Dvl3 in the maintenance of differentiated muscle and the development of the Xenopus sclerotome. Together, these data demonstrate that that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution. The planar cell polarity (PCP) signaling pathway is essential for embryonic development because it governs diverse cellular behaviors, and the "core PCP" proteins, such as Dishevelled and Frizzled, have been extensively characterized. By contrast, the "PCP effector" proteins, such as Intu and Fuz, remain largely unstudied. These proteins are essential for PCP signaling, but they have never been investigated in a mammal and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice display neural tube defects, polydactyly, and skeletal dysmorphologies that stem from defective ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we establish a central role for Fuz in membrane trafficking, showing that Fuz is essential for apical trafficking of ciliogenesis factors in ciliated cells and also for exocytosis in secretory cells. We identify a novel, Rab-related small GTPase as an interaction partner for Fuz, and this GTPase also is essential for ciliogenesis and secretion. These results are significant because they provide novel insights into the mechanisms by which developmental regulatory systems like PCP signaling interface with fundamental cellular systems such as the vesicle trafficking machinery.<br>text

博士<br>國立成功大學<br>基礎醫學研究所<br>97<br>Learning and memory usually involve various synaptic proteins and neurotrophic factors in the limbic system. Among these molecules, synaptic protein such as synaptotagmin (Syt), a Ca2+-dependent synaptic vesicle protein, the brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB) have been discovered to play some roles in hippocampus or amygdala associated learning and memory. Recent studies indicate that both treadmill exercise and voluntary wheel running can improve aversive memory and spatial memory in rodents. Whether treadmill exercise training can improve learning and memory by upregulating some of these molecules and whether different types of exercise affect different learning/memory behavior tasks, BDNF-TrkB signaling and Syt expression differentially remains unclear. To answer these questions, BALB/c mice were trained by treadmill exercise or voluntary wheel running for 4 weeks. The ability of learning and memory after exercise was evaluated by passive avoidance (PA) test and Morris water maze. Hippocampal or amygdalar TrkB and Syt I protein expression after exercise were determined by Western blotting and BDNF was evaluated by ELISA. Our results showed that 1) both treadmill exercise and wheel running improve spatial learning and memory in Morris water maze, but only treadmill exercise improves aversive memory in PA test; 2) Both two exercises transiently increased the hippocampal BDNF level and persistently increased the hippocampal protein levels of full-length TrkB and Syt; 3) Only treadmill exercise transiently increased the amygdalar BDNF level and persistently increased the amygdalar protein levels of Syt; 4) Local administration of K252a into the hippocampus or basolateral amygdala to blunt TrkB signaling abolished treadmill exercise-enhanced PA performance and related protein expression; 5) Local administration of Syt I lentiviral-shRNA into the basolateral amygdala to inhibit Syt I expression also abolished treadmill exercise-enhanced PA performance and amygdalar Syt I expression. In conclusion, these data suggest that the upregulation of TrkB and Syt I may contribute to the exercise-facilitated aversive memory. Furthermore, moderate treadmill exercise and voluntary wheel running have differential effects on the aversive learning and memory, possibly due to the diverse influences on synaptic plasticity-related proteins in the amygdala.