Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 180-194.

Submitted by Cristiane Dambrós (cristiane@ourinhos.unesp.br) on 2016-07-19T15:15:31Z No. of bitstreams: 1 UNESP - Tese - Cristiane Dambros.pdf: 5772395 bytes, checksum: b6037520e0eca710e93ca6fb14626d1a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-20T17:15:30Z (GMT) No. of bitstreams: 1 dambros_c_dr_rcla.pdf: 5772395 bytes, checksum: b6037520e0eca710e93ca6fb14626d1a (MD5) / Made available in DSpace on 2016-07-20T17:15:30Z (GMT). No. of bitstreams: 1 dambros_c_dr_rcla.pdf: 5772395 bytes, checksum: b6037520e0eca710e93ca6fb14626d1a (MD5) Previous issue date: 2016-01-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este texto tem como finalidade contribuir com reflexões acerca das transformações e da complexidade do mundo rural, como objeto de análise optou-se por pequenas propriedades rurais localizadas nos municípios de Brotas/SP e Rio Claro/SP. Defendemos a teses que, se a paisagem é resultado da ação humana no espaço, quanto mais diversa e multifuncional ela for, maior será a possibilidade de manutenção dos recursos hídricos, ou seja, as pequenas propriedades rurais de Brotas/SP e Rio Claro/SP que são multifuncionais (maior diversidade paisagística), apresentam maior grau de conservação da água em contraste com as pequenas propriedades rurais não multifuncionais. Para a realização deste estudo foi necessário a estruturação de uma metodologia, esta resultado da compilação de outras três: Multifuncionalidade da Paisagem, Sistema GTP e Sistema de Agricultura. Os dados necessários para o estudo foram bibliográficos, coleta, sistematização e análises de dados primários na forma de 40 entrevistas por questionário em Brotas e Rio Claro e dados secundários através de fontes confiáveis. A paisagem rural, considerando os fatos visíveis e invisíveis, identificada nas pequenas propriedades rurais e compreendidadas no contexto municipal, foi o parâmetro usado na reflexão e explicação da complexidade do mundo rural. A partir do diagnóstico considera-se que as áreas rurais não podem mais ser percebidas apenas como produtoras de alimentos, mas como territórios que abarcam múltiplas funções agrícolas e não-agrícolas, impulsionados pela necessidade ou por políticas públicas específicas. Em suma, estamos falando de uma reestruturação do espaço rural, onde a busca por iniciativas que complementem a renda, a elaboração de políticas públicas que condizem com a realidade do agricultor, a ação coletiva e a cooperação serão elementos primordiais para coesão e perpetuação dos agentes locais. / This text aims to contribute reflections on the changes and the complexity of the rural world, and as the object of analysis we chose to small farms located in the municipalities of Brotas/SP and Rio Claro/SP. We defend the thesis that if the landscape is the result of human action in space, the more diverse and multifunctional it is, the greater the possibility of maintenance of water resources, ie small farms Brotas / SP and Rio Claro / SP that are multifunctional (greater landscape diversity), have a higher degree of water conservation in contrast to small non multifunctional farms. For this study it was necessary to structure a methodology, this result of the compilation of three other: multifunctionality landscape, GTP System and Farming System. The necessary data for the study were bibliographic, collection, systematization and primary data analysis in the form of 40 interviews by questionnaire and Brotas Rio Claro and secondary data through reliable sources. The rural landscape, considering the visible and invisible facts, identified in small farms and compreendidadas in the municipal context, was the parameter used in reflection and explanation of the complexity of the countryside. From the diagnosis it is considered that rural areas can no longer be seen only as producers of food, but as territories that span multiple agricultural and non-agricultural functions, driven by need or by specific public policies. In short, we are talking about a restructuring of rural areas, where the search for initiatives that complement the income, the development of public policies that are consistent with the reality of the farmer, collective action and cooperation are key elements of cohesion and perpetuation of agents sites.

Guanine nucleotide exchange factors (GEFs) regulate and organize diverse cellular processes through their role in converting GTPases from the inactive GDP bound state to the active GTP bound state. An increasing number of GEFs undergo autoregulatory mechanisms through complex intramolecular interactions. Relief of autoinhibition involves specific phosphorylation or binding to lipid and/or effector proteins at sites distal from the catalytic domain, and is often coupled to membrane recruitment. In Cytohesin Arf GEFs, the catalytic Sec7 domain is autoinhibited by a linker region and C-terminal helix flanking a Pleckstrin Homology (PH) domain. Upon binding of the PH domain to low abundance phosphoinositides, the GTPase Arf6-GTP can both relieve autoinhibition and recruit Cytohesins to the plasma membrane. This thesis focuses on determining the molecular mechanism underlying both these functions. The structural mechanisms by which Arf6-GTP binding relieves autoinhibition were studied using biochemical and crystallographic studies. The crystal structure of the Grp1 PH domain in complex with Arf6 revealed that Arf6-GTP binding relieves autoinhibition through competitive sequestration of the inhibitory elements into grooves formed at the periphery of the interface. Importantly, the interaction orients all known membrane targeting components to a common surface. Detailed biochemical studies showed a common mode of binding among Cytohesin family members in which phosphoinositide head group binding primes the interaction with Arf6, and membrane recruitment of both stimulatory and substrate Arf enhances the effect. To assess changes in the Sec7 domain conformation upon activation, Size Exclusion Chromatography in line with Small Angle X-Ray Scattering (SEC-SAXS) was performed. The unique nature of this data led to the development of a novel data analysis and processing strategy. A graphically based, python-extensible software package was created for data normalization, buffer correction, Guinier Analysis, and constant background subtraction. As an unbiased substitute for traditional buffer subtraction, a method to reconstruct the protein scattering through singular value decomposition (SVD) and linear combination of the basis vectors was developed. These methods produced exceptional data quality and allowed versatility for application to other data collection techniques or systems, especially those lacking confident buffer matching or low signal. SEC-SAXS confirmed the overall structure of autoinhibited Grp1 in solution and showed only slight overall changes upon activation by deletion of the autoinhibitory Cterminal helix. Fusion of Arf6 with Grp1 produced a consistently elongated shape in the active state that was incompatible with the autoinhibited or theoretical active positions of the Sec7 domain. Monte Carlo and rigid body modeling using known structural domains revealed a requirement for Sec7-PH linker flexibility in addition to Sec7 domain mobility. These data support an integrated structural model whereby phosphoinositides and Arf-GTP support nucleotide exchange at membranes through allosteric activation, membrane recruitment, and large-scale rearrangement of the Sec7 domain. Overall, these findings offer insight into Cytohesin function that can be applied to assess relief of autoinhibition in the context of other GEFs and GTPases.

The dynamic processes that occur at specific times and locations in cells and/or whole organisms during cellular division, migration, morphogenesis and development are critical. When these molecular events are not properly regulated, disease states can develop. Tools that can allow us to better understand the specific events that, when misregulated, result in disease development can also allow us to determine better ways to combat such misregulation. Specifically, tools that could allow us to better visualize cellular processes or those that allow us to control cellular functioning in a spatiotemporal manner could present great insight into the detailed inner workings of cells and/or whole organisms. Where chemistry and biology intersect presents a powerful starting point for the development of such tools. The first half of this thesis addresses tools to allow the better visualization of cellular events, in particular the intriguing process of bioluminescence and the work that has been done to better understand and optimize its utilization, particularly in living organisms. The novel work presented here details a parallel approach to improve our ability to observe cellular functioning specifically by improving bioluminescence imaging through the generation and characterization of mutant luciferase proteins that can better utilize novel small molecule luciferin substrates. The second half of this thesis discusses methods that have been developed to better control cellular events through the control of protein activity, specifically a family of proteins called the Rho GTPases. This family’s activation at specific times and locations is essential to proper cellular function and exemplifies the need for spatiotemporal control. Described are methods to control the activation states of the Rho GTPases to probe their cellular roles in a temporal and spatial manner using photosensitive small molecules. Taken together, the findings described herein demonstrate the application of chemistry to allow for the better observation and control of cellular processes, toward the ultimate goal of improving our understanding of the regulatory processes involved in the control of key factors leading to disease states.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17800号 / 生博第288号 / 新制||生||37(附属図書館) / 30607 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 根岸 学, 教授 松田 道行, 教授 垣塚 彰 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Les petites protéines G sont des protéines capables de fixer du GDP ou du GTP, ce qui va induire des changements de conformation au sein de la protéine qui lui permettront d'interagir avec des partenaires cellulaires distincts, et ainsi de jouer un rôle "d'interrupteur moléculaire". Le cycle GDP/GTP des petites protéines G ne fonctionne pas seul, il est régulé par un facteur d'échange GDP/GTP (GEF) et une protéine activatrice de la GTPase (GAP). Les petites protéines G sont impliquées dans des processus cellulaires fondamentaux et divers, comme la différentiation et la prolifération cellulaire, l'organisation et la dynamique du cytosquelette, et les transports intracellulaires. Un certain nombre de structures de petite protéine G sont maintenant connues, et ont permis de définir le repliement général des petites protéines G et les changements de conformation au cours du cycle GDP/GTP. Le premier projet porte sur l'étude structurale par diffraction des rayons X de la petite protéine G Rap2A, homologue de l'oncogène Ras dans un complexe non catalytique avec le GTP. Cette étude a permis de mettre en évidence la présence d'une nouvelle interaction au niveau du site nucléotidique entre la tyrosine 32 et le phosphate gamma du GTP. Et, nous avons montré que les changements de conformation de Rap2A au cours de son cycle GDP/GTP sont caractérisés par deux transitions désordre/ordre. Le second projet porte sur l'étude structurale par diffraction des rayons X de la petite protéine G Arf6 en complexe avec du GDP. Cette étude a montré que deux protéines qui possèdent une forte homologie de séquence peuvent avoir des structures assez différentes pour être distinguées. Les principaux partenaires des formes GDP des petites protéines G sont les GEF, ce qui suggère une base structurale pour la spécificité des GEF. En conclusion, nous discutons des bases structurales qui permettent aux petites protéines G d'être distinguées les unes des autres.

Cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC form the bacteriocin colicin V (ColV) secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter with nucleotide-binding motifs in the C-terminal domain (CTD). To study the role of CvaB-CTD in the ColV secretion, a truncated construct of this domain was made and over-expressed. Different forms of CvaB-CTD were obtained during purification, and were identified as monomer, dimer, and oligomer on gel filtration. Nucleotide binding was shown critical for the CvaB-CTD dimerization: oligomers could be converted into dimers by nucleotide bindings; the removal of nucleotide from dimers resulted in transient monomers followed by CTD oligomerization and aggregation; no dimer form could be cross-linked from the nucleotide-binding deficient mutant D654H. The spatial proximity of the Walker A site and ABC signature motif in CTD dimer was identified through disulfide cross-linking of mixed CvaB-CTD with mutants A530C and L630C, while mutations did not dimerize individually. Those results indicated that the CvaB-CTD formed a nucleotide-dependent head-to-tail dimer. Molecular basis of differential nucleotide bindings was also studied through bioinformatics prediction and biochemical verification. Through sequence alignment and homology modeling with bound ATP or GTP, it was found that the Ser503 and Gln504 on aromatic stacking region (Y501DSQ-loop) of CvaB-CTD provided two additional hydrogen-bonds to GTP, but not to ATP. Site-directed mutations of the S503A and/or Q504L were designed based on the model. While site-directed mutagenesis studies of Walker A&B sites or the ABC signature motif affected little on the GTP-binding preference, the double mutation (S503A/Q504L) on the Y501DSQ-loop increased both ATP-binding and ATPase activity at low temperatures. The double mutant showed slight decrease of GTP-binding and about 10-fold increase of the ATP/GTP-binding ratio. Similar temperature sensitivity in nucleotide-binding and activity assays were identified in the double mutant at the same time. Mutations on the Y501DSQ-loop did not affect the ColV secretion level in vivo. Together, the Y501DSQ-loop is structurally involved in the differential binding of GTP over ATP.

FtsZ, the principal cell-division protein, polymerizes in GTP-dependent manner in vitro (Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994; Rivas et al., 2000). FtsZ polymerization at the mid-cell site of bacterium leads to formation of a guiding scaffold, the Z-ring, for bacterial cytokinesis (Bi and Lutkenhaus, 1991; Sun and Margolin, 1998). GTP-induced polymerization process of FtsZ can be monitored in vitro Using 90º light scattering (Mukherjee and Lutkenhaus, 1999) and polymers formed can be visualized using transmission electron microscopy (Lu and Erickson, 1998) or quntitated in terms of the amount of FtsZ polymer pelleted during ultracentrifugation (Mukherjee and Lutkenhaus, 1998). The research work presented in this thesis focused on structure-function correlative analysis of Mycobacterium tuberculosis FtsZ(MtFtsZ0 and FtsZ proteins of Mycobacterium leprae (M1FtsZ), Mycobacterium smegmatis(MsFtsZ), and Streptomyces coelicolor (ScFtsZ) (as it is from Actinomycetes family to which mycobacteria belong) in vitro. It was initiated with investigation on the biochemical properties of Mycobacterium leprae FtsZ (M1FtsZ) in vitro. In comparison with those of MtFtsZ. Subsequently, the role of C-terminal stretch of amino acid residues of MtFtsZ in polymerization was investigated. Finally, a comparative analysis of the biochemical properties of MtFtsZ, MsFtsZ, and ScFtsZ was carried out in order to find out whether a correlation exists between the time taken by the FtsZ of a bacterium to polymerise and the generation time of the organism. The thesis is presented in five chapters. First Chapter gives an exhaustive introduction on the structure-function aspects of FtsZ. Second Chapter deals with materials used in this research work and details of various experimental methods [cloning and expression of FtsZ (White et. Al., 2000), decision and point mutagenesis, preparation of His-tag free MtFtsZ and M1FtsZ by thrombin cleavage method, 90º light scattering (Mukherjee and Lutkenhaus, 1999), White, et al., 2000), transmission electron microscopy (Lu and Erickson, 1998), pelleting assay for polymeric FtsZ (Mukherjee and Lutkenhaus, 1998), GTP-binding by UV-crosslinking (RayChaudhuri and Park, 1992; de Boer et al.,) GTPase assay(RayChaudhuri and Park, 1992); de Boer et al., 1992), Circular Dichroism (Saxena and Wetlaufer, 1971) and ANS fluorescence emission spectroscopy (Semisotnov, et al., 1991)]. The Chapters three to five contain all the data related to the research work, the outlines of which are given below. Chapter 3. Biochemical Characterisation of FtsZ Protein of Mycobacterium leprae In Comparison with the Biochemical Properties of FtsZ Protein of Mycobacteriulm tulberculosis In Vitro The major finding in this part of thesis work is on the demonstration that single reciprocal point mutation partially revives polymerization-inactive M1FtsZ and Inactivates polymerization-active MtFtsZ in vitro. In brief, soluble, recombinant M1FtsZ did not show detectable polymerization in vitro, in contrast to MtFtsZ, which showed appreciable polymerization, under standard conditions, when monitored using 90º light scattering assay and transmission electron microscopy. This was a surprising result, as M1FtsZ and MtFtsZ has 96% protein sequence identity. Mutation f T172 in the N-terminal domain of M1FtsZ to A172, as it exists in MtFtsZ, showed dramatic levels of polymerization in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in M1FtsZ, abolished polymerization in vitro. Further, M1FtsZ showed weak GTPase activity, in contrast to MtFtsZ, which showed appreciable GTPase activity. While T172A mutation enhanced GTPase activity of MtFtsZ in vitro. Circular dichroism spectroscopy and ANS fluorescence emission spectroscopy showed that there were no major secondary or tertiary structural changes in these point mutants. These observations demonstrate that the residue at position 172 plays a critical role in the polymerization of M1FtsZ and MtFtsZ, without appreciably affecting their respective GTpPase activity. Further, this result might have implications on evolution of a slow polymerizing FtsZ in slow growing bacteria. Further details of evolution related questions are addressed in Chapter 5. Chapter 4. Role of Carboxy Terminal Residues in the Biochemical Properties of FtsZ Protein of Mycobacterium tuberculosis In Vitro The major finding in this part of thesis work is the demonstration that the C-terminal end residues are critically required for polymerization of MtFtsZ in vitro, which is in direct contrast to the dispensability of C-terminal residues of Escherichia coli FtsZ(EcFtsZ), Bacillus subtilis FtsZ (BsFtsZ), and Pseudomonas aeruginosa (PaFtsZ) for polymerization. FtsZ protein from several bacterial species namely, Methanococcus jannaschii (MjFtsZ), Bacillus subtillis(BsFtsZ), Pseudomonas aeruginosa (PaFtsZ), and Aquifex aeolicus (AaFtsZ) (Lowe and Amos, 1998; Oliva et al., 2007), and Mycobacterium tuberculosis H37Rv (mtFtsZl Leung et al., 2004), whose crystal structures have been solved so far, were found to possess an N-terminal domain and a C-terminal domain that were connected to each other through a helix. The extreme C-terminal portion of all these FtsZ proteins is constituted by an unstructured tail (Lowe and Amos, 1998; Oliva et al., 2007l Leung et al., 2004), which is not found in the respective crystal structure of the protein. We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (mtFtsZ) have any role in MtFtsZ polymerization in vitro. Deletion of C-terminal 66 residues (313-379) was found to abolish polymerization. Replacement of the C-terminal 66 residues with the extreme C-terminal 13-residue stretch (DDDDVDVPPFMRR) did not restore polymerization. Although the terminal R in DDDDVDVPPFMRR is dispensable for full-length MtFtsZ polymerization, the terminal R in DDDDVDVPPFMR is indispensable for polymerization. Neither replacement of this R, in the terminal R deletion mutant DDDDVDVPPFMR, with K/H/D/A residues enabled polymerization. GTP binding and GTPase activities of the mutants were partially affected. The indispensable nature of C-terminal residues for MtFtsZ polymerization in vitro is contrary to the dispensability of the equivalent extreme C-terminal residues of Escherichi coli, Pseudomonas aeruginosa, and Bacillus subtilis FtsZ (Wang et. Al., 1997; Cordell et al., 2003; Singh et al., 2007) for in vitro polymerization. The essentiality of C-terminal extreme residues of BtFtsZ for polymerization offers direction to design anti MtFtsZ polymerization agents. Chapter 5. An attempt to find correlation between Biochemical properties of FtsZ and Generation Time of the Bacterium The clue that there might be a correlation between FtsZ polymeristion and generation time of the bacterium came from the observation mentioned in chapter 3. The presence of polymerization-aversive T172 in the FtsZ of extremely slow-growing M. leprae 913.5 days generation time, Levy, 1970) and polymerization-favouring A172 in the FtsZ of M. tuberculosis(18hrs generation time, Patterson and Youmans, 1970). For a bacterium, which has short generation time, it might be conducive to have an FtsZ that will also polymerise fast. Conversely, for a bacterium, which has long generation, it might be conducive to have an FtsZ molecule that will polymerise slow. In this respect, a preliminary comparative study was carried out between the generation time of bacterial species, E. coli, Mycobacterium smegmatis, Streptomyces coelicolor, M leprae, and M. tuhberculosis and their respective FtsZ (EcFtsZ, MsFtsZ, M1FtsZ and MtFtsZ). Detailed biochemical characterization of EcFtsZ and MtFtsZ has already been reported in the literature. In this thesis work, biochemical characterisation of M1FtsZ(Chapter 3), ScFtsZ and MsFtsZ (in this Chapter) were carried out. E. coli, which has a generation time of 18-55 min(labrum, 1953), possesses FtsZ (EcFtsZ) that reaches steady state of polymerization in about 10 sec under standard conditions in vitro (Beamhill and Thompson, 1994), using 90º light scattering assay (Mukherjee and Lukenhaus, 1999). On the other hand, M. tuberculosis, which has a generation time of 18hrs in vivo (Patterson and Youmans, 1970) and 24 hrs in vitro (Hiriyanna and Ramakrishnan, 1986) possesses FtsZ (MtFtsZ) that reaches steady state of polymerization in about 6 min post-addiction of GTP in vitro (White et al., 2000). Further, M. leprae, which takes 13.5 days tp divide once in vivo (levy, 1970), possesses an FtsZ (M1FtsZ) that does not even show polymerization under standard conditions in vitro (Chapter 3 of this thesis). The organisms Mycobacterium smegmatis and Streptomyces coelicolor have generation times that fall in between those of the other three organisms mentioned above. While M. smegmatis divides once in 2-3 hrs (Husson, 1998), S. coelicolor has a variable generation time depending on growth condition, which can be as fast as once in 2.31 hours, depending upon growth conditions (Cox, 2004). We found ScFtsZ and MsFtsZ takes around 4 min to reach polymerization saturation after addition of GTP, EcFtsZ( 10 sec), MtFtsZ (10 min) and M1FtsZ (dose not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation after addition of GTP, EcFtsZ (10sec), MtFtsZ (10 min) and M1FtsZ (does not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation time and the generation time of the respective bacterium. But when we compared polymerization time of ScFtsZ and MsFtsZ (4 min both case) with MtFtsZ ( 6 min), we found that there is no linear correlation with generation time of these bacteria and the time taken by their FtsZ to reach steady state of polymerization. Many more bacterial FtsZ proteins need to be characterized to conclusively state wthether there exist a correlation between generation time of bacteria and the time taken for their FtsZ to reach steady state of polymeristion. Such correlation would simply reveal the fact that the primary structure of an FtsZ protein might have evolved to suit the generation time of the bacterium.