Thesis (Ph.D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-12, Section: B, page: 7351. Adviser: Tanja Kortemme.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Geeta J. Narlikar.

Resumo: O objetivo deste trabalho é investigar a localização de energia e o aprisionamento do breather no modelo de Peyrard Bishop com o potencial original de Morse, potencial simétrico de Morse e potenciais quárticos em cadeias homogêneas e não homogêneas usando o Teorema do limite anticontinuum. No caso não homogêneo, a impureza é introduzida pela profundidade do potencial. Foi observado que o modelo SPB apresenta pequenas amplitudes e menor densidade de energia comparada com o modelo PB. Para potenciais quárticos simétricos e assimétricos foi verificado numericamente que não temos transição de fase usando o conceito de amplitude média das vibrações. Entretanto, um potencial híbrido formado por um quártico e Morse pode fornecer uma grande amplitude de vibração das fitas de DNA . Finalmente, no sistema não homogêneo, se verificou a hipótese de aprisionamento por meio de uma simulação de interação do breather móvel com a região de TATA box para uma cadeia longa de DNA, correspondente à seqüencia de nucleotídeos que codifica a insulina. Este resultado deve ajudar a estender a aplicação dos breathers discretos a sistemas biológicos que levam em conta reações bioquímicas localizadas (como, por exemplo, reações enzimáticas) em macromoléculas biológicas. O modelo PB pode ser modificado para explicar a existência de movimentos localizados de grande amplitude. O decaimento da função potencial para valores grandes mostraram uma vibração localizada no equilíbrio de grande amplitude. Isso é feito alterando o potencial on site. / Abstract: The objective of this work is to investigate the energy localization and the breather trapping in the Peyrard-Bishop model with the original Morse potential, symmetric Morse potential and quartics potentials in homogeneous and inhomogeneous chains. In the inhomogeneous case, the impurity is introduced by the depth of the potential. It was observed that the SPB model shows small amplitude and lower density of energy compared with the PB model. Quartics potentials, for symmetric and asymmetric cases, do not show phase transition. It is verified by numerical calculation using the concept of mean amplitude of the vibrations. Meanwhile, a potential hybrid formed by a quartic and Morse is ideal for DNA applications. Finally, it was verified the trapping hypotheses through the simulation of interaction of mobile breather with the region of TATA box for a long chain of DNA corresponding to the nucleotide sequence that encodes the insulin. This result should help to extend the application of discrete breathers the biological systems that take into account located biochemical reactions (such as enzymatic reactions) in biological macromolecules. In addition, the PB model can be modified to explain the existence of highly localized large amplitudes motions of the base pairs in DNA. We do this change the on site potential. / Orientador: Elso Drigo Filho / Coorientador: José Roberto Ruggiero / Banca: Gerald Weber / Banca: Regina Maria Ricotta / Banca: Jayme Vicente de Luca Filho / Banca: Masayoshi Tsuchida / Doutor

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-24Bitstream added on 2014-06-13T18:49:56Z : No. of bitstreams: 1 costa_lc_me_sjrp_parcial.pdf: 205173 bytes, checksum: bf8330ba8ebec2e33d2e94ec3f85ca8c (MD5) Bitstreams deleted on 2015-06-25T13:01:07Z: costa_lc_me_sjrp_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:25Z : No. of bitstreams: 1 000624262_20151231.pdf: 180856 bytes, checksum: 0c0db07749af7e17dd04bbdebcc70b88 (MD5) Bitstreams deleted on 2016-01-04T10:26:22Z: 000624262_20151231.pdf,. Added 1 bitstream(s) on 2016-01-04T10:28:15Z : No. of bitstreams: 1 000624262.pdf: 814952 bytes, checksum: c2e88a5f2759a15bbe1e835ebd0266af (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Mastoparanos são uma família de peptídeos líticos, extraídos do saco de veneno de vespas sociais, que apresentam moderada a intensa atividade antimicrobiana e alguns são hemolíticos e citotóxicos. Embora o mecanismo de ação destes peptídeos não seja bem compreendido ainda, aceita-se que envolva desestabilização da fase lipídica da membrana celular. Acredita-se que a carga líquida do peptídeo e sua hidrofobicidade média contribuam na modulação da atividade lítica e na seletividade. Nesta dissertação apresentamos um estudo de quatro peptídeos mastoparanos que possuem resíduos ácidos e básicos resultando em uma carga elétrica líquida variando de +1 a +4. Este estudo envolve a análise conformacional destes peptídeos em vesículas zwitteriônicas e aniônicas por dicroísmo circular. Suas atividades líticas foram avaliadas usando espectroscopia de fluorescência monitorando a recuperação da intensidade de fluorescência devido à liberação de marcador fluorescente encapsulado em vesículas. As constantes de partição destes peptídeos em vesículas zwitteriônicas foram também determinadas por espectroscopia de fluorescência usando um método de análise que é independente de modelo. Com o objetivo de entender a influência de resíduos ácidos e a carga líquida dos peptídeos e sua hidrofobicidade na interação peptídeo-lipídio, foram estimadas as contribuições energéticas eletrostáticas e não eletrostática. Para alcançar este objetivo, os resultados obtidos nas isotermas de ligação por fluorescência foram associados com medidas de potencial zeta. Foi observado que a afinidade destes peptídeos em vesículas zwitteriônicas decresce com a carga líquida do peptídeo e as curvas de dose-resposta são mais cooperativas para os dois peptídeos com as cargas mais baixas. Os peptídeos com maior carga apresentaram uma maior afinidade... / Mastoparans are a family of lytic peptides extracted from the venom sac of wasps which present moderate to intense antimicrobial activity and some of them are hemolytic and cytotoxic. Although the mechanism of action of these peptides are still not completely understood, it is accepted that it involves the destabilization of the lipidic phase of the cell membrane. It is believed that both the peptide net electrical charge and its mean hydrophobicity contribute to modulate their lytic activity and selectivity. In this dissertation we present a study of four mastoparan peptides which have acidic and basic residues resulting in net electrical charges ranging from +1 to +4. This study involves the conformational analysis of these peptides in zwitterionic and anionic lipid vesicles by circular dichroism. Their lytic activities were evaluated using fluorescence spectroscopy by the release of fluorescent dye entrapped in these two types of vesicles. The partition constants of these peptides to zwitterionic vesicles were also determined by fluorescence spectroscopy using a method of analysis which is model independent. Aiming to understand the influence of acidic residues and of the peptides net charge and hydrophobocity on the peptide-lipid interaction, the electrostatic and non electrostatic energetic contributions were estimated. To achieve this goal it was used the association of the results of fluorescence binding isotherms and zeta potential measurements. It was observed that the affinity and lytic activity of these peptides in zwitterionic vesicles decrease with the peptides net electrical charges and the dose-response curves are more cooperative for the two peptides with lower net charges. The more charged peptides exhibit higher affinity and lytic activity in anionic vesicles. The most charged peptide displayed the higher selectivity for the vesicles studied. The present work... (Complete abstract click electronic access below)

Resumo: Mastoparanos são uma família de peptídeos líticos, extraídos do saco de veneno de vespas sociais, que apresentam moderada a intensa atividade antimicrobiana e alguns são hemolíticos e citotóxicos. Embora o mecanismo de ação destes peptídeos não seja bem compreendido ainda, aceita-se que envolva desestabilização da fase lipídica da membrana celular. Acredita-se que a carga líquida do peptídeo e sua hidrofobicidade média contribuam na modulação da atividade lítica e na seletividade. Nesta dissertação apresentamos um estudo de quatro peptídeos mastoparanos que possuem resíduos ácidos e básicos resultando em uma carga elétrica líquida variando de +1 a +4. Este estudo envolve a análise conformacional destes peptídeos em vesículas zwitteriônicas e aniônicas por dicroísmo circular. Suas atividades líticas foram avaliadas usando espectroscopia de fluorescência monitorando a recuperação da intensidade de fluorescência devido à liberação de marcador fluorescente encapsulado em vesículas. As constantes de partição destes peptídeos em vesículas zwitteriônicas foram também determinadas por espectroscopia de fluorescência usando um método de análise que é independente de modelo. Com o objetivo de entender a influência de resíduos ácidos e a carga líquida dos peptídeos e sua hidrofobicidade na interação peptídeo-lipídio, foram estimadas as contribuições energéticas eletrostáticas e não eletrostática. Para alcançar este objetivo, os resultados obtidos nas isotermas de ligação por fluorescência foram associados com medidas de potencial zeta. Foi observado que a afinidade destes peptídeos em vesículas zwitteriônicas decresce com a carga líquida do peptídeo e as curvas de dose-resposta são mais cooperativas para os dois peptídeos com as cargas mais baixas. Os peptídeos com maior carga apresentaram uma maior afinidade... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: Mastoparans are a family of lytic peptides extracted from the venom sac of wasps which present moderate to intense antimicrobial activity and some of them are hemolytic and cytotoxic. Although the mechanism of action of these peptides are still not completely understood, it is accepted that it involves the destabilization of the lipidic phase of the cell membrane. It is believed that both the peptide net electrical charge and its mean hydrophobicity contribute to modulate their lytic activity and selectivity. In this dissertation we present a study of four mastoparan peptides which have acidic and basic residues resulting in net electrical charges ranging from +1 to +4. This study involves the conformational analysis of these peptides in zwitterionic and anionic lipid vesicles by circular dichroism. Their lytic activities were evaluated using fluorescence spectroscopy by the release of fluorescent dye entrapped in these two types of vesicles. The partition constants of these peptides to zwitterionic vesicles were also determined by fluorescence spectroscopy using a method of analysis which is model independent. Aiming to understand the influence of acidic residues and of the peptides net charge and hydrophobocity on the peptide-lipid interaction, the electrostatic and non electrostatic energetic contributions were estimated. To achieve this goal it was used the association of the results of fluorescence binding isotherms and zeta potential measurements. It was observed that the affinity and lytic activity of these peptides in zwitterionic vesicles decrease with the peptides net electrical charges and the dose-response curves are more cooperative for the two peptides with lower net charges. The more charged peptides exhibit higher affinity and lytic activity in anionic vesicles. The most charged peptide displayed the higher selectivity for the vesicles studied. The present work... (Complete abstract click electronic access below) / Orientador: João Ruggiero Neto / Coorientador: Marcia Perez dos Santos Cabrera / Banca: Bibiana Monson de Souza / Banca: Marcelo Andrés Fossey / Mestre

Orientador: Elso Drigo Filho / Banca: Makoto Yoshida / Banca: Gerald Weber / Banca: Jorge Chahine / Banca: João Ruggiero Neto / Resumo: O objetivo deste trabalho é investigar um modelo mecânico para a molécula de DNA. O modelo consiste de duas cadeias de osciladores harmônicos representando as tas do DNA. Esses osciladores são ligados por um potencial de Morse que simula as interações tipo pontes de hidrogênio, como no modelo original de Peyrard e Bishop. Entretanto, neste trabalho, os movimentos de rotação e de vibração de cada par de base podem ocorrer ao mesmo tempo. Neste contexto, propriedades estruturais e termodinâmicas são discutidas. / Abstract: The objective of this work is to investigate a mechanical model for the DNA molecule. The model consist in two chains of harmonic oscillators representing the ribbons of DNA linked by a Morse potential which represents the hydrogen bonds as in the Peyrard-Bishop's model. However, in this work, the rotation and vibration motion of each base pairs can be occur at the same time. In this context, thermodynamic and structural properties are discussed. / Doutor

Orientador: Marcio Francisco Colombo / Coorientador: Chien Ho / Banca: Marinônio Lopes Cornélio / Banca: Marcelo Andrés Fossey / Banca: Hamilton Cabral / Banca: Marcelo Matos Santoro / Resumo: A influência de ânions sobre as características estruturais e funcionais de hemoglobinas (Hb) vem sendo alvo de pesquisas de nosso laboratório nos últimos anos. Estudos anteriores mostraram que a ligação preferencial de ânions como o Cl- e fosfatos orgânicos (2,3-DPG, IHP) entre outros à desoxi-Hb humana (HbA), modula o equilíbrio entre dois estados alostéricos desoxigenados possíveis, estados To e Tx, livre e complexado com ânions, respectivamente. Medidas funcionais utilizando o método de estresse osmótico, que determina o número de moléculas de água que se liga a superfície da Hb, acompanhando a oxigenação, mostraram que o estado To é um estado com hidratação e afinidade ao oxigênio intermediárias ao Tx e ao oxi-R. Assim, a oxigenação da Hb demanda um modelo considerando estes três estados, To, Tx e R, modulados pela presença de ânions em solução. O íon cloreto é um dos ânions de maior importância fisiológica. A presença de sítios específicos para sua ligação, bem como seu sistema de ligação para a Hb, contudo, são controversos. Evidências estruturais e funcionais apontaram os resíduos arginina 141 e 92 da cadeia alfa como possíveis sítios de ligação de cloreto à Hb. Para investigar essa hipótese, realizamos estudos funcionais variando atividade de água e pH (efeito Bohr), em função da presença de cloreto com hemoglobinas mutantes para essas posições. As hemoglobinas selecionadas foram Chesapeake (R92L), J-Cape Town (R92Q), desArg (141Δ) e Chesapeake desArg (R92L,141Δ). Aspectos estruturais dessas Hbs foram obtidos através de espectroscopia 1H RMN. Todas as Hbs estudadas apresentaram afinidade maior ao oxigênio quando comparadas à HbA, para todas as condições experimentais. Estudos de variação da atividade de água em função da concentração de cloreto mostraram que a única... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The influence of anions on structural and functional properties of hemoglobins (Hb) has been studied by our research group for the last few years. Former studies have shown that the preferential binding of anions, such as Cl and organic phosphates (2,3-DPG, IHP) among others to human deoxy-Hb (HbA), modulates the equilibrium between two possible deoxy states: To and Tx, free and complexed with anions respectively. Functional measurements using the osmotic stress method, which allows the determination of the number of water molecules that binds to the protein surface upon the change in protein conformation induced by oxygenation, showed that To state has intrinsic affinity and hydration intermediate of those of Tx and oxy-R . Hence, Hb oxygenation requires a three state model, considering To, Tx and R, modulated by the presence of anions in solution. Chloride is one of the most important physiological ions and the presence of specific binding sites of this anions is controversial. Structural and functional evidence have pointed to Arginines 141 and 92 as possible binding sites for chloride to Hb. To investigate this hypothesis, we have performed functional studies, changing the water activity and pH (Bohr effect), in function of chloride concentration with recombinants Hbs for these positions. The selected hemoglobins are Chesapeake (R92L), J-Cape Town (R92Q), desArg (141Δ) eandChesapeake desArg (R92L,141Δ). Structural features were obtained through 1H NMR spectroscopy. All Hbs studied have higher affinity than HbA for all experimental conditions. Water activity studies in function of chloride concentration showed that the only Hb that can be adjusted with the three state model is Hb desArg, whereas the other Hbs don't present the Tx state, fact confirmed by the number of water molecules bound to each... (Complete abstract click electronic access below) / Doutor

Neurotensin (NT) is a tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. Neurotensin receptor 1 (NTS1), a high affinity receptor for NT, is a member of the GPCR superfamily and is a putative target for the treatment of conditions such as Schizophrenia, Parkinson’s Disease and drug addiction. Overexpression and purification are typically limiting steps in the high resolution structure determination of GPCRs. In this study, through the optimisation of the E.coli strain used for overexpression of rat NTS1 (NTS1) and the inclusion of phospholipids in the purification buffers to prevent delipidation, an approximate 3-fold improvement in active receptor yield was obtained relative to existing protocols. Preliminary electron microscopy (negative stain and cryo) confirmed a monodisperse receptor population. Purified NTS1 is now being produced at a sufficient level for high resolution structural studies, including 3D crystallisation and further electron microscopy studies. The existing construct for the expression of NTS1 in E.coli, termed NTS1B, was modified to contain a fusion to the genes encoding either the eCFP or eYFP fluorescent proteins. These constructs were used for the E.coli expression of NTS1 tagged with either fluorescent protein at the C-terminus. Tagged receptor was successfully expressed at levels of up to 0.29 ± 0.03 mg per l of culture. Successful purification and proteolytic removal of the MBP and TrxA-His10 fusion partners was achieved whilst retaining both fluorescence and ligand binding capability (K_d = 0.91 ± 0.17 nM). Purified, fluorescent receptor was reconstituted into brain polar lipid (BPL) liposomes in an active conformation which was both fluorescent and able to bind NT. Experimentation with alternative lipid compositions suggested that specific lipids are required in order to maintain ligand-binding activity. FRET between the eCFP- and eYFP-tagged receptors was observed in reconstituted samples. The FRET efficiency was comparable to that observed in vivo for other GPCRs, including the yeast α-factor receptor, which is believed to be dimeric. This suggests that NTS1 could also be multimeric. In contrast, no FRET was observed in detergent samples. Therefore, a functioning in vitro system has been developed which enables the study of NTS1 multimerisation in lipid bilayers and future studies will attempt to implement single molecule fluorescence techniques. In addition, fluorescent derivatives of NT were successfully synthesised and purified. Radioligand competition assays and fluorescence correlation spectroscopy (FCS) confirmed that the fluorescent peptides bound to purified NTS1 in specific competition with unlabelled NT. Surface plasmon resonance (SPR) was used to confirm the ligand binding activity of purified NTS1. A novel approach was utilised which involved the measurement of the binding of detergent-solubilised NTS1 to immobilised, N-terminally biotinylated NT on the sensor surface. The use of a rigorous control, which consisted of immobilised ‘scrambled sequence’ NT, demonstrated a specific interaction. Analysis of the kinetics revealed a multiphasic interaction with a K_d in the nanomolar range. In summary, improvements to the expression and purification of NTS1, the generation of fluorescent constructs as useful tools in the study of receptor multimerisation and the optimisation of lipid-reconstitution protocols have opened up several preliminary lines of study which show considerable potential for future research.

Correct regulation of transcription is essential for maintaining a healthy cellular state. During transcription RNA polymerase II (Pol II) proceeds in a regulated manner through several transitions to ensure appropriate control of synthesis and enable correct processing of the pre-RNA. Shortly after initiation Pol II is caused to pause by the binding of factors, DSIF and NELF. To enable transition of Pol II into the elongation phase CDK9/cyclin T phosphorylates the C-terminal domain (CTD) of Pol II, DSIF and NELF. This phosphorylation releases the paused state and provides an alternative set of post-transcriptional modifications on the CTD to generate a binding platform for elongation, histone modifying and termination factors. CDK9/cyclin T is itself regulated within multicomponent complexes. A small activated complex, containing Brd4, recruits CDK9/cyclin T to active sites of transcription, thereby promoting the elongation of transcription. The role of CDK9/cyclin T in the regulation of transcription has resulted in its validation as a drug target against several disease states including cancer, HIV and cardiac hypertrophy. In this thesis, I present the crystallographic structures of a series of 2-amino-4-heteroaryl-pyrimidine compounds and the roscovitine derivative, (S)-CR8, bound to CDK9/cyclin T and CDK2/cyclin A. In combination with thermal denaturation data and kinetic analysis, these structures have suggested chemical modifications that might be made to increase the CDK9 specificity of these compounds. I have also validated the use of a mutated form of cyclin T for use in the development of CDK9/cyclin T inhibitors. In addition, I present both structural and kinetic analysis of the Brd4-CDK9/cyclin T interaction. I show that C-terminal fragments of Brd4 enhance the in vitro kinase activity of CDK9/cyclin T against the Pol II CTD. Furthermore, I demonstrate that this enhancement may be inhibited by Plk1-mediated phosphorylation of Brd4. Finally, I show that Brd4 binds to a site that spans CDK9 and cyclin T and I propose detailed molecular models of the Brd4-cyclin T interaction.

The bacterial flagellar motor is one of the few rotary motors found in nature, and an excellent example of a complex molecular machine. Flagellar motors in the model organism Escherichia coli are products of the coordinated expression of ∼50 different genes. The E. coli flagellar motor is powered by the proton-motive force (pmf), an electrochemical gradient across the cell membrane. Motor torque is gen- erated by proton flow through membrane-embedded stator units which bind to the basal body of the motor. This thesis aimed to investigate the relationship between the pmf and the flag- ellar motor. A novel pmf control system was developed, based on the light-driven proton pump proteorhodopsin (pR). This system enabled pmf -dependent changes in motor behaviour to be precisely monitored in vivo. Expression of pR in E. coli was shown to be sufficient to drive the flagellar motor at wild-type speeds. Using the pR-based pmf control system, the motor was shown to respond to changes in pmf on a timescale of milliseconds. Surprisingly, motor speed increase was observed when pmf was increased above the physiological norm. Reduction of pmf to low levels enabled individual steps in motor rotation to be observed. Motor response to loss of pmf was investigated. Motors were shown to exhibit a two-stage speed decrease after disruption of pmf , with motor speed falling to ∼20 % of its initial value within milliseconds, reaching a complete stop after 1 s. Extended periods of pmf loss was shown to lead to disengagement of stators from the motor, with motor speed increasing in a stepwise fashion after pmf restoration. The integrity of the motor at different pmf levels was investigated by using TIRF microscopy to directly image positioning of fluorescently tagged motor components. The stator protein MotB was shown to physically leave and rejoin the motor after pmf disruption and restoration, with MotB dispersal following motor stop.