Use of hydrogen bonding as internuclear connecting information to better determine molecular structure or dynamics.

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

A majority of kinase inhibitors predominantly occupy the highly conserved adenine-binding pocket located in the kinase catalytic cleft, and therefore the target selectivity of these molecules is a major concern. In order to design highly specific next-generation drugs, it is essential to exploit the less-conserved binding pockets, which lie adjacent to the adenine-binding pocket. Small peptides that can function as adenosine triphosphate (ATP) competitive inhibitors would prove useful in identifying and validating new druggable surfaces in the kinase catalytic cleft. These peptides, being larger than small molecules, have the potential to target the ATP binding pocket as well as surfaces that lie adjacent to this pocket. Such peptides recognizing novel binding pockets can assist the drug discovery process in several ways. In this thesis, we describe the isolation and characterization of a novel class of cyclic peptides, referred to as lariats, against Abl kinase, a drug target important in chronic myeloid leukemia and other disorders. Using a yeast two-hybrid approach, we first isolated two related lariats, named A1 and A2, from a pool of five million lariats, which interact with the catalytic domain of Abl kinase. In vitro studies indicated that the synthetic A1 lariat competitively inhibits ATP binding by targeting the catalytic cleft that lies between the N- and C- lobes of the kinase catalytic domain. To obtain tighter-binding variants of the A1 lariat, we developed an affinity maturation protocol consisting of two steps. In the first step, we defined acceptable and tolerable substitutions at each position of the A1 lariat using site-saturation mutagenesis (SSM). In the second step, we designed specific mutations to the A1 lariat based on the SSM results and evolved higher affinity variants. Synthetic and recombinant higher affinity lariats exhibited a strong inhibition of Abl kinase activity in vitro and Bcr-Abl kinase activity in vivo, respectively, illustrating the potential of lariats as chemical genetic tools. Resistance mutation profiling showed that the lariats are not affected by the activating mutations located in the activation loop of kinase, and instead bind preferentially to the kinase active conformation. Selectivity analysis indicated that the lariats do not recognize Src family kinases, which share a high structural similarity with Abl kinase in their active conformation. These findings, coupled with preliminary results from modeling studies, strongly suggest that the lariats have identified novel allosteric drug-binding pockets in the kinase catalytic cleft.

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-08-21T14:06:33Z No. of bitstreams: 2 Dissertação - Ohana Benevides Rodrigues - 2018.pdf: 55370195 bytes, checksum: 561eb17a68ab26adaad81c0fca8020af (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-22T12:50:18Z (GMT) No. of bitstreams: 2 Dissertação - Ohana Benevides Rodrigues - 2018.pdf: 55370195 bytes, checksum: 561eb17a68ab26adaad81c0fca8020af (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-22T12:50:18Z (GMT). No. of bitstreams: 2 Dissertação - Ohana Benevides Rodrigues - 2018.pdf: 55370195 bytes, checksum: 561eb17a68ab26adaad81c0fca8020af (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-06-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Liquid Argon Time Projection Chambers (LArTPCs) are ideal detectors for precision neutrino physics, but their technology is not completely mastered so far. In order to achieve a complete domain over the technology, the Liquid Argon In A Testbeam (LArIAT) experiment was built. LArIAT consists of a LArTPC placed at a dedicated calibration test beamline at Fermilab. In 2016 LArIAT performed the first pion-argon cross section measurement in history. The Aerogel Cherenkov detectors and the Muon Range Stack (MuRS) detector are auxiliary detectors whose purpose is to separate incoming pions and muons at the Time Projection Chamber (TPC) and separate the through-going muons and pions from the TPC, respectively. These detectors' data were not used on this first analysis due to the lack of people working on their data reconstruction. Therefore, on this work we used as tool the Geant4 Monte Carlo software to characterize the MuRS detector and quantify its purity and efficiency on muon/pion separation. / Câmaras de projeção temporal de argônio líquido (do inglês, LArTPC), são detectores considerados ideais para a física de neutrinos de alta precisão por permitirem a reconstrução de trajetórias de partículas com precisão na ordem de milímetros. Apesar de seu potencial, a tecnologia ainda não é perfeitamente dominada. Com este objetivo em mente, o experimento Liquid Argon In A Testbeam (LArIAT) foi construído. O LArIAT consiste em uma câmara de projeção temporal colocada em uma linha de feixe dedicada. Em 2016, o LArIAT apresentou a primeira medida de seção de choque entre píons e argônio líquido da história. Na ocasião da publicação deste resultado, dois dos detectores acessórios do experimento ainda não tinham seus dados reconstruídos: o detector Cherenkov de aerogel e o Muon Range Stack (MuRS). O último é o foco deste trabalho. O objetivo fundamental do MuRS é permitir a distinção entre píons e múons cujas trajetórias não estão completamente contidas dentro do LArTPC. Para investigar a capacidade do detector de distinguir múons e píons e quantificar sua eficiência e pureza, este trabalho simulou a interação de píons e múons com o detector via Monte Carlo utilizando o Geant4.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-16T13:10:45Z No. of bitstreams: 1 Romulo Augusto Lins de Campos.pdf: 5930168 bytes, checksum: 7553330ff526456aa81970434c7c3475 (MD5) / Made available in DSpace on 2017-02-16T13:10:45Z (GMT). No. of bitstreams: 1 Romulo Augusto Lins de Campos.pdf: 5930168 bytes, checksum: 7553330ff526456aa81970434c7c3475 (MD5) Previous issue date: 2011-03-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Potentiometric sensors are known for their high sensitivity and selectivity. Such sensors have versatility, low cost and robustness, which ensures their applicability in a wide variety of complex chemical samples. The ion-selective electrodes for polymeric membrane are the best known potentiometric sensors, has by these traits. However, modified carbon paste electrodes have gained prominence due a great advance in their studies. This paper proposes an electrode chemically modified carbon paste for potentiometric determination of copper (II), using a new ionophore lariat-crown ether synthesized from precursor aminopropyl triethoxysilane is a silylating agent known in the literature. Studies were conducted to evaluate the influence of pH on the potential, the best buffer as supporting electrolyte, and the experimental design of mixtures to obtain the best composition of EQMPC. The proposed electrode showed a sensitivity of 38.5 mV/decade of activity with a detection limit of 2.04 x 10-5 mol L-1, and as a supporting electrolyte buffer biftalate/HCl pH = 3.0 proving to be promising for application in the determination of Cu(II). The electrode showed no significant analytical sensitivity to Co(II), Ni(II) and Zn(II) being these their main interferences. / Sensores potenciométricos são conhecidos por sua alta sensibilidade e seletividade. Tais sensores apresentam versatilidade, baixo custo e robustez, o que garante a esses sensores aplicabilidade em uma grande variedade de amostras químicas complexas. Os eletrodos íon-seletivos de membrana polimérica são os sensores potenciométricos mais conhecidos, por possuirem essas características. Contudo, os eletrodos modificados de pasta de carbono têm ganhado forte destaque devido ao avanço em seus estudos. Neste trabalho é proposto um eletrodo quimicamente modificado de pasta de carbono para determinação potenciométrica de cobre (II), utilizando um novo ionóforo lariat-éter coroa sintetizado a partir de precursor aminopropil trietoxisilano que é um agente sililante conhecido na literatura. Foram realizados estudos para avaliar a influência do pH sobre o potencial, o melhor tampão como eletrólito de suporte, além do planejamento experimental de misturas para se obter a melhor composição do EQMPC. O eletrodo proposto apresentou uma sensibilidade de 38,5mV/década de atividade com limite de detecção de 2,04 x 10-5 mol L-1, tendo como eletrólito de suporte um solução-tampão biftalato/HCl pH = 3,0 demonstrando ser promissor para aplicação na determinação de íons Cu(II). O eletrodo não apresentou sensibilidade analítica significativa aos íons Co(II), Ni(II) e Zn(II) sendo esses seus principais interferentes.