<p dir="ltr">The process of ubiquitination is an important regulatory process that helps modulate eukaryotic signaling inside the cell. This process is described as the formation of an isopeptide linkage between the C-terminal residue of ubiquitin to the lysine residue of a target protein, a process catalyzed by the E1, E2, and E3 enzyme cascade. As mentioned, this process has only been observed in eukaryotes, although recently, prokaryotic pathogens have been shown to possess enzymes that affect the ubiquitination pathway in their hosts. While some of these enzymes have well known mechanisms, there are still many that are unknown and are novel when compared to other well documented enzymes. The studies conducted in this Thesis involve biochemical, structural, and bioinformatic studies involving both prokaryotic and eukaryotic enzymes as an approach to develop methodologies that help study processes involving ubiquitin. Among the insights provided in this document, the importance of a unique insertion found in the <i>Legionella </i>effector MavC, that is important for substrate recognition. The use of disulfide bridge formation to obtain Ub-DUB complexes through the design of a simple cysteine mutation on Ub. Finally, utilizing AlphaFold, in combination with other bioinformatic processes, to help study large, protein family data sets, like the USP family DUBs that is studied in this Thesis. Altogether, these findings show that through a combination of multiple techniques, we can have a more streamlined way of studying these new, exciting protein pathways.</p>

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

A obesidade à um tema recorrente na literatura cientÃfica da atualidade. Isso se deve ao aumento exponencial de sua prevalÃncia em todas as camadas da sociedade. A popularidade deste tema fez tambÃm com que assuntos associados a ele emergissem e ganhassem maior notabilidade em publicaÃÃes da Ãrea da saÃde. O uso da saliva como mÃtodo diagnÃstico avanÃou consideravelmente nos Ãltimos anos. DesequilÃbrios na quantidade e na qualidade da saliva podem tanto gerar afecÃÃes bucais quanto ser indicativo de alguma alteraÃÃo sistÃmica importante. Este trabalho objetivou estudar o perfil de proteÃnas salivar e saliva total humana em pacientes com sobrepeso e obesidade. A amostra foi constituÃda por sessenta pacientes com obesidade e sobrepeso (grupo experimental) e sessenta pacientes com peso adequado (grupo controle), tendo sido avaliado o fluxo salivar, o diÃrio de dieta e o perfil proteico. Saliva total nÃo estimulada foi coletada e armazenada a â 80ÂC. Posteriormente foi adicionado o inibidor enzimÃtico e as amostras foram centrifugadas a 15.000 rpm por 15 minutos a 4ÂC, sendo o sobrenadante separado para realizaÃÃo da dosagem de proteÃnas. A concentraÃÃo de proteÃnas totais salivares foi determinada pelo mÃtodo do Ãcido BicinconÃnico, usando uma curva de albumina sÃrica bovina (BSA). Ao analisar o fluxo salivar nÃo estimulado foi possÃvel observar que o grupo de estudo apresentou mÃdia menor que o grupo controle, sendo essa diferenÃa estatisticamente significante (p=0,006). O grupo controle apresentou uma mÃdia de concentraÃÃo total de proteÃnas maior que o grupo experimental, sendo essa diferenÃa estatisticamente significante (p=0,002). Os resultados deste estudo sugerem haver padrÃes diferenciados na composiÃÃo salivar entre os grupos avaliados.

<p>    </p> <p>DNA-encoded chemical libraries (DELs) are collections of DNA-linked small molecules, where each synthetic small molecule is covalently attached to a unique DNA barcode that encodes its identity. This technology harnesses the power of organic chemistry and genetics, which extends the application of molecular evolution and natural selection to the discovery of specific small molecules binders to protein targets of interest. Rather than discretely screening individual molecules, up to billions of DNA-encoded small molecules can be assessed collectively by a selection assay in a single tube. As a result, the high sensitivity, low cost, and unprecedented level of molecular complexity of DELs allow rapid generation of novel bioactive compounds. While powerful, this approach has its own limitations, including limited target scope and selection strategies. Currently, DEL targets have been largely limited to biochemically purified proteins and used in affinity-based selections assays. In the first area of this work, we address both these limitations by capitalizing on the power of affinity labeling. This allows DELs to be applied to protein targets within and on living cells and expands the power of DNA-encoding to the identification of small molecules with specific biological functions beyond binding. </p> <p>In the second area, we harnessed affinity labeling and DNA sequence analysis to develop multiplexed small molecule ligand binding assays. This method is the initial demonstration of split-and-pool ligand binding assays using DNA-linked small molecule probes. We used this approach in a high-throughput screening campaign to identify selective inhibitors by screening 1000 compounds against 5 bromodomain proteins concurrently. In addition, this approach was utilized to rank order the affinity of a 96-member library of DNA-linked ligands to a protein simultaneously, which significantly increases the throughput of ligand binding assays while keeps the cost low. </p> <p>Lastly, we developed proximity-induced selection assays to enrich ligands from DELs. This approach involves uncaging or installation of a biotin purification tag on the DNA construct either through photo-deprotection of a protected biotin group using a light emitting protein tag or by amine acylation using an engineered biotin ligase. Compared to affinity labeling-based selection approaches, this approach results in improved recovery of ligands and, at the same time, removes the onerous requirement of protein purification. The enzyme-mediated proximity labeling approach should serve as a convenient tool for molecular discovery with DELs. </p>

<p>Basic leucine zipper (bZIP) transcription factors can have an oncogenic role in cancer development. Nuclear factor erythroid related 2-factor 2 (Nrf2) is a bZIP transcription factor that traditionally is thought of as a cellular protector. In normal cells, Nrf2 is only activated after exposure to reactive oxygen species or electrophiles and induces expression of antioxidant and detoxification genes. However, in many cancers (<i>e.g.,</i> lung, pancreatic, and breast) Nrf2 is constitutively activated and is associated with poor overall survival and intrinsic resistance to anticancer therapies. Nrf2 heterodimerizes with transcription factors small musculoaponeurotic fibrosarcoma Maf (sMAF) proteins (e.g., MafG) in the nucleus and binds DNA, inducing transcription of Nrf2 target genes, conferring chemotherapeutic resistance to cancer cells. c-Myc another bZIP transcription factor is often overexpressed in a variety of cancers and acts like a protooncogenic transcriptional regulator. Mutations that drive abnormal MYC expression are the most common cariogenic event in tumor progression. c-Myc heterodimerizes with Max, its obligate bHLH-LZ heterodimerization partner, to form an active transcriptional state and induces DNA transcription. Hence inhibiting the interaction between c-Myc-Max and Nrf2-MafG will not only prevent bZIP heterodimerization but also DNA binding and downstream functions of c-Myc and Nrf2 that promote carcinogenesis. Stapled peptides, with their ability to target large surface area interactions, have shown promise for specifically inhibiting protein-protein interactions. Stapled peptides have improved cell permeability and oral bioavailability when compared to biologics. We have designed and synthesized stapled peptide for Nrf2/MafG interaction inhibition and stapled peptides for c-Myc/Max heterodimerization inhibition. Nrf2/MafG inhibition using the synthesized stapled peptide N1S, was demonstrated by luciferase and fluorescence polarization assays. Overall, we hypothesize that stapled peptides will be an effective therapeutic strategy resulting in decreased chemotherapeutic resistance and cancer cell proliferation.</p>

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

<p>Peptides are a fundamental building-block of living systems and play crucial roles at both functional and structural level. Therefore, they have attracted increased attention as a platform to design and engineer new self-assembled systems that span the nano-to-meso scales. The rules of peptide design and folding enable the construction of suitable building-blocks to develop soft materials for biomaterial applications. Herein we present the use of the alpha-helical secondary structure to create two distinct structural motifs, namely coiled-coils and helical bundles. These peptide components can differ in size and incorporate a host of different functional moieties, the effects of which are described through their hierarchical assembly. </p> <p>First, we describe the self-assembly of coiled coil oligomers (trimer and tetramer) of the GCN4 leucine zipper peptide. The trimeric coiled coil was modified with varying number of aromatic groups (one to three) along each helical backbone, to facilitate higher order assemblies into banded nano- to micron-sized structures, the formation of which could be controlled reversibly as a function of pH. In addition, the electrostatic and aromatic interactions of the peptide material were harnessed for non-covalent binding of small drug molecules, followed by their subsequent pH-triggered release. Furthermore, these nanostructures are compatible with MCF-7 breast cancer cells, making them suitable drug-delivery agents for chemotherapeutics. In the absence of aromatic modifications, the coiled-coil trimer assembles into higher-order nanotubes that can be harnessed for selective encapsulation of high molecular weight biomolecules. With an increase in oligomerization from three to four, along with a single aromatic group modification on each helix, the tetrameric coiled-coil mutant successfully demonstrates a metal-assisted two-tier structural assembly into microbarrels and spheres.</p> <p>Second, we present the higher-order assembly of short tetrameric and pentameric helical bundle proteins, covalently stabilized by a belt of disulfide bridges, with metal-binding ligands at each helix termini. The addition of metals like Zn(II) and Cu(II) promote the assembly of the bundles into a 3D globular matrix, which upon thermal annealing transforms into microspheres. Additionally, these microspheres also demonstrate the metal-assisted inclusion of His-tagged fluorophores. Thus, peptide-based materials can be constructed by self-assembly of alpha-helical building blocks into systems with sophisticated, diverse morphologies and dynamic chemical properties, that can be further modulated to enhance performance for medical applications. </p>

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 140-150).

Orientadores: Maria Helena Andrade Santana, Ricardo de Lima Zollner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-27T03:55:49Z (GMT). No. of bitstreams: 1 Victorino_IgorRicardodeSouza_M.pdf: 6760377 bytes, checksum: 05fc12e1f97a8b092dbd91b47b06441d (MD5) Previous issue date: 2000 / Resumo: Os lipossomas têm sido utilizados como imunoadjuvantes antigênicos, em estudos que enfatizam a sua atuação no sistema imunológico. Extratos peptídicos ou protéicos de várias fontes foram encapsulados em lipossomas ou associados à sua superficie, e aplicados em imunoterapias e vacinas. Apesar disso, poucos são os estudos voltados para a performance do processo de preparação desses lipossomas, eficiência da associação proteína/lipídio e estabilidade das vesículas. Este trabalho trata da avaliação do encapsulamento das proteínas: Albumina de Soro Bovino (BSA), Mioglobina (Mio) e Cito cromo C (Cit C), simulando extratos alergênicos provenientes de fungos e ácaros, no que se refere à faixa de peso molecular das suas proteínas. Os lipossomas foram preparados com os lipídios L-a-distearoilfosfatidi1colina (DSPC) e Colesterol (Col) na razão molar 70:30 respectivamente. As proteínas foram encapsuladas individualmente e em forma de misturas de composições molares BSA:Mio:Cit C de 20:40:40, 40:20:40 e 40:40:20, respectivamente. Além disso, a mistura de proteínas de composição 40:20:40 (BSA:Mio:Cit C) foi também associada covalentemente à superficie das vesículas. Os lipossomas foram caracterizados pelo teor de fósforo e diâmetro médio. O desempenho do encapsulamento foi analisado através dos perfis e eficiências de encapsulamento das proteínas individuais e em misturas, além da estabilidade das vesículas em tensoativo penta etileno glicol mono-n-dodecil éter (C12Es), e de plasma humano. Para as misturas, analisou-se a exclusão de proteínas durante o encapsulamento e a associação à superficie dos lipossomas. Os resultados indicam que a eficiência de encapsulamento depende mais das interações proteína:lipídio que do tamanho das proteínas. As eficiências de encapsulamento variaram entre 0,03% e 3,55% para as proteínas individuais e entre 1,04% e 3,94% para as misturas. Não houve alteração na estabilidade dos lipossomas em C12Es com a presença das proteínas. Em plasma, essas vesículas permaneceram estáveis por aproximadamente 20 horas. Na associação à superficie dos lipossomas, predominou a presença de BSA em relação às outras proteínas / Abstract: Liposomes has been studied as antigenic immunoadjuvants with emphasis on their performance in the imunological system. Peptides or proteins of various sources were encapsulated in liposomes or associated to their surface, and applied in immunotherapy and vaccines. In spite of this, there are few studies about the performance of preparation process ofthese liposomes, efficacy ofthe association proteinllipid and the stability ofvesic1es. This work concems with the evaluation of the encapsulation of the proteins Bovine Serum Albumin (BSA), Myoglobin (Myo) and Cythochrome C (Cyt C), simulating allergen extracts from molds and mites, related to the range of molecular weight of their proteins. Liposomes were prepared with lipid L-a-disteraoylphosphatidylcholine (DSPC) and Cholesterol (Chol) at molar ratio 70:30 respectively. The proteins were encapsulated individualyand in mixtures with molar composition BSA:Myo:Cyt C of20:40:40, 40:20:40 and 40:40:20 respectively. Furthermore, the mixture 40:20:40 (BSA:Myo:Cyt C) was also covalent1y associated to the surface of vesic1es. Liposomes were characterized by their phosphate contents and mean diameter. The performance of the protein encapsulation was evaluated through the profiles and efficiency of the encapsulation and throught the stability of vesic1es in the presence of the surfactant penta ethylene glycol mono-n-dodecyl ether (C12ES) and human plasma. For the mixtures, the exclusion of proteins duringentrapment or association to the liposome surface was also evaluated. The experimental results indicate that the efficacy of encapsulation is more dependent of the interactions proteinllipid than the size of the proteins. The efficiencies of encapsulation changed between 0.03% and 3,55% for individual proteins and were between 1,04% and 3,94% for the mixtures. The presence ofproteins does not altered the stability of liposomes in C12ES . In human plasma the vesic1es remained with stability about 20 hours. For the mixtures, the presence ofthe BSA protein predominated in the vesicles / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Orientador: Fernanda Klein Marcondes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T15:57:25Z (GMT). No. of bitstreams: 1 Lima_PatriciaOliveirade_D.pdf: 849171 bytes, checksum: 8d1338b60a2ebc4b9e9be0660be72427 (MD5) Previous issue date: 2014 / Resumo: Além de doenças orais, o estresse e o ciclo menstrual também têm sido relacionados à produção de compostos sulfurados voláteis (CSV), principais gases responsáveis pela halitose. O objetivo deste trabalho foi investigar a relação entre alterações emocionais, associadas a atividades acadêmicas, e produção de CSV, por meio da determinação do nível de estresse, fluxo salivar, concentrações salivares de cortisol, IgA secretória (IgAs), proteínas totais, beta-defensina ¿ 2 (?-defensin-2), atividade de alfa-amilase e expressão das proteínas mucinas 5B, 7 e lactoferrina, na cavidade oral, em mulheres (na fase menstrual do ciclo reprodutivo) e homens dos 4 anos do curso de Graduação em Odontologia da Faculdade de Odontologia de Piracicaba (UNICAMP). Os dados da análise psicológica mostraram que o estresse associado às atividades acadêmicas varia entre os anos do curso. Mulheres apresentaram maiores concentrações bucais de CSV e metil mercaptana (CH3SH), menor fluxo salivar, maiores concentrações salivares de proteínas totais e IgAs e menor expressão salivar de mucinas 5B e 7 em relação aos homens. Em relação ao ano do curso, homens e mulheres cursando o terceiro ano do curso apresentaram maiores valores de CSV, sulfeto de hidrogênio (H2S), alfa-amilase e mucina 5B, em relação a alunos cursando o primeiro ano. Não houve diferença entre os quatro anos da graduação nas concentrações bucais de CH3SH e dimetil sulfeto, concentrações salivares de proteínas totais, IgAs e cortisol e valores de fluxo salivar. Alunos do terceiro ano apresentaram menores concentrações salivares de ?-defensina -2, em relação a alunos do primeiro ano. Alunas cursando o terceiro e quarto anos do curso de graduação apresentaram maior expressão salivar de lactoferrina, em relação às alunas do primeiro e segundo anos. Houve correlação significativa entre os valores de CSV e H2S, em ambos os gêneros. Nas mulheres, observou-se correlação direta entre estresse e CSV e estresse e alfa-amilase e, nos homens, entre estresse e MUC5B. Os resultados confirmam dados anteriores, reforçando a influência do estresse sobre a produção de CSV e sugerem que a ?-defensina -2, mucina 5B e lactoferrina podem estar envolvidas na associação entre estresse e produção de CSV / Abstract: Oral diseases, stress and menstrual cycle have been related to the production of volatile sulfur compounds (VSC), main gases responsible for halitosis. The aim of this study was to evaluate the relationship between emotional alterations associated with academic activities and production of VSC. The following parameters were determined: stress levels, salivary flow, salivary concentrations of cortisol, secretory IgA (SIgA), total protein and beta-defensin-2 (?-defensin -2), alpha-amylase activity, and the expression of the proteins mucin 5B, 7 and lactoferrin, in the oral cavity. Women, during menstrual phase of the reproductive cycle, and men, enrolled at Piracicaba Dental School, University of Campinas, participated in the study. The data of psychological analysis showed that the stress associated with academic activities varies between years of the course. Women showed higher oral concentrations of VSC and methyl mercaptan (CH3SH), lower salivary flow, higher concentrations of total protein and SIgA and lower salivary expression of mucins 5B and 7 compared to men. Men and women in the third year of undergraduate course presented higher values of VSC, hydrogen sulfide (H2S), alpha-amylase and mucin 5B compared to students in the first year. There was no difference on the oral concentrations of CH3SH and dimethyl sulphide; salivary proteins, SIgA and cortisol concentrations and salivary flow values between the four years of the undergraduate course. Men scholars in the third year of the undergraduate course showed lower salivary concentrations of ?-defensin-2 compared to the first year. Women scholars in the third and fourth years of the undergraduate course presented higher lactoferrin expression compared to students in the first and second years. There was significant correlation between VSC and H2S in both gender. In women, it was observed correlation between stress and VSC and between stress and alpha-amylase. In men, stress and MUC5B presented positive correlation. The results confirm previous data strengthening the stress influence on the VSC production. Moreover, suggest that the proteins beta-defensin-2, mucin 5B and lactoferrin may be involved in the association between stress and VSC production / Doutorado / Fisiologia Oral / Doutora em Odontologia