Thesis (M.S.)--Rochester Institute of Technology, 2006. / Typescript. Includes bibliographical references (leaves 47-48).

Thesis (Ph. D.)--Illinois State University, 1997. / Title from title page screen, viewed June 5, 2006. Dissertation Committee: H. Tak Cheung (chair), Sean Arkins, Herman E. Brockman, Paul A. Garris, Brian J. Wilkinson. Includes bibliographical references (leaves 113-121) and abstract. Also available in print.

Thesis (Ph.D.)--Duquesne University, 2005. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. ) and index.

Foram purificados dois isoinibidores de α-amilase (IAs) do feijão preto, Phaseolus vulgaris, cv. Rico 23, através de extração aquosa, precipitação fracionada com sulfato de amônio, diálise contra água destilada e cromatografias em colunas de troca iônica, interação hidrofóbica e peneira molecular. Foram analisadas as caracteristicas químicas dos inibidores (I-1 e I-2), apresentando: 9,9 e 12,1% de carboidratos, 58 e 51 KDaltons de peso molecular, 4,70 e 4,65 como pontos isoelétricos e 13,4 e 36,7 de hidrofobicidade superficial, respectivamente. Também foram analisados o conteúdo de amino ácidos, os mapas trípticos, bem como as condições de dissociação molecular e mudanças conformacionais por agentes químicos e temperatura, avaliadas em SDS-PAGE, calorimetria diferencial e emissão de fluorescência. A separação das subunidades peptídicas foi realizada por cromatografia em DEAE-celulose-uréia 6M, procedendo-se o seqüenciamento parcial de 3 peptídeos isolados. A confrontação das seqüências obtidas com a seqüência da var. Greensleaves revelou homologias de até 59%. Porém, diferenças marcantes entre o I-1 e o I-2 demonstram que essas proteínas devem ser sintetizadas a partir de DNAs distintos. As proteínas isoladas são comprovadamente isoinibidores pertencentes a uma nova família de inibidores de α-amilase específica dos Phaseolus vulgaris. / Two α-amylase isoinhibitors, I-1 and I-2, were purified from black beans Phaseolus vulgaris, cv. Rico 23. Both are glycoproteins acting on mammalian and insect α-amylases, having similar isoelectric points, 4.70 and 4.65, and aminoacid composition, but showing different molecular weights, 58 and 51 KDaltons, and surface hydrophobicity, 13.4 and 36.7, respectivily. Conformational changes due to dissociating agents and temperature were detected by SDS-PAGE, DSC and by following the fluorescence emission. The peptides dissociated by urea-SDS-β-mercaptoethanol were isolated on a urea-DEAE-celulose column. Three of the isolated peptides were sequenced showing up to 59% homology with the deduced sequence from the inhibitor of the var. Greensleaves. In spite of the similarity, they seem to be sythesizes by different DNAs.

Orientador: Zanoni Dias / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Computação / Made available in DSpace on 2018-08-22T15:29:14Z (GMT). No. of bitstreams: 1 Almeida_AndreAtanasioMaranhao_D.pdf: 2248939 bytes, checksum: b57ed5328b80a2fc7f36d1509558e756 (MD5) Previous issue date: 2013 / Resumo: Alinhamento de seqüências é, reconhecidamente, uma das tarefas de maior importância em bioinformática. Tal importância origina-se no fato de ser uma operação básica utilizada por diversos outros procedimentos na área, como busca em bases de dados, visualização do efeito da evolução em uma família de proteínas, construção de árvores filogenéticas e identificação de motifs preservados. Seqüências podem ser alinhadas aos pares, problema para o qual já se conhece algoritmo exato com complexidade de tempo O(l2), para seqüências de comprimento l. Pode-se também alinhar simultaneamente três ou mais seqüências, o que é chamado de alinhamento múltiplo de seqüências (MSA, do inglês Multiple Sequence Alignment ). Este, que é empregado em tarefas como detecção de padrões para caracterizar famílias protéicas e predição de estruturas secundárias e terciárias de proteínas, é um problema NP - Difícil. Neste trabalho foram desenvolvidos métodos heurísticos para alinhamento múltiplo de seqüências de proteína. Estudaram-se as principais abordagens e métodos existentes e foi realizada uma série de implementações e avaliações. Em um primeiro momento foram construídos 342 alinhadores múltiplos utilizando a abordagem progressiva. Esta, que é uma abordagem largamente utilizada para construção de MSAs, consiste em três etapas. Na primeira delas é computada a matriz de distâncias. Em seguida, uma árvore guia é gerada com base na matriz e, finalmente, o MSA é construído através de alinhamentos de pares, cuja ordem é definida pela árvore. Os alinhadores desenvolvidos combinam diferentes métodos aplicados a cada uma das etapas. Para a computação das matrizes de distâncias foram desenvolvidos dois métodos, que são capazes também de gerar alinhamentos de pares de seqüências. Um deles constrói o alinhamento com base em alinhamentos locais e o outro utiliza uma função logarítmica para a penalização de gaps. Foram utilizados ainda outros métodos disponíveis numa ferramenta chamada PHYLIP. Para a geração das árvores guias, foram utilizados os métodos clássicos UPGMA e Neighbor Joining. Usaram-se implementações disponíveis em uma ferramenta chamada R. Já para a construção do alinhamento múltiplo, foram implementados os métodos seleção por bloco único e seleção do par mais próximo. Estes, que se destinam a seleção xiii do par de alinhamentos a agrupar no ciclo corrente, são comumente utilizados para tal tarefa. Já para o agrupamento de um par de alinhamentos, foram implementados 12 métodos inspirados em métodos comumente utilizados - alinhamento de consensos e alinhamento de perfis. Foram feitas todas as combinações possíveis entre esses métodos, resultando em 342 alinhadores. Eles foram avaliados quanto à qualidade dos alinhamentos que geram e avaliou-se também o desempenho dos métodos, utilizados em cada etapa. Em seguida foram realizadas avaliações no contexto de alinhamento baseado em consistência. Nesta abordagem, considera-se MSA ótimo aquele que estão de acordo com a maioria dos alinhamentos ótimos para os n(n ? 1)/2 alinhamentos de pares contidos no MSA. Alterações foram realizadas em um alinhador múltiplo conhecido, MUMMALS, que usa a abordagem. As modificações foram feitas no método de contagem k-mer, assim como, em outro momento, substituiu-se a parte inicial do algoritmo. Foram alterados os métodos para computação da matriz de distâncias e para geração da árvore guia por outros que foram bem avaliados nos testes realizados para a abordagem progressiva. No total, foram implementadas e avaliadas 89 variações do algoritmo original do MUMMALS e, apesar do MUMMALS já produzir alinhamentos de alta qualidade, melhoras significativas foram alcançadas. O trabalho foi concluído com a implementação e a avaliação de algoritmos iterativos. Estes se caracterizam pela dependência de outros alinhadores para a produção de alinhamentos iniciais. Ao alinhador iterativo cabe a tarefa de refinar tais alinhamentos através de uma série de ciclos até que haja uma estabilização na qualidade dos alinhamentos. Foram implementados e avaliados dois alinhadores iterativos não estocásticos, assim como um algoritmo genético (GA) voltado para a geração de MSAs. Nesse algoritmo genético, implementado na forma de um ambiente parametrizável para execução de algoritmos genéticos para MSA, chamado ALGAe, foram realizadas diversas experiências que progressivamente elevaram a qualidade dos alinhamentos gerados. No ALGAe foram incluídas outras abordagens para construção de alinhamentos múltiplos, tais como baseada em blocos, em consenso e em modelos. A primeira foi aplicada na geração de indivíduos para a população inicial. Foram implementados alinhadores baseados em blocos usando duas abordagens distintas e, para uma delas, foram implementadas cinco variações. A segunda foi aplicada na definição de um operador de cruzamento, que faz uso da ferramenta M-COFFEE para realizar alinhamentos baseados em consenso a partir de indivíduos da população corrente do GA, e a terceira foi utilizada para definir uma função de aptidão, que utiliza a ferramenta PSIPRED para predição das estruturas secundárias das seqüências. O ALGAe permite a realização de uma grande variedade de novas avaliações / Abstract: Sequence alignment is one the most important tasks of bioinformatics. It is a basic operation used for several procedures in that domain, such as sequence database searches, evolution effect visualization in an entire protein family, phylogenetic trees construction and preserved motifs identification. Sequences can be aligned in pairs and generate a pairwise alignment. Three or more sequences can also be simultaneously aligned and generate a multiple sequence alignment (MSA). MSAs could be used for pattern recognition for protein family characterization and secondary and tertiary protein structure prediction. Let l be the sequence length. The pairwise alignment takes time O(l2) to build an exact alignment. However, multiple sequence alignment is a NP-Hard problem. In this work, heuristic methods were developed for multiple protein sequence alignment. The main approaches and methods applied to the problem were studied and a series of aligners developed and evaluated. In a first moment 342 multiple aligners using the progressive approach were built. That is a largely used approach for MSA construction and is composed by three steps. In the first one a distance matrix is computed. Then, a guide tree is built based on the matrix and finally the MSA is constructed through pairwise alignments. The order to the pairwise alignments is defined by the tree. The developed aligners combine distinct methods applied to each of steps. Then, evaluations in the consistency based alignment context were performed. In that approach, a MSA is optimal when agree with the majority along all possible optimal pairwise alignments. MUMMALS is a known consistency based aligner. It was changed in this evaluation. The k-mer counting method was modified in two distinct ways. The k value and the compressed alphabet were ranged. In another evaluation, the k-mer counting method and guide tree construction method were replaced. In the last stage of the work, iterative algorithms were developed and evaluated. Those methods are characterized by other aligner's dependence. The other aligners generate an initial population and the iterative aligner performs a refinement procedure, which iteratively changes the alignments until the alignments quality are stabilized. Several evaluations were performed. However, a genetic algorithm for MSA construction stood out along this stage. In that aligner were added other approaches for multiple sequence alignment construction, such as block based, consensus based and template based. The first one was applied to initial population generation, the second one was used for a crossover operator creation and the third one defined a fitness function / Doutorado / Ciência da Computação / Doutor em Ciência da Computação

Over the past several years, the fields of synthetic biology and machine learning have demonstrated marked advances in the scale of their capabilities and the success of their applications. The work presented in this thesis focuses on the translation of recent advances in machine learning toward new applications in synthetic biology. In particular it is argued that the needs of synthetic biology researchers and practitioners are well met by a class of generative machine learning models, and that the scale of synthetic biology capabilities allows for their successful application across multiple domains of interest. In Chapter 1, a novel algorithm utilizing Markov Random Fields is used to, for the first time, design functional synthetic overlapping pairs of genes with potential applications for improved biological robustness and biosafety. In Chapter 2, motivated by a desire to extend the scope of protein sequence modeling to a greater range and diversity of protein sequences, a variant of a variational autoencoder model is used to project hundreds of millions of protein sequences into a continuous latent space with potentially useful representation features. Finally, in Chapter 3, we move beyond the realm of protein sequences to define a probabilistic species-specific model of regulatory sequences and explore this model’s utility for the challenging task of gene expression prediction for non-model bacterial organisms. Machine learning models presented in this thesis represent novel applications of models traditionally applied to data in the domains of images, text or sound toward addressing challenging problems in biology. Particular attention is devoted to the challenging task of utilizing large amounts of unlabeled data present in metagenomic sequences and the genomes of poorly characterized bacteria in the hope of improving researchers’ abilities to manipulate complex biological phenomena.

Cleavage and polyadenylation are two necessary messenger RNA (mRNA) maturation steps for gene expression. The Cleavage and Polyadenylation Specificity Factor (CPSF) complex, which recognizes the AAUAAA polyadenylation signal and executes the cleavage reaction, is indispensable for these two processes. In this thesis, I describe my study of the regulation and functions of two non-canonical isoforms of the CPSF subunit WDR33. In addition, I provide detailed analyses on our current knowledge of CPSF subunits’ functions and their influences on a diverse collection of biological processes and conditions. In Chapter1, I provide a general introduction to cleavage and polyadenylation, WDR33, innate immune response via molecular pattern recognition, and the cGAS-STING pathway. Chapter 2 presents my original research on non-canonical WDR33 isoforms, termed WDR33v2 (V2) and WDR33v3 (V3). I determined that their mRNAs are produced by alternative polyadenylation. Both V2 and V3 proteins lack multiple WD repeats, but they can interact with and stabilize each other. This is a novel mode of protein-protein interaction, which I termed WD repeat complementation (WDRC). Unexpectedly, I found that even though V2 and V3 are isoforms of a polyadenylation factor, they are not themselves polyadenylation factors. Regulated by the NF-κB pathway, they are interestingly immune factors involved in the cGAS-STING pathway that induces immune responses against cytosolic double-stranded DNA. V2 decreases STING disulfide oligomerization and suppresses STING-mediated interferon β induction, but facilitates STING-mediated autophagy. Binding of V3 to V2 via WDRC prevents V2’s regulation of STING, suggesting that V3 is a V2 inhibitor. My findings thus further our understanding of STING-mediated immune responses. More broadly, these findings also demonstrate that isoforms produced by alternative mRNA processing can be functionally unrelated. In light of the versatility of the WDR33 gene, I performed a literature review in Chapter 3 on both the canonical and non-canonical functions of CPSF. I first summarize the general functions of CPSF subunits. Subsequently, I discuss their involvements in a variety of biological processes and conditions. This discussion reveals that different processes involve different CPSF subunits. Although CPSF is responsible for only two simple biochemical reactions, it has profound influences on cellular homeostasis. Together, my thesis studies reveal new insights into the molecular mechanism of the cGAS-STING pathway, underscore the importance of alternative mRNA processing, and provide the latest analyses of the functional significances of CPSF.

In eukaryotes, sequences that code for the amino acid structure of proteins represent a small fraction of the total sequence space in the genome. These are referred to as coding sequences, whereas the remaining majority of the genome is designated as noncoding. Studies of translation, the process in which a ribosome decodes a coding sequence to synthesize proteins, have primarily focused on coding sequences, mainly due to the belief that translation outside of canonical coding sequences occurs rarely and with little impact on a cell. However, recently developed techniques such as ribosome profiling have revealed pervasive translation in a diverse set of noncoding sequences, including long noncoding RNAs (lncRNAs), introns, and both the 5’ and 3’ UTRs of mRNAs. Although proteins with amino acid sequences derived partially or entirely from noncoding regions may be functional, they will often be nonfunctional or toxic to the cell and therefore need to be removed. Translation outside of canonical coding regions may further expose the noncoding genome to selective pressure at the protein level, leading to the generation of novel functional proteins over evolutionary timescales. Despite the potentially significant impact of these processes on the cell, the cellular mechanisms that function to detect and triage translation in diverse noncoding regions, as well as how peptides that escape triage may evolve into novel functional proteins, remain poorly understood.This thesis will describe novel findings that offer new insight into the process of noncoding translation mitigation revealed by a combination of high-throughput systems-based approaches and validated by biochemical and genetic approaches. Chapter 1 will discuss general concepts in the translation of noncoding sequences and the relevant cellular systems and impacts on human health. Chapter 2 will discuss the results of a high-throughput reporter assay investigating translation in thousands of noncoding sequences from diverse sources. The results discussed in this chapter revealed two factors involved in the mitigation of proteins derived from noncoding sequences: C-terminal hydrophobicity and proteasomal degradation. Chapter 3 will build on Chapter 2 and discuss the results of a genome-wide CRISPR/Cas9 knockout screen that identified the BAG6/TRC35/RNF126 membrane protein chaperone complex as a key cellular pathway in the detection and degradation of proteins with translated noncoding sequences. Having identified the BAG6 complex as targeting a specific reporter of translation of the 3’ UTR in the AMD1 gene, a series of knockout cell lines validated these results and demonstrated the participation of two additional genes, SGTA and UBL4A. Through coimmunoprecipitation western blots and rescue assays with flow cytometry as a readout, we confirmed physical interaction between BAG6 and the 3’ UTR of AMD1, and a similar experiment confirmed interaction between BAG6 and a readthrough mutant of the SMAD4 tumor suppressor gene. Finally, by combining our high-throughput reporter library with our BAG6 knockout cell line, we demonstrated that BAG6 targets hydrophobic C-terminal tails in many noncoding sequences of diverse origin. Finally, Chapter 4 will discuss the evolutionary perspective of noncoding translation through analyses of the sequence content of human and mouse genomes. The findings of this chapter demonstrate a significant trend for increased uracil content in noncoding regions of the genome, which frequently results in the translation of hydrophobic amino acids. We also find that many functional translated noncoding peptides localize to membranes, providing a theoretical link between the shuttling of translated noncoding sequences to a protein complex involved in membrane protein quality control and the emergence of newly evolving proteins from the noncoding genome.

No / The minerals involved in the formation of metazoan skeletons principally comprise glassy silica, calcium phosphate or carbonate. Because of their ancient heritage, glass sponges (Hexactinellida) may shed light on fundamental questions such as molecular evolution, the unique chemistry and formation of the first skeletal silica-based structures, and the origin of multicellular animals. We have studied anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi; Porifera, Class Hexactinellida), which are remarkable for their size, durability, flexibility and optical properties. Using slow-alkali etching of biosilica, we isolated the organic fraction, which was revealed to be dominated by a hydroxylated fibrillar collagen that contains an unusual [Gly-3Hyp-4Hyp] motif. We speculate that this motif is predisposed for silica precipitation, and provides a novel template for biosilicification in nature.

Yes / In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a 'catalytic' effect upon the channel gating kinetics. / Deutsche Forschungsgemeinschaft, the Wellcome Trust (083547/ Z/07/Z and 092970/Z/10/Z) and the British Heart Foundation (PG/09/016/ 26992).