Dissertations / Theses on the topic 'Promoter Recognition'

This thesis presents an innovative human promoter recognition model HPR-PCA. Principal component analysis (PCA) is applied on context feature selection DNA sequences and the prediction network is built with the artificial neural network (ANN). A thorough literature review of all the relevant topics in the promoter prediction field is also provided. As the main technique of HPR-PCA, the application of PCA on feature selection is firstly developed. In order to find informative and discriminative features for effective classification, PCA is applied on the different n-mer promoter and exon combined frequency matrices, and principal components (PCs) of each matrix are generated to construct the new feature space. ANN built classifiers are used to test the discriminability of each feature space. Finally, the 3 and 5-mer feature matrix is selected as the context feature in this model. Two proposed schemes of HPR-PCA model are discussed and the implementations of sub-modules in each scheme are introduced. The context features selected by PCA are III used to build three promoter and non-promoter classifiers. CpG-island modules are embedded into models in different ways. In the comparison, Scheme I obtains better prediction results on two test sets so it is adopted as the model for HPR-PCA for further evaluation. Three existing promoter prediction systems are used to compare to HPR-PCA on three test sets including the chromosome 22 sequence. The performance of HPR-PCA is outstanding compared to the other four systems.

Master of Engineering
This thesis presents an innovative human promoter recognition model HPR-PCA. Principal component analysis (PCA) is applied on context feature selection DNA sequences and the prediction network is built with the artificial neural network (ANN). A thorough literature review of all the relevant topics in the promoter prediction field is also provided. As the main technique of HPR-PCA, the application of PCA on feature selection is firstly developed. In order to find informative and discriminative features for effective classification, PCA is applied on the different n-mer promoter and exon combined frequency matrices, and principal components (PCs) of each matrix are generated to construct the new feature space. ANN built classifiers are used to test the discriminability of each feature space. Finally, the 3 and 5-mer feature matrix is selected as the context feature in this model. Two proposed schemes of HPR-PCA model are discussed and the implementations of sub-modules in each scheme are introduced. The context features selected by PCA are III used to build three promoter and non-promoter classifiers. CpG-island modules are embedded into models in different ways. In the comparison, Scheme I obtains better prediction results on two test sets so it is adopted as the model for HPR-PCA for further evaluation. Three existing promoter prediction systems are used to compare to HPR-PCA on three test sets including the chromosome 22 sequence. The performance of HPR-PCA is outstanding compared to the other four systems.

Cholera is an acute diarrheal infection caused by the bacterium Vibrio cholerae. An estimated 2.9 million of cases and about 100000 cholera deaths occur annually all over the world. Upon ingestion of the V. cholerae, the bacterium travels to small intestine where it colonizes and produces the cholera toxin. Cholera toxin raises intracellular cyclic AMP and leads to chloride secretion and the subsequent secretory diarrhoea. Cholera toxin production is regulated through the master virulence regulator ToxT. Trascriptional activation of toxT is activated by membrane-localized ToxR, in association with another membrane protein named TcpP. Both ToxR and TcpP work as a two-component regulatory system merged in single proteins: they receive an external signal through its periplasmic C-terminal domain and bind to the toxT promoter by their cytoplasmic N-terminal domains. This project thesis aims at characterizing part of the system by studying ToxR-DNA complexes, since two ToxR molecules are supposed to bind the promoter to recruit TcpP and hence the RNA polymerase for transcription activation. Using X-ray crystallography, we have solved the structure of three complexes of the ToxR DNA binding domain with 20-bp, 40-bp and 25-bp oligonucleotides at 2.0 A, 2.6 A and 3.2 A resolution, respectively. According to the three structures, ToxR is able to bind to an extensive region of the toxT promoter that goes from the position -97 to the position -45. Considering an integrated model of the three structures, there are four ToxR molecules binding the toxT promoter: two molecules bind the DNA in tandem, one molecule binds the ToxR degenerate box and the last one is binding what is supposed to be the TcpP binding site. The structure determination of the three complexes is important to define with more accuracy the ToxR binding site in the toxT promoter. This site is characterized by eleven bases with a high A-T rich region sequence followed by a CATA/CATG/TGTA box, where the last two bases perform direct and specific contacts with the protein. In the three structures, ToxR shows a winged helix-turn-helix (w-HTH) fold. The wing interacts with the minor groove in an A-T rich region sequence while the recognition helix enters in the major groove at the region with a sequence corresponding to a CATA box. We have compared ToxR with the other w-HTH family proteins and we have found a new structural element, the secondary wing, which displays interactions with the DNA. We have analyzed the protein-DNA contacts in the three structures, and also the protein-protein interactions in the ToxR-DNA40 structure, thus validating the data published on ToxR defective mutations. Finally, we put forward a model of toxT promoter activation at molecular level, based on our crystal structures and on what is known in literature and from our collaborators. We propose that ToxR acts as co-activator in the first steps of toxT transcription activation at different levels. First, it would capture the DNA and hold it close to the cytoplasmic membrane, since both ToxR and TcpP are membrane proteins. Second, it would play a key-role in relieving H-NS from the toxT promoter: H-NS binds the DNA and transcription is repressed, but ToxR is able to replace it in a region that goes from the position -97 to the position -45. Third, ToxR would not be recruiting the RNA polymerase directly, but creating the suitable conditions for the action of TcpP. ToxR recruits TcpP probably through a hand-holding mechanism since one of the ToxR binding site is very close to the TcpP's binding site.
El cólera es una infección diarreica aguda causada por la bacteria Vibrio cholerae. La producción de la toxina colérica se controla a través del regulador maestro de virulencia ToxT, cuya activación se lleva a cabo por las proteínas de membrana ToxR y TcpP. Este proyecto de tesis tiene como objetivo el estudio de los complejos formados por ToxR junto con el ADN, dado que se conoce que ToxR se une al promotor toxT para reclutar TcpP y consecuentemente la ARN polimerasa, produciendo la activación de la transcripción. Mediante cristalografía de rayos X hemos resuelto la estructura de tres complejos del dominio de unión a ADN de ToxR con oligonucleótidos de 20, 40 y 25 pares de bases a resoluciones de 2.0 A, 2.6 A y 3.2 A, respectivamente. De acuerdo con las tres estructuras, ToxR es capaz de unirse a una amplia región del promotor toxT que se expande desde la posición -97 hasta la posición -45. Teniendo en cuenta el modelo integrado de las tres estructuras, cuatro moléculas de ToxR se unen al promotor toxT en tándem e invertidas. En las tres estructuras, ToxR muestra un tipo de plegamiento winged helix-turn-helix (w-HTH). El ala (wing) interactúa con el surco menor del ADN, mientras que la hélice de reconocimiento penetra en el surco mayor. Comparando ToxR con el resto de proteínas de la familia w-HTH, hemos encontrado un nuevo elemento estructural, el ala secundaria (secondary wing), que interacciona con el ADN. La determinación de la estructura de los tres complejos es importante para definir con mayor precisión el sitio de unión de ToxR en el promotor toxT. Este sitio se caracteriza por once bases con una secuencia rica en A-T seguida de una caja CATA/CATG/TGTA, donde las dos últimas bases contactan directamente y específicamente con la proteína. Proponemos que ToxR actuaría como co-activador de la transcripción de toxT a diferentes niveles: (i) podría ser responsable de capturar el ADN y mantenerlo cerca de la membrana citoplasmática, (ii) podría jugar un papel clave en el desplazamiento de H-NS, (iii) podría reclutar TcpP y estabilizar su interacción con el promotor.

Hepatitis delta virus (HDV) is the smallest known mammalian RNA virus, containing a genome of ~ 1700 nt. Replication of HDV is extremely dependent on the host transcription machinery. Previous studies indicated that RNA polymerase II (RNAPII) directly binds to and forms an active preinitiation complex on the right terminal stem-loop fragment (R199G) of HDV genomic RNA, and that the polypyrimidine tract-binding protein-associated splicing factor (PSF) directly binds to the same region. Further studies demonstrated that PSF also binds to the carboxyl-terminal domain (CTD) of RNAP II. In my thesis, co-immunoprecipitation assays were performed to show that PSF stimulates the interaction of RNAPII with R199G. Results of co-immunoprecipitation experiments also suggest that both the RNA recognition motif 2 (RRM2) and N-terminal proline-rich region (PRR) of PSF are required for the interaction between PSF and RNAPII, while the two RNA recognition motifs (RRM1 and RRM2) might be required for the interaction of PSF with R199G. Furthermore, in vitro run-off transcription assays suggest that PSF facilitates the HDV RNA transcription from the R199G template. Together, the above experiments suggest that PSF might act as a transcription factor for the RNAPII transcription of HDV RNA by linking the CTD of RNAPII and the HDV RNA promoter. My experiments provide a better understanding of the mechanism of HDV RNA-dependent transcription by RNAP II.

Die Detektion sogenannter Vitalitäts-assoziierte molekularer Muster (vita-PAMPs), signalisiert dem Immunsystem die Präsenz lebender Mikroorganismen und ruft verstärkte Immunantworten hervor. Die vorliegende Studie untersuchte die Erkennung bakterieller Vitalität durch humane Antigen-präsentierenden Zellen (APC), sowie die Effekte auf die adaptive Immunität. Transkriptomanalysen von humanen Monozyten zeigten eine selektive transkriptionelle Antwort auf lebendige im Vergleich zu hitzegetöteten Bakterien. Die inflammatorischen Zytokine IL-12 und TNF wurden nahezu ausschließlich in Reaktion auf lebende Bakterien exprimiert. Die Detektion bakterieller Vitalität ist in Menschen, Schweinen, Mäusen und Fischen konserviert. In Mäusen wurde bakterielle RNA als bakterielles vita-PAMP identifiziert. In menschlichen Monozyten führte die Supplementierung toter Bakterien mit bakterieller RNA oder mit synthetischen Liganden des Toll-like-Rezeptors (TLR) 7 und TLR8, jedoch nicht mit anderen TLR-Liganden, zur Rekonstitution der TNF- und IL-12-Antwort. Umgekehrt hemmte silencing von TLR8- vita-PAMP-induzierte Zytokinproduktion. T-follikuläre Helferzellen (TFH) spielen eine zentrale Rolle in der Initiierung humoraler Immunantworten. Hier wurde die Erkennung lebender Bakterien, bakterieller RNA oder synthetischer TLR8-Liganden durch APC als Stimulus für robuste TFH-Zelldifferenzierung identifiziert. Die TFH-Differenzierung ist abhängig von der Erkennung bakterieller RNA über TLR8 und der Produktion von IL-12. Immunisierung von Schweinen mit einem bakteriellen Lebendimpfstoff löste deutlich robustere TFH-Zell- und Antikörperreaktionen als eine Immunisierung mit der hitzegetöteten Version des gleichen Impfstoffs aus. Zusammenfassend haben wir TLR8 als den ersten bekannten vita-PAMP-Rezeptor identifiziert und seine zentrale Rolle für TLR8-vermittelte “Vitalitätserkennung” für die Induktion von TFH-Zellen und Impfreaktionen demonstriert.
The detection of the so-called vitality-associated molecular patterns (vita-PAMPs) signals to the immune system the presence of living microorganisms and triggers an increased immune response. The present study investigated the detection of bacterial vitality by human antigen-presenting cells (APC), as well as the effects of this recognition events on adaptive immunity. Transcriptome analysis of human monocytes showed a selective transcriptional response to live versus heat-killed bacteria. Among others, the inflammatory cytokines IL-12 and TNF were expressed almost exclusively in response to live bacteria. Moreover, the detection of bacterial vitality is conserved in humans, pigs, mice and fish. In mice, bacterial RNA was identified as a bacterial vita-PAMP. In human monocytes, supplementation of dead bacteria with bacterial RNA or with synthetic ligands of toll-like receptor (TLR) 7 and TLR8, but not with other TLR ligands, resulted in the reconstitution of the TNF and IL-12 responses. Conversely, silencing of TLR8 inhibited vita-PAMP-dependent cytokine production. T-follicular helper cells (TFH) play a central role in the initiation of humoral immune responses. Here, the recognition of living bacteria, bacterial RNA or synthetic TLR8 ligands by APCs was identified as a stimulus for robust TFH cell differentiation. TFH differentiation is dependent on the recognition of bacterial RNA via TLR8 and the production of IL-12. Immunization of pigs with a live bacterial vaccine elicited significantly more robust TFH cell and antibody responses than immunization with the heat-killed version of the same vaccine. In summary, we identified TLR8 as the first known vita-PAMP receptor in human and demonstrated its pivotal role in TLR8-mediated “viability recognition” for the induction of TFH cells and vaccine reactions.

It is now widely accepted that human behaviour accounts for a large portion of total global emissions, and thus influences climate change to a large extent. Changing human behaviour when it comes to mode of transportation is one component which could make a difference in the long term. In order to achieve behavioural change, we investigate the use of a persuasive multiplayer game. Transportation mode recognition is used within the game to provide bonuses and penalties to users based on their daily choices regarding transportation. To easily identify modes of transportation, an approach to transport recognition based on accelerometer and gyroscope data is analysed and extended. Preliminary results from the machine learning tests show that the classification true-positive rate for recognizing 10 different classes can reach up to 95% when using a history set (66% without). Preliminary results from testers of the game indicate that using games may be successful in causing positive change in user behaviour.

Del av Erasmus Mundus PERCCOM. Redovisning skedde på anordnad summer school av partner-universitet där hela konsortiet närvarade.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2004.
David Collard, Committee Member ; Robert Guldberg, Committee Member ; Cheng Zhu, Committee Member ; Elliot Chaikoff, Committee Member ; Harish Radhakrishna, Committee Member ; Andres J. Garcia, Committee Chair. Includes bibliographical references.

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Universidade do Vale do Rio dos Sinos
A expressão dos genes em procariotos é desencadeada quando a enzima RNApolimerase interage com uma região adjacente ao gene, chamada de promotor, onde se encontram os principais elementos regulatórios do processo de transcrição. Apesar do crescente avanço das técnicas experimentais em biologia molecular, caracterizar e identificar um número significante de promotores, presentes em um dado genoma, continua sendo uma tarefa demorada e cara. Abordagens in silico são bastante utilizadas para reconhecer essas regiões em procariotos. Entretanto, além do alto número de falsos positivos obtidos, elas enfrentam a inexistência de um número adequado de promotores conhecidos para identificar padrões conservados entre as espécies. Logo, um método criterioso e confiável para predizêlos em qualquer organismo procariótico ainda é um desafio. Esta dissertação propõe um protocolo de uso de hidden Markov models (HMMs) que emprega Estimação de Limiar de Decisão (ELD) e Análise de Discriminação (AD) neste problema. Quatro espécie
Gene expression on prokaryotes initiates when the RNA-polymerase enzyme interacts with DNA regions called promoters. In these regions are located the main regulatory elements of the transcription process. Despite the improvement of in vitro techniques for molecular biology analysis, characterizing and identifying a great number of promoters on a genome is a complex task. In silico approaches are usually employed to recognize theses regions on prokaryotes. Nevertheless, the main drawback is the absence of a large set of promoters to identify conserved patterns among the species. Hence, a in silico method to predict them on any species is a challenge. This work proposes a protocol to use hidden Markov models (HMMs) methodology with Decision Threshold Estimation and Discrimination Analysis on this problem. Four prokaryotic species are investigated (Escherichia coli, Bacillus subtilis, Helicobacter pylori e Helicobacter hepaticus). The influence of different aspects in the recognition and prediction are examined:

Understanding DNA sequences has been an ongoing endeavour within bioinfor- matics research. Recognizing the functionality of DNA sequences is a non-trivial and complex task that can bring insights into understanding DNA. In this thesis, we study deep learning models for recognizing gene regulating regions of DNA, more specifi- cally promoters. We first consider DNA modelling as a language by training natural language processing models to recognize promoters. Afterwards, we delve into current models from the literature to learn how they achieve their results. Previous works have focused on limited curated datasets to both train and evaluate their models using cross-validation, obtaining high-performing results across a variety of metrics. We implement and compare three models from the literature against each other, us- ing their datasets interchangeably throughout the comparison tests. This highlights shortcomings within the training and testing datasets for these models, prompting us to create a robust promoter recognition testing dataset and developing a testing methodology, that creates a wide variety of testing datasets for promoter recognition. We then, test the models from the literature with the newly created datasets and highlight considerations to take in choosing a training dataset. To help others avoid such issues in the future, we open-source our findings and testing methodology.
Graduate

Thesis (Ph. D.)--University of Wisconsin-Madison, 1989.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The methylotrophic yeast Pichia pastoris is widely used for recombinant protein production due to its ability to grow to high cell densities as well as possession of an inducible methanol utilization pathway (MUT). The expression of genes encoding enzymes of the MUT pathway is very tightly regulated. These genes are turned on when methanol but not glucose is used as the sole carbon source. Thus, P. pastoris cells can be grown to high densities in glucose containing medium and expression of genes of MUT pathway can be turned on by changing the carbon source to methanol. This strategy is widely used for recombinant protein production wherein the gene of interest is cloned downstream of the methanol-inducible promoter of the gene encoding the first enzyme of the MUT pathway, alcohol oxidase I (AOXI). Despite production of a large number of recombinant proteins using the AOXI promoter, the mechanism of transcriptional activation of AOXI is not very well understood. It is only recently that a zinc finger protein known as Mxr1p (methanol expression regulator 1) was shown to play a key role in the regulation of AOXI as well as other genes of methanol utilization pathway (1) P. pastoris strains that do not express Mxr1p (mxr1) are unable to grow on peroxisomal substrates such as methanol and oleic acid. Methanol-inducible expression of genes involved in MUT pathway as well as those involved in peroxisome biogenesis (peroxins,) is severely impaired in mxr1 strains. While Mxr1p is constitutively expressed in cells cultured on glucose as well as methanol, it is cytosolic in glucose-grown cells, but nuclear in methanol-grown cells (1). The exact nucleotide sequence to which Mxr1p binds and regulates the expression of genes of MUT pathway is not known. The aim of this thesis is to map the Mxr1p binding sites in the promoters of methanol-inducible genes of P. pastoris. As a first step towards understanding the mechanism of transcriptional regulation of AOXI and other methanol inducible genes of P. pastoris by Mxr1p, the N-terminal region comprising of 150 amino acids, including the zinc finger DNA binding domain of Mxr1p was cloned into an E. coli expression vector and the recombinant protein was purified from E. coli cells. This recombinant protein (referred to as Mxr1p in this study) was used in an electrophoretic mobility shift assay (EMSA) to identify Mxr1p binding sites in the AOXI promoter. EMSA was carried out with sixteen different oligonucleotides spanning AOXI promoter region between -940 and -114 bp. Such studies led to the identification of six Mxr1p binding sites in AOXI promoter. Using a combination of DNase I footprinting as well as EMSA with chimeric double stranded oligonucleotides, the minimal Mxr1p binding site was identified as a 20 bp DNA sequence containing a core 5’CYCC 3’ sequence. Using methylation interference as well as extensive mutagenesis studies, nucleotides critical for Mxr1p binding were identified. Comparative analysis of Mxr1p binding sites identified in our study with the AOXI promoter deletion studies of Hartner et al (2) suggested that the Mxr1p binding sites identified in our study are likely to function as methanol-inducible enhancers in vivo, since deletion of AOXI promoter regions comprising Mxr1p binding sites results in a significant loss of methanol-inducible promoter activity. Thus, Mxr1p binding sites are likely to function as Mxr1p response elements (MXREs) in vivo. Mxr1p is considered to be the P. pastoris homologue of S. cerevisiae Adr1p (alcohol dehydrogenase II [ADH2] synthesis regulator). Adr1p is a key regulator of S. cerevisiae genes involved in the metabolism of glycerol, ethanol and oleic acid. The DNA binding domains of Adr1p and Mxr1p share 82% similarity and 70% identity. We therefore examined whether Mxr1p can bind to the Adr1p binding site of ADH2 promoter(ADH2-UAS1). Our studies indicate that Mxr1p does not bind to ADH2-UAS1. Interestingly, a single point mutation restores Mxr1p binding to ADH2-UAS1. Since Mxr1p is involved in the regulation of a number of genes including AOXI, we examined whether promoters of other Mxr1p-regulated genes also harbour MXREs similar to those identified in AOXI promoter. The promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8) were chosen for this purpose. A detailed analysis of Mxr1p binding to these promoter sequences led to the conclusion that DHAS and PEX8 promoters also harbour Mxr1p binding sites similar to those of AOXI promoter. Based on these studies, we have derived a consensus sequence for Mxr1p binding. This study is the first report on detailed characterization of Mxr1p binding sites in three methanol-inducible promoters of P. pastoris and thus provides the molecular framework by which this transcription factor functions as a master regulator of genes involved in methanol utilization pathway of P. pastoris. Our study provides the blue print for mapping Mxr1p binding sites in the promoters of other Mxr1p-regulated genes.

碩士
國立中興大學
分子生物學研究所
87
The events that some of the X. campestris (Xc) promoters, studied by promoter probing vector in vivo, are recognized very poorly by E. coli RNA polymerase (RNAP), lead us to define the characteristic of promoters recognition by Xc RNA polymerase. In order to achieve this study, purification of Xc RNAP was done firstly. Using a step salt gradient of KCl, RNAP holoenzyme (Es70) and core enzyme (E) were partially purified through Heparin-Sepharose CL-6B chromatographic steps. The holoenzyme, which was preliminary isolated at 0.3 M of KCl, was then purified by DEAE A25. Whereas, the core enzyme isolated at 0.4 M was then purified by Bio-Rex 70. The transcriptional activities of these purified RNAPs, as well as the reconstituted Es32 , had been examined by run-off in vitro transcription system using T7A1 and dnaK promoter as template, respectively. With respect to a fixed amount of core polymerase (1 pmol), the amount of sigma 32 required for reconstitution was roughly 3 pmol. Taken together, the results indicated that purified Es70 and Es32 are capable to direct transcription in vitro. In vitro transcription studies using T7A1 and rpoD promoters and that of Xc and E. coli RNAP under various reaction conditions were performed. Neither Xc nor E. coli RNAP showed differences under different RNAP concentrations and preincubation time factors with T7A1 promoter. In contrast to E. coli enzyme, Xc RNAP was demonstrated to have different behavior under different DNA and NaCl concentrations, and temperature factors. RNAP from Xc11 gave a best fit of transcription under 300 mM of NaCl, 32°C and 2 pmol of DNA template (1:20, RNAP:DNA). Conversely, the conditions for E. coli RNAP is 50 mM of NaCl, 37°C and 2 pmol of DNA template. When Xc11 rpoD promoter was applied as template, the NaCl concentration for Xc RNAP was 100mM and 200 mM for E. coli enzyme. These results revealed that Xc and E. coli RNAP are somewhat different in promoter selectivity. The promoter specific binding site of Xc RNAP to the rpoD promoter at 28°C was demonstrated by DNase I footprint analysis. The protected region in the non-template strand was from base +18 to -40, which is suggested to be binding site of Xc RNAP open complex. The DNaseI hypersensitive sites were -26 and -36, and sensitive sites -22 and -31 nt upstream of the transcription start site. The TATAAT and TTCCGG sequences recognized by Xc RNAP were proposed to be the -10 and -35 region, respectively. The spacing between these two regions is 20 bp, which is not well consistent with the E. coli sigma 70 consensus. Taken together, the results showed that the promoter structure recognized by purified Xc RNAP are similar, but not identical to those recognized by E. coli Es70.

碩士
中華大學
土木工程學系
104
Word recognition is the core language education for junior classes in elementary schools. Given that course plans are usually tight, most teachers have little options but teaching in rather conventional ways, which features one-way give-and-take patterns and demanding repetitive character printing practice. The insipidity fails to draw attention of the students, thus, students’ learning performance compromises. If teachers develop interesting strategies which meet cognitive development of the juniors, instead of the said pattern, it’s believed that students will be more interested in word recognition learning and furthermore their autonomous learning capabilities can be as well promoted. Once students have established adequate vocabulary, they will be able to set up firmer base to learn reading and writing. Therefore aims to discuss ways to improve junior students’ motivation and interest in word recognition education. The research surveys a junior class with 28 pupils in an elementary school in Hsinchu County by analyzing their problems of learning and providing appropriate suggestion based on the TRIZ theory. Conclusions are drawn as following: 1. Group competition and games help promote students’ interest of learning; 2. Multiple materials such as riddles and nursery rhymes motivate students to learn and help develop concept of categorization of characters and their radicals.

Historical empathy (HE) is refers to deep inquiry in which academic and emotional responses to historical content are shaped through source analysis of the actions, motives, perspectives, and beliefs of people in the past. There are limited studies about whether students demonstrate HE through analysis of underrepresented historical figures. Additionally, studies are limited on how students’ social identities influence demonstration of HE. Consequently, there is a gap in the literature with regard to whether source analysis of underrepresented historical figures, as well as students’ social identities, impact demonstration of HE and critical race consciousness (CRC). Elizabeth Jennings is an example of an underrepresented historical figure. She was an African American teacher who was forcibly ejected from a streetcar due to her race in 1854. Jennings sued the streetcar company and won. Although Jennings set an important precedent for African Americans to use the legal system to challenge antebellum segregation ordinances, she remains a relatively obscure historical figure. The purpose of this study was to examine whether or not an instructional unit about Elizabeth Jennings called “The Elizabeth Jennings Project” (EJP) promotes conditions conducive for student demonstration of HE and/or CRC. A case study of one middle and two high school classes was conducted at one private, non-secular school in an urban area of the Northeast. Instructional methods that best promoted HE included in-class discussion and debate. Students provided insights about their social identities during focus group sessions with regard to how the EJP fostered HE and CRC.

碩士
開南大學
觀光運輸學院碩士在職專班
105
In recent years, the government has encouraged the transformation of traditional industries into a tourist factory with manufacturing, history, culture and recreation. It is an emerging tourist attraction, providing visitors with leisure and entertainment, industry knowledge and local culture. It is fascinated by its experience. , Such as DIY courses, game activities, so that consumers can operate in a safe space, personally experience or feel the production and life-related things, enjoy the fun of exploration. In this study, we will use the five facets proposed by Schmitt (1999): sensory experience, emotional experience, thinking experience, action experience and association experience. Based on the domestic sightseeing factory, the scale is made from June to July in 2017, A total of 398 valid questionnaires were collected and the effective recovery rate was 99.5%. The results were analyzed by descriptive statistics, independent sample t test, single factor mutation analysis, correlation analysis (P <0.05). The questionnaire was collected from the visitors of Guo Yuan-yang Cake Museum. And the results of the study show that: experience activities and enhance brand awareness and visitors to buy a significant positive relationship, that through the visitors' Experience, can give brand awareness to enhance the positive, enhance the purchase intention.

Significant advancements in genome sequencing techniques and other high-throughput initiatives have resulted in the availability of complete sequences of genomes of a large number of organisms, which provide an opportunity to study detailed biological information encoded therein. Identification of functional roles of proteins can aid in comprehension of various cellular activities in an organism, which is traditionally achieved using techniques pertaining to the field of molecular biology, protein chemistry and macromolecular crystallography. The established experimental methods for protein structure and function determination, although accurate and resourceful, are laborious and time consuming. Computational analyses of sequences of gene products and exploration of evolutionary relationships can give clues on protein structure and/or function with reasonable accuracy which can be used to direct experimental studies on proteins of interest, effectively. Moreover, with growing volumes of data, there has been a growing disparity in the number of well-characterized and uncharacterized proteins, further necessitating the use of computational methods for investigating evolutionary and structure-function relationships. The remarkable progress made in the development of computational techniques (Chapter 1) has immensely contributed to the state-of-the-art biological sequence analysis and recognition of protein structure and function in a reliable manner. These methods have largely influenced the exploration of protein sequence-structure-function space. One of the relevant applications of computational approaches is in the understanding of functional make-up of human pathogens, their complex interplay with the host and implications in pathogenesis. In this thesis, sensitive profile-based search procedures have been utilized to address various aspects in the context of three pathogens- Mycobacterium tuberculosis, Plasmodium falciparum and Trypanosoma brucei, which are causative agents of potentially life- threatening diseases. The existing drugs approved for the diseases, although of immense value in controlling the disease, have several shortcomings, the most important of them being the emergence of drug resistance that render the current treatment regimens futile. Thus, the identification of practicable targets and new drugs or new combination therapies become an important necessity. Analyses on structural and functional repertoire of proteins encoded in the pathogenic genomes can provide means for rational identification of therapeutic intervention strategies. This thesis begins with the computational analyses of proteins encoded in M. tuberculosis genome. M. tuberculosis is a primary aetiological agent of tuberculosis in humans, and is o responsible for an estimated 1.5 million deaths every year. The complete genome of the pathogen was sequenced and made available more than a decade ago, which has been valuable in determination of functional roles of its gene products. Yet, functions of many M. tuberculosis proteins remain unknown. Computational prediction of protein function is an on- going process based on ever growing information made available in public databases as well as the introduction of powerful homology recognition techniques. Hence, a continuous refinement is essential to make the most of the sequence data, ensuring its accuracy and relevance. With the use of multiple sequence and structural profile-based search procedures, an enhanced structural and functional characterization of M. tuberculosis proteins, totalling to 95% of the genome was achieved (Chapter 2). Following are the key findings. o Domain definitions were obtained for a total of 3566 of 4018 proteins. Amino acid residue coverage of >70% was achieved for 2295 proteins which constitute more than half of the proteome. o Domain assignments were newly identified for 244 proteins with domain-unassigned regions. Structure prediction for these proteins corroborated all the remote homologyrelationships recognized using profile-based methods, enhancing the reliability of the predictions. o Comparison on domain compositions of proteins between M. tuberculosis and human host, revealed presence of pathogen-specific domains that are not homologous to proteins in human. Such proteins in M. tuberculosis are mainly virulence factors involved in host-pathogen interactions such as immune-dominance and aiding entry and survival in human host macrophages, hence forming attractive targets for drug discovery. o Putative structural and functional information for proteins with no recognizable domains were inferred by means of fold recognition and an iterative profile-based search against sequence database. o Attributing putative structures and functions to 955 conserved hypothetical proteins in M. tuberculosis, 137 of which are reportedly essential to the pathogen, provide a basis to re-investigate their involvement in pathogenesis and survival in the host. Proteins with no detectable homologues were recognized as M. tuberculosis H37Rv-specific, which can serve as promising drug targets. An attempt was made to identify porin-like proteins in M. tuberculosis, considering MspA porin from M. smegmatis as a template. The difficulty in recognition of putative porins in M. tuberculosis is indicative of novel outer membrane channel proteins, not characterized yet, or high representation of ion-channels, symporters and transporters to compensate for the functional role of porins. In addition, MspA-like proteins were not readily recognized in other slow-growing mycobacterial pathogens that are known to infect human host, apart from M. tuberculosis. This indicates probable acquisition of physiological adaptations, i.e. absence of porins, to confer drug-resistance, in the course of their co-evolution with human hosts. Evolutionary relationships recognized between sequence (Pfam) and structural (SCOP) families aided in association of potential structures and/or functions for 55 uncharacterized Pfam domains recognized in M. tuberculosis. Such associations deliver useful insights into the structure and function of a protein housing the uncharacterized domain. The functional inferences drawn for M. tuberculosis proteins based on the predictions can provide valuable basis for experimental endeavours in understanding mechanisms of pathogenesis and can significantly impact anti-tubercular drug discovery programmes. An interesting outcome benefitted from the exercise of exploring relationships between Pfam and SCOP families, was the identification of evolutionary relationship between a Pfam domain of unknown function DUF2652 and class III nucleotidyl cyclases. A detailed investigation was undertaken to assess this relationship (Chapter 3). Nucleotidyl cyclases synthesize cyclic nucleotides which are critical second messengers in signalling pathways. The DUF2652 family predominantly comprises of bacterial proteins belonging to three lineages- Actinobacteria, Bacteroidetes and Proteobacteria. Thus, recognition of evolutionary relationship between these bacterial proteins and nucleotide cyclases is of particular interest due to the indispensability of cyclic nucleotides in regulation of varied biological activities in bacteria. Use of fold recognition program suggested presence of nucleotide cyclase-characteristic topological motif (βααββαβ) in all the members of the DUF2652 family. Detailed analyses on structural and functional features of the uncharacterized set of bacterial proteins corresponding to 50 bacterial genomes, using profile- based alignments, revealed presence of key features typical of nucleotidyl cyclases, including metal-binding aspartates, substrate-specifying residues and transition-state stabilizing residues. Depending on the features, 20 proteins of Actinobacteria lineage, predominantly mycobacteria, of unknown structure and function were identified as putative nucleotide cyclases, 23 proteins of Bacteroidetes lineage were associated with guanylyl cyclases, while 8 uncharacterized proteins of Proteobacteria were recognized as nucleotide cyclase-like proteins (7 adenylyl and one guanylyl cyclase). Sequence similarity-based clustering of the predicted nucleotide cyclase-like proteins with established nucleotide cyclases indicated the apparent evolutionarily distinctness of the subfamily of class III nucleotidyl cyclases predicted. Furthermore, analysis of evolutionarily conserved gene clusters of the predicted nucleotide cyclase-like proteins indicated functional associations that support the predictions on their participation in cellular signalling events. The inferences made can be experimentally investigated further to ascertain the involvement of the uncharacterized bacterial proteins in signalling pathways, which can help in understanding the pathobiology of pathogenic species of interest. The next objective was the recognition of biologically relevant protein-protein interactions across M. tuberculosis and human host (Chapter 4). M. tuberculosis is well known for its ability to successfully co-evolve with human host in terms of establishing infection, survival and persistence. The current knowledge on the mechanisms of host invasion, immune evasion and persistence in the host environment can be attributed, and is limited, to the experimental studies pursued by numerous groups. Chapter 4 presents an approach for computational identification of biologically feasible protein-protein interactions across M. tuberculosis and human host. The approach utilizes crystal structures of intra-organism protein-protein complexes which are transient in nature. Identification of homologues of host and pathogen proteins in the database of known protein-protein interactions, formed the initial step, followed by identification of conserved interfacial patch and integration of information on tissue-specific expression of human proteins and subcellular localization of human and M. tuberculosis proteins. In addition, appropriate filters were used to extract biologically feasible host-pathogen protein-protein interactions. This resulted in recognition of 386 interactions potentially mediated by 59 M. tuberculosis proteins and 90 human proteins. A predominance of host-pathogen interactions (193 protein-protein interactions) brought about by M. tuberculosis proteins participating in cell wall processes, was observed, which is in concurrence with the experimental studies on immuno-modulatory activities brought about by such proteins. These set of mycobacterial proteins were predicted to interact with diverse set of host proteins such as those involved in ubiquitin conjugation pathways, metabolic pathways, signalling pathways, regulation of cell proliferation, transport, apoptosis and autophagy. The predictions have the potential to complement experimental observations at the molecular level. Details on couple of interesting cases are presented in the chapter, one of which is the probable mechanism of immune evasion adopted by M. tuberculosis to inhibit lysozyme activity in macrophages, and second is the mechanism of nutrient uptake from host. The set of M. tuberculosis proteins predicted to mediate interactions with host proteins have the potential to warrant an experimental follow-up on probable mechanisms of pathogenesis and also serve as attractive targets for chemotherapeutic interventions. proteins known to participate in P. falciparum metabolism. Pathway holes, where evidence on metabolic step exists but the catalysing enzyme is not known, have also been addressed in the study, several of which have been suggested to play an important role in growth and development of the parasite during its intra-erythrocytic stages in human host. A subsequent objective was the recognition P. falciparum proteins potentially capable of remodelling erythrocytes to suit their niche (Chapter 7). Exploitative mechanisms are brought about by the parasite to remodel erythrocytes for growth and survival during intra-erythrocytic stages of its life-cycle, the understanding of which is limited to experimental studies. To achieve physicochemically viable protein-protein interactions potentially mediated by proteins of human erythrocytes and P. falciparum proteins, a structure-influenced protocol, similar to the one demonstrated in Chapter 4, was employed. Information on subcellular localization and protein expression is crucial especially for parasites like P. falciparum, which reside in One of the major shortcomings with current treatment regimen for tuberculosis is the emergence of multidrug (MDR) and extensively drug-resistant (XDR) strains that render first-line and second-line drug treatments futile. This entails a need to explore target space in M. tuberculosis as well as explore the potential of existing drugs for repurposing against tuberculosis. A drug repurposing strategy i.e. exploring within-target-family selectivity of small molecules, has been implemented (Chapter 5) to contribute towards time and cost-saving anti-tubercular drug development efforts. With the use of profile-based search procedures, evolutionary relationships between targets (other than proteins of M. tuberculosis) of FDA-approved drugs and M. tuberculosis proteins were investigated. A key filter to exclude drugs capable of acting on human proteins substantially reduced the chances of obtaining anti-targets. Thus, total of 130 FDA-approved drugs were recognized that can be repurposed against 78 M. tuberculosis proteins, belonging to the functional categories- intermediary metabolism and respiration, information pathways, cell wall and cell processes and lipid metabolism. The catalogue of structure and function of M. tuberculosis proteins and their involvement in host-pathogen protein-protein interactions compiled from chapters 2 and 4 served as a guiding tool to explore the functional importance of targets identified. Many of the potential targets identified have been experimentally shown to be essential for growth and survival of the pathogen earlier, thus gaining importance in terms of pharmaceutical relevance. Polypharmacological drugs or drugs capable of acting of multiple targets were also identified (92 drugs) in the study. These drugs have the potential to stand tolerance against development of drug resistance in the pathogen. Comparative sequence and structure-based analysis of M. tuberculosis proteins homologous to known targets yielded credible inferences on putative binding sites of FDA-approved drugs in potential targets. Instances where information on binding sites could not be readily inferred from known targets, potentially druggable sites have been predicted. Comparison with earlier experimental studies that report anti-tubercular potential of several approved drugs enhanced the credibility of 74 of 130 FDA-approved drugs that can be readily prioritized for clinical studies. An additional exercise was pursued to identify prospective anti-tubercular agents by means of structural comparison between ChEMBL compounds and 130 FDA-approved drugs. Only those compounds were retained that showed considerably high structural similarity with approved drugs. Such compounds with minor changes in terms of physicochemical properties provide a basis for exploration of compounds that may exhibit higher affinities to bind to M. tuberculosis targets. The set of approved drugs recognized as repurpose-able candidates against tuberculosis, in concert with the structurally similar compounds, can significantly impact anti-tubercular drug development and drug discovery. The next part of the thesis focuses on Plasmodium falciparum, an obligate intracellular protozoan parasite responsible for malaria. The parasite genome features unusual characteristics including abundance of low complexity regions and pronounced sequence divergence that render protein structure and function recognition difficult. The parasite also manifests remarkable plasticity in its metabolic organization throughout its developmental stages in two hosts-human and mosquito; thus obtaining an exhaustive list of metabolic proteins in the parasite gains importance. Considering the utility of multiple sensitive profile-based search approaches in enhanced annotation of M. tuberculosis genome, a similar exercise was employed to recognize potential metabolic proteins in P. falciparum (Chapter 6). A total of 172 metabolic proteins were identified as participants of 78 metabolic pathways, over and above 609heterogeneous environmental conditions at different stages in their lifecycle. Inclusion of such data aided in extraction of 208 biologically relevant protein-protein interactions potentially mediated by 59 P. falciparum proteins and 30 erythrocyte proteins. Host-parasite protein-protein interactions were predicted pertaining to several major strategies spanning intra-erythrocytic stages in P. falciparum pathogenesis including- gaining entry into the host erythrocytes (category: RBC invasion, protease), redirecting parasitic proteins to erythrocyte membrane (category: protein traffic), modulating erythrocyte machinery (category: rosette formation, putative adhesin, chaperone, kinase), evading immunity (category: immune evasion) and eventually egress (category: merozoite egress) to infect other uninfected erythrocytes. Elaborate means to analyse and evaluate the functional viability of a predicted interaction in terms of geometrical packing at the interfacial region, electrostatic complementarity of the interacting surfaces and interaction energies is also demonstrated. The protein-protein interactions, thus predicted between human erythrocytes and P. falciparum, have the potential to provide a useful basis in understanding probable mechanisms of pathogenesis, and indeed in pinning down attractive targets for antimalarial drug discovery. The emergence of drug resistance against all known antimalarial agents, currently in use, necessitates discovery and development of either new antimalarial agents or unexplored combination of drugs that may not only reduce mortality and morbidity of malaria, but also reduce the risk of resistance to antimalarial drugs. In an attempt to contribute towards the same, Chapter 8 explores the established concept of within-target-family selectivity of small molecules to recognize antimalarial potential of the approved drugs. Eighty six FDA-approved drugs, predominantly constituted by antibacterial agents, were identified as feasible candidates for repurposing against 90 P. falciparum proteins. Most of the potential parasite targets identified are known to participate in housekeeping machinery, protein biosynthesis, metabolic pathways and cell growth and differentiation, and thus are pharmaceutically relevant. During intra-erythrocytic growth of P. falciparum, the parasite resides within the erythrocyte, within a protective encasing, known as parasitophorous vacuole. Hence a drug, intended to target a parasite protein residing in an organelle, must be sufficiently hydrophilic or hydrophobic to be able to permeate cell membranes and reach its site of activity. On the basis of lipophilicity of the drugs, a physical property determined experimentally, 57 of 86 FDA-approved drugs were recognized as feasible candidates for use against P. falciparum during the course of blood-stages of infection, which can be prioritized for antimalarial drug development programmes. The final section of the thesis focuses on the protozoan parasite Trypanosoma brucei, a causative agent of African sleeping sickness (Chapter 9). This disease is endemic to sub-Saharan regions of Africa. Despite the availability of completely sequenced genome of T. brucei, structure and function for about 50% of the proteins encoded in the genome remain unknown. Absence of prophylactic chemotherapy and vaccine, compounded with emergence of drug-resistance renders anti-trypanosomal drug discovery challenging. Thus, considering the utility of frameworks established in earlier chapters for recognition of protein structure, function and drug-targets, similar steps were undertaken to understand functional repertoire of the parasite and use drug repurposing methods to accelerate anti-trypanosomal drug discovery efforts. Structures and functions were reliably recognized for 70% of the gene products (5894) encoded in T. brucei genome, with the use of multiple profile-based search procedures, coupled with information on presence of transmembrane domains and signal peptide cleavage sites. Consequently, a total of 282 uncharacterized T. brucei proteins could be newly coined as potential metabolic proteins. Integration of information on stage-specific expression profiles with Trypanosoma-specific and T-.brucei-specific proteins identified in the study, aided in pinning down potential attractive targets. Additionally, exploration of evolutionary relationships between targets of FDA-approved drugs and T. brucei proteins, 68 FDA-approved drugs were predicted as repurpose-able candidates against 42 potential T. brucei targets which primarily include proteins involved in regulatory processes and metabolism. Several targets predicted are reportedly essential in assisting the parasite to switch between differentiation forms (bloodstream and procyclic) in the course of its lifecycle. These targets are of high therapeutic relevance, hence the corresponding drug-target associations provide a useful resource for experimental endeavours. In summary, this thesis presents computational analyses on three pathogenic genomes in terms of enhancing the understanding of functional repertoire of the pathogens, addressing metabolic pathway holes, exploring probable mechanisms of pathogenesis brought about by potential host-pathogen protein-protein interactions, and identifying feasible FDA-approved drug candidates to repurpose against the pathogens. The studies are pursued primarily by taking advantage of powerful homology-detection techniques and the ever-growing biological information made available in public databases. Indeed, the inferences drawn for the three pathogenic genomes serve an excellent resource for an experimental follow-up. The set of protocols presented in the thesis are highly generic in nature, as demonstrated for three pathogens, and can be utilized for genome-wide analyses on many other pathogens of interest. The supplemental data associated with the chapters is provided in a compact disc attached with this thesis.