This thesis focuses on tactile perception and aims at a comprehensive analysis of its characteristics over the time-course of various goal-directed movements. Tactile perception is assessed by means of discrimination and detection paradigms, as well as event-related potentials (ERPs). The main question investigated throughout the thesis is: ‘What changes in tactile perception, if any, take place over the time course of a goal-directed movement?’ In Chapter 2, the mechanisms related to such identified changes are examined: a facilitatory one – attention, and an inhibitory one – suppression. The experiment in Chapter 3 tests, at a brain level, amongst several explanations of the experimental results outlined in Chapter 2: timing-based, effector-based, and modality-based attentional/suppressive influences. In Chapter 4, other naturalistic movements are investigated (i.e., the movements involved in juggling and throwing/catching a basketball). The results indicate a lack of facilitation in the processing of tactile information during the preparatory phase of the movement. Furthermore, differential changes are identified in tactile perception over the execution phase of the movement: At a behavioural level, tactile sensitivity significantly declines over the execution phase of the movement (though the detection of incoming tactile stimulation is enhanced), while at a neuronal level the same period exhibits significantly enhanced responses to somatosensory stimulation. The experiments reported here thus bring evidence in favour of a dissociation between detecting and discriminating what is felt while moving. These results suggest that the quality of what is felt while moving may not be important for movement and, at the same time, that different pathways in the brain may be responsible for detecting and discriminating what is felt over the time course of a goal-directed movement. Based on these findings, in Chapter 5, the implications of these results are discussed and directions for further research are outlined.

This investigation compares the effectiveness of two methods of teaching production work in beginning typewriting. One method is defined as the traditional approach, which adheres to suggestions and materials for teaching found in current typewriting textbooks. Students are paced, drilled, and timed on straight copy to build speed and accuracy, but not on production work; they usually type from perfectly arranged copy; and they circle their errors for at least half the course. The other method, developed at North Texas State University by Payne and Anderson, is defined as the teacher-directed approach. Students are intensively paced, drilled, and timed by the teacher on short, simple jobs or parts of jobs; they usually type from unarranged copy; they learn to erase errors on production work during the first production unit; and they are evaluated on the basis of the number of mailable items produced during a specific time period.

Orientador: Ana SílviaLopes Davi Médola / Banca: Luciano Guimarães / Banca: Terezinha Fortes Mestrinelli / Resumo: O presente estudo tem como objeto a comunicação dirigida e investiga as estratégias discursivas da campanha "Triângulos" do HSBC no Brasil. São analisados os procedimentos enunciativos que regem a organização textual das peças publicitárias veiculada em folders disponibilizados nos pontos de venda das agências bancárias do HSBC, no final de 2004. O instrumental teórico-metodológico que baliza as reflexões aqui apresentadas é decorrente da semiótica discursiva de linha francesa, a chamada Escola de Paris, a partir das postulações elaboradas por A. J. Greimas e Jean- Marie Floch. Após apresentar o percurso de constituição do banco enquanto instituição inserida em uma economia globalizada, o trabalho demonstra o papel das formas discursivas na comunicação dirigida e suas implicações nas relações interculturais. As análises realizadas demonstram, a partir do texto, a configuração do contexto da relação comunicativa, depreendendo os valores e ideologias presentes nos discursos organizados com o objetivo central de promover o consumo dos serviços oferecidos pelo HSBC, por meio de estratégias de manipulação baseadas na sedução e na provocação. / Abstract: The present study has as object the directed communication and investigates the discursives strategies of the campaign "Triangles" of the HSBC in Brazil. The enunciative procedures are analyzed which conduct the textual organization of the advertising pieces that are propagated in folders availables at points of sales of HSBC the agencies at the end of 2004. The theoretician-methodological instrumental which signalize the presented reflections is due to discursive semiotics of French line, the professed School of Paris, from postulations elaborated by J. Greimas and Jean-Marie Floch. After introduces the course of bank forming, while inserted institution in a worldwide economy, this work demonstrates the part taken by discursive features in directed communication and its implications in the intercultural relations. The performed analyses demonstrate from the text on, the configuration of the context of the communicative relation, gathering the values and ideologies presented in the speeches organized with the main objective to encourage the use of services offered by HSBC, by means of strategies of manipulation based on seduction and temptation. / Mestre

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

by Au Lai Yin, Cathy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 50-66). / Chapter I. --- ABSTRACT --- p.i / Chapter II. --- ACKNOWLEDGMENTS --- p.iii / Chapter III. --- TABLE OF CONTENTS --- p.iv / Chapter IV. --- LIST OF TABLES --- p.viii / Chapter V. --- LIST OF FIGURES --- p.x / Chapter VI. --- INTRODUCTION --- p.1 / Chapter VII. --- LITERATURE REVIEW --- p.3 / Chapter A. --- Mycobacterial tuberculosis Infections --- p.3 / Chapter B. --- Diagnostic Criteria forM .tuberculosis Infections --- p.3 / Chapter C. --- Mycobacteriological Laboratory Investigations for M. tuberculosis --- p.4 / Chapter 1. --- Conventional methods --- p.4 / Chapter 2. --- Rapid methods --- p.4 / Chapter D. --- Polymerase chain reaction (PCR) - the Principle --- p.5 / Chapter E. --- Application of PCR for Detection of M. tuberculosis --- p.6 / Chapter 1. --- Choice of target sequences --- p.6 / Chapter 2. --- Choice of method for the detection & identification of PCR-amplified product --- p.7 / Chapter 3. --- Studies on pure cultures --- p.9 / Chapter a. --- Detection limit - target DNA --- p.9 / Chapter b. --- Detection limit - Colony forming units --- p.9 / Chapter c. --- Detection limit - Number of cells --- p.10 / Chapter 4. --- Studies on clinical specimens --- p.10 / Chapter 5. --- Problems --- p.12 / Chapter a. --- Availability of target DNA --- p.13 / Chapter (i) --- Cell breakage efficiency --- p.13 / Chapter (ii) --- Target sequence --- p.14 / Chapter b. --- Inhibitory factors for Taq polymerase --- p.14 / Chapter c. --- Contamination --- p.15 / Chapter VIII. --- MATERIALS AND METHODS --- p.16 / Chapter A. --- Bacterial Strains and Strain Maintenance --- p.16 / Chapter 1. --- Reference Strains --- p.16 / Chapter 2. --- Clinical isolates --- p.16 / Chapter B. --- Growth media and culture conditions --- p.17 / Chapter C. --- Restriction Fragment Length Polymorphism (RFLP) --- p.17 / Chapter 1. --- Extraction of chromosomal DNA from M. tuberculosis --- p.18 / Chapter 2. --- Digestion of chromosomal DNA by PVU II --- p.19 / Chapter 3. --- Separation of digested DNA fragment by electrophoresis --- p.19 / Chapter 4. --- Southern Blotting --- p.19 / Chapter 5. --- Preparation of DNA probes by Polymerase Chain Reaction --- p.20 / Chapter 6. --- Hybridization --- p.21 / Chapter 7. --- Detection --- p.21 / Chapter D. --- Assessment of number of organisms --- p.22 / Chapter 1. --- Viable cell count --- p.22 / Chapter 2. --- Direct cell count --- p.22 / Chapter E. --- Assessment of the presence of IS6110/986 in M. tuberculosis isolates --- p.23 / Chapter F. --- Human leukaemic monocytic cell line (THP-1) --- p.23 / Chapter 1. --- Growth media and maintenance --- p.23 / Chapter 2. --- Culture Conditions --- p.24 / Chapter 3. --- Uptake of M. tuberculosis --- p.24 / Chapter G. --- Cell breakage and DNA extraction methodologies --- p.25 / Chapter H. --- Polymerase chain reaction (PCR) methodologies --- p.28 / Chapter 1. --- Primer and probe --- p.28 / Chapter 2. --- PCR conditions --- p.28 / Chapter 3. --- Detection --- p.29 / Chapter I. --- Patients and Clinical specimens --- p.30 / Chapter 1. --- Patients recruitment --- p.30 / Chapter 2. --- Clinical specimens --- p.30 / Chapter IX. --- RESULTS --- p.32 / Chapter A. --- "Development or Selection of a ""Standardized"" PCR Protocol for the Detection of M. tuberculosis Using Pure Cultures In Vitro" --- p.32 / Chapter 1. --- Selection of organisms for verification of the PCR protocol --- p.32 / Chapter 2. --- Optimization of the PCR conditions --- p.32 / Chapter 3. --- Detection limit of target DNA using the PCR procedure --- p.33 / Chapter B. --- Initial Screening of Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 &22a Based on Detection Limits of Colony Forming Units and Number of Cells --- p.34 / Chapter C. --- Comparison of Method 1 and Method 2 Based on Detection Limits of Colony Forming Units and Number of Cells Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis with variable copies of IS6110/986 --- p.34 / Chapter D. --- Detection of M. tuberculosis Isolates Within Macrophages --- p.35 / Chapter 1. --- Uptake of M. tuberculosis cells by THP-1 --- p.35 / Chapter 2. --- Comparison of the Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 & 22a Phagocytized by Activated THP-1 Macrophages --- p.35 / Chapter 3. --- Comparison of Method 1 and Method 2 Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis Phagocytized by Activated THP-1 Macrophages --- p.36 / Chapter E. --- Analysis of Clinical Specimens Using Method 1 & 2 with the Optimized PCR Protocol --- p.36 / Chapter 1. --- Bronchial Aspirate & Bronchoaveolar Lavage Fluid --- p.36 / Chapter 2. --- Pleural Fluid --- p.37 / Chapter 3. --- Tissue --- p.37 / Chapter 4. --- Sputum --- p.38 / Chapter 5. --- Cerebrospinal Fluid --- p.38 / Chapter X. --- DISCUSSION --- p.39 / Chapter A. --- Selection of IS6110/986 for DNA amplification --- p.39 / Chapter B. --- Optimization of PCR conditions reflected by detection limit of target DNA --- p.40 / Chapter C. --- Selection of cell breakage methods based on detection limits of CFU and/or number of mycobacterial cells --- p.41 / Chapter D. --- Application of Methods 1 & 2 and the optimized PCR protocol for clinical specimens --- p.43 / Chapter 1. --- Bronchial aspirates and bronchoaveolar lavage fluids --- p.43 / Chapter 2. --- Pleural fluids --- p.44 / Chapter 3. --- Tissues --- p.45 / Chapter 4. --- Sputa --- p.46 / Chapter 5. --- Cerebrospinal fluids --- p.46 / Chapter XI. --- CONCLUSION --- p.48 / Chapter XII. --- LITERATURE CITED --- p.50 / Chapter XIII --- TABLES --- p.67 / Chapter XIV. --- FIGURES --- p.85

Thesis (Ed.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The purpose of this study was to determine whether a match between a participant's learning style and type of online instruction improved learner performance on tests measuring comprehension and retention. Learning style was measured by the Self-Directed Leamer Readiness Scale (SDLRS) and the Grasha-Riechmann Student Learning Style Scale (GRSLSS) and online instruction varied among online courses, recorded online courses, and computer-based tutorials. The setting for the study was a high tech machine vision company in Massachusetts and online users of its products were the participants. Three groups of learners participated in the study: employees, high school students, and customers. All three groups were comprised of engineers or engineering students. All 106 participants completed a survey that measured their preference for self-directed and collaborative learning style with the standard instruments SDLRS and GRSLSS. Participants completed 323 pre- and post-tests for 46 live online courses, recorded online courses, and computer-based tutorials during the data collection phase of the study. Those participants learning in their preferred learning style had the highest mean improvement from pre- to post-tests. Those participants with average or below average scores for self-directed and collaborative learning style showed the least improvement. The results of this study supported the hypothesis that matching the type of activity, collaborative or self-directed, to the learner's preferred learning style improved performance. The study included ten research questions. / 2031-01-01

Chan Kam Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 175-182). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Drosophila as a model for studying development --- p.1 / Chapter 1.2 --- The formation of the body axis in Drosophila --- p.2 / Chapter 1.3 --- The maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.11 / Chapter 1.5 --- The formation of the biocid protein gradient from anterior pole to posterior pole of the embryo --- p.13 / Chapter 1.6 --- The bed protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.15 / Chapter 1.7 --- The formation of the bed protein gradient in embryo --- p.17 / Chapter 1.8 --- Components required for bcd mRNA localization at anterior pole of oocyte --- p.21 / Chapter 1.8.1 --- Cis-acting elements --- p.21 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.9 --- The properties of exuperantia protein --- p.25 / Chapter 1.9.1 --- The function of exu protein --- p.25 / Chapter 1.9.2 --- Exuperantia is a phosphoprotein --- p.26 / Chapter 1.9.3 --- Phosphorylation pattern of exuperantia protein is stage-specific --- p.28 / Chapter 1.9.4 --- Reversible phosphorylation is one of the major mechanisms to control protein activity in all eukaryotic cells --- p.29 / Chapter 1.9.5 --- The relationship between the exu protein phosphorylation and the bcd mRNA localization --- p.30 / Chapter 1.10 --- Aim of project --- p.31 / Chapter CHAPTER 2 --- Preparation of the exuperantia genomic DNA and complement DNA (cDNA) mutant Constructs / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- DNA preparation methods --- p.35 / Chapter 2.2.1.1 --- Preparation of double-stranded DNA by polyethylene glycol6000 --- p.35 / Chapter 2.2.1.2 --- Preparation of M13mp8 single-stranded DNA --- p.37 / Chapter 2.2.1.3 --- "Preparation of double-stranded DNA by Biol prep (Modified from Maniatis et al.,1989)" --- p.38 / Chapter 2.2.2 --- "Preparation of DH5α，JM109, TG1 competent cells" --- p.39 / Chapter 2.2.3 --- Bacteria transformation --- p.40 / Chapter 2.2.4 --- Restriction enzyme digestion --- p.40 / Chapter 2.2.5 --- Phenol/chloroform extraction --- p.41 / Chapter 2.2.6 --- Purification of DNA fragment by electro-elution --- p.42 / Chapter 2.2.7 --- DNA ligation --- p.43 / Chapter 2.2.8 --- DNA dephosphorylation --- p.43 / Chapter 2.2.9 --- In vitro site-directed mutagenesis --- p.44 / Chapter 2.2.9.1 --- The Sculptor´ёØ in vitro mutagenesis --- p.44 / Chapter 2.2.9.2 --- The GeneEditor´ёØ in vitro site-directed mutagenesis --- p.47 / Chapter 2.2.10 --- The double-stranded or single-stranded DNA sequencing by T7 DNA polymerase sequencing system --- p.50 / Chapter 2.2.11 --- Denatured polyacrylamide gel electorphoresis --- p.51 / Chapter 2.2.11 --- Nucleotide sequence of the sequencing primers and the mutageneic oligonucleotides --- p.54 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Design exuperantia mutant constructs --- p.55 / Chapter 2.3.1.1 --- Comparison of exu protein amino acids sequence with different Drosophila species --- p.56 / Chapter 2.3.2 --- The exu genomic mutant constructs --- p.63 / Chapter 2.3.3 --- The exu cDNA mutant constructs --- p.63 / Chapter 2.4 --- Discussion --- p.76 / Chapter CHAPTER 3 --- Epitope tagging of exuperantia protein with c-myc eptiope / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Materials and methods --- p.84 / Chapter 3.2.1 --- Preparation of the c-myc eptiope DNA fragment --- p.84 / Chapter 3.2.2 --- End-filling of 5'overhang DNA fragment by Klenow fragment --- p.86 / Chapter 3.2.3 --- In vitro translation of protein by TNT® Quick coupled transcription and translation system --- p.86 / Chapter 3.2.4 --- Immunoprecipitation of recombinant exu protein --- p.87 / Chapter 3.2.5 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 3.2.5.1 --- SDS-PAGE preparation --- p.88 / Chapter 3.2.5.2 --- SDS-PAGE electrophoresis --- p.90 / Chapter 3.2.6 --- Western blot analysis --- p.90 / Chapter 3.2.6.1 --- Transfer the protein to a nitro-cellulose membrane by semi-dried blotting --- p.90 / Chapter 3.2.6.2 --- Western blot blocking and antibody recognition --- p.91 / Chapter 3.3 --- Results --- p.92 / Chapter 3.3.1 --- Construction of the plasmid containing exu cDNA tagging with a c-myc epitope --- p.92 / Chapter 3.3.2 --- In vitro translation of c-myc epitope tagged exu protein --- p.102 / Chapter 3.3.3 --- Immunoprecipitation of c-myc labeled exu protein by a polyclonal rabbit anti-exu antibody and monoclonal mouse anti-myc antibody --- p.104 / Chapter 3.4 --- Discussion --- p.109 / Chapter CHAPTER 4 --- In vitro phosphorylation of exuperantia Protein / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Materials and methods --- p.113 / Chapter 4.2.1 --- Exogenous kinase phsophorylation reactions --- p.113 / Chapter 4.2.2 --- Separation of the phosphorylated exu protein variants by SDS- PAGE --- p.114 / Chapter 4.3 --- Results --- p.115 / Chapter 4.3.1 --- Western blot analysis of in vitro translated exu protein variants --- p.115 / Chapter 4.3.2 --- Phosphorylation of in vitro translated exu protein variants by exogenous cAMP-dependent protein kinase --- p.118 / Chapter 4.3.3 --- Phosphorylation of in vitro translated exu protein variants by exogenous cGMP-dependent protein kinase --- p.123 / Chapter 4.3.4 --- Phosphorylation of in vitro translated exu protein variants by exogenous protein kinase C --- p.128 / Chapter 4.4 --- Discussion --- p.133 / Chapter CHAPTER 5 --- Introduction of the exuperantia genomic constrcuts into the germline of Drosophila by P element-mediated transformation / Chapter 5.1 --- Introduction --- p.136 / Chapter 5.2 --- Materials and methods --- p.138 / Chapter 5.2.1 --- Construction of a genomic construct for production of transgenic flies --- p.138 / Chapter 5.2.2 --- Preparation of double-stranded DNA by ultra-centrifugation --- p.142 / Chapter 5.2.3 --- P-element mediated transformation --- p.143 / Chapter 5.2.3.1 --- Eggs collection --- p.143 / Chapter 5.2.3.2 --- Dechorionating the eggs --- p.143 / Chapter 5.2.3.3 --- Orientating the eggs --- p.144 / Chapter 5.2.3.4 --- Microinjection --- p.145 / Chapter 5.2.4 --- Collecting virgin female Drosophila --- p.146 / Chapter 5.2.5 --- Setup a crossing experiment --- p.146 / Chapter 5.2.6 --- Preparation of total ovaries and testes extracts exu protein from Female and male Drosophila --- p.147 / Chapter 5.2.7 --- Immunohistochemical distribution of exuperantia protein --- p.147 / Chapter 5.3 --- Results --- p.150 / Chapter 5.3.1 --- Insertion of the mutated exu fragments into the Drosophila Transformation vector (pCaSpeR) --- p.150 / Chapter 5.3.2 --- Introduction of the mutated exu gene into the genome of Drosophila by P-element mediated transformation --- p.153 / Chapter 5.3.3 --- Western blot analysis of the exu protein in the exu (ES2.1) transgenic fly --- p.160 / Chapter 5.3.4 --- Immunohistochemical distribution of exu protein in exuES21 mutants --- p.162 / Chapter 5.3.5 --- Rescue test of exuES2.1 trangenic flies --- p.165 / Chapter 5.4 --- Discussion --- p.168 / Chapter CHAPTER 6 --- General Discussion --- p.171 / References --- p.173 / Chapter Appendix I: --- List of reagents --- p.183 / Chapter Appendix II: --- Publication --- p.187

In this experiment it was investigated how scent affect the memory. Encoding information in the same context as retrieving it has been suggested to be beneficial for memory, earlier research has mostly explored how environmental contextual cues affects the memory. In this research the contextual cue was created by a presentation of a scent. The participants were presented with two lists of words, during the encoding of the first list all the participants were presented with a scent, half of the group was directed to forget the first list straight after encoding and the other half to keep remembering the list. For the second list no one was presented with a scent. In the retrieval of both lists half of each group was reinstated with the scent they were presented with at encoding and the other half without the scent (control group). The data were analysed with univariate ANOVAs and significant effects were followed up with independent-samples t-test. The results were that participants that were reinstated with the scent were thought to remember more than the others, however there was only a significant difference in the forget condition with reinstatement, where they remembered less than in the other conditions.

The thermophilic enzyme, Thermus aquaticus (Taq) DNA polymerase, is an essential tool in molecular biology because of its ability to synthesis DNA in vitro and its inherent thermal stability. Taq DNA polymerase is widely used in the polymerase chain reaction (PCR), an essential technique in a broad range of different fields from academic research to clinical diagnostics. The use of PCR-based tests in diagnostic testing is ever increasing; however, many of the samples being tested contain substances that inhibit PCR and prevent target amplification. Many attempts have been made to engineer polymerases not only to increase resistance to overcome the problem of inhibition, but also to enhance other characteristics such as fidelity, processivity and thermostability. Heparin, found in blood samples, and phytate, found in faecal samples, are two examples from a number of known PCR inhibitors. The mode of action of most PCR inhibitors is not well understood, but inhibition is thought to occur by enzyme binding or through the chelation of Mg2+ ions essential for PCR. In this project, a system of directed evolution by compartmentalised self-replication (CSR) was established and successfully employed to screen a mutant library for Taq DNA polymerase variants with enhanced resistance to the inhibitors heparin and phytate. CSR is a recently-established high-throughput method for the creation of novel polymerases, based on a feedback loop whereby polymerase variants replicate their own encoding gene. A mutant library of 106 variants was produced by random mutagenesis error-prone PCR, in which only the polymerase domain of Taq was mutagenised. Firstly, the CSR system was established and tested by performing a screen in the presence of heparin to select for heparin-resistant variants. Characterisation of selected variants revealed that a single round of CSR had produced a Taq variant (P550S, T588S) with a 4-fold increase in heparin resistance. The IC50 was increased from 0.012U/ml heparin to 0.050U/ml heparin. The study with heparin was followed by a phytate screen, in which two rounds of CSR were performed with an initial round of error-prone PCR followed by re-diversification (recombination) of the mutant library using the staggered extension process (StEP). The two rounds of CSR yielded a Taq variant with a 2-fold increase in phytate-resistance compared to the wild-type, with IC50 increased from 360μM phytate to 700μM phytate. The best phytate mutant (P685S, M761V, A814T) was further characterised and it was found that the catalytic activity, thermostability and fidelity of the mutant were comparable to the wildtype enzyme. The position of resistance-conferring mutations of the novel Taq variants evolved in this study provided some evidence for the inhibitors’ predicted modes of action in the case 2 of both phytate and heparin. As phytate’s mode of action is poorly understood, further investigations were performed to elucidate its role in PCR inhibition. A thorough investigation into the importance of relative phytate and Mg2+ levels on PCR was conducted and revealed for the first time convincing evidence that the primary mode of phytatemediated PCR inhibition is by chelation. Further work led to the successful crystallisation of Taq in the presence of phytate, although subsequent X-ray diffraction data to 2.5Å did not reveal phytate bound within the enzyme structure. Site-directed mutagenesis studies were used to probe cross-over between heparin and phytate-conferring mutations. Thus, in addition to providing valuable information for novel Taq variants with a potential application in fecal-based PCR diagnostic tests, this project has begun to provide insight into the fundamental aspects of the mode of action of phytate as a polymerase and PCR inhibitor.

Typescript (photocopy). / Digitized by Kansas Correctional Industries