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The regulation of the immune system is complex, with many factors involved in controlling immune cell development, activation and homeostasis. These factors include neuropeptides as well as classic immunoregulatory molecules such as cytokines, chemokines and hormones. Neuropeptides and tachykinins in particular are known to be involved in immune response modulation through a cascade of events including vasodilation, plasma extravasation, the activation of immune cells, the secretion of pro-inflammatory cytokines and the recruitment of more immune cells. Furthermore, there is growing evidence that tachykinins play a role in hematopoiesis with Substance P as the proposed effector molecule. In 2000, our lab discovered a new tachykinin with remarkable structural similarity to SP and SP-like neurokinin receptor binding affinity. This molecule was designated Hemokinin-1 due to its expression in hematopoietic cells and its function in B cell development. Further gene expression analysis of HK-1 reveals a wide expression pattern although HK-1 transcripts are found predominantly in peripheral tissues while SP is mainly expressed in neuronal tissue. Based on this differential expression pattern, it has been suggested that HK-1 may act as the peripheral tachykinin and may have functions distinct from SP. In addition, given the crossreactivity of the SP antibodies to HK-1, it is important to determine whether HK-1 is the actual mediator of some functions previously attributed to SP. In this thesis, we examine the differential expression pattern of HK-1 to determine molecular mechanisms of regulation of HK-1 transcription and ultimately provide clues to its function in the immune system. In our analysis of the HK-1 promoter, we found a major difference in the basic transcriptional control of HK-1 and SP at the level of transcription initiation and identified several transcription factors including CREB and NFκB involved in regulating TAC4 gene expression in immune cells. Data presented in this thesis also reveal that the HK-1 gene is a direct target of Early B-cell Factor, a transcription factor known to activate B cell-specific genes as well as genes involved in adipogenesis and neuronal development. Our results show EBF regulates HK-1 gene expression in differentiating B cells as well as a monocytic cell line. Our data indicate EBF may also be responsible for the high levels of HK-1 transcript in the olfactory epithelium, suggesting a bridge between the nervous system and the immune system.

In today's changing society, companies need to work with continuous change and improvement to evolve with the market. It is therefore important for managers to learn how to manage teams because they are expected to streamline selected products or processes in the business. Employees and teams need to develop in line with the changing environment in this process feedback is a tool and an important pillar. Leaders must learn to manage and transform feedback into something useful. The purpose of this study was to create an understanding of how systems-based feedback in the form of employee surveys can be a support for the leader in team development. We wanted to identify relevant behaviors and actions of the leader that may increase the work with feedback. Further examined how the NPS method can assist in monitoring system-based feedback. In our theoretical framework we outline the theories that underpin the study, which lands in a self-constructed analysis model containing four themes; feedback process, the group becomes team, lead the team and employee input. To gain a deeper understanding of the different themes we chose to do a qualitative study in which we later interviewed six respondents from two different companies. Respondents gave us different insights into the use of feedback. This eventually resulted in six aspects that relate to behaviors and practices of how a leader can use and utilize system-based feedback, in the form of employee surveys.

In transgenic research the precise control of transgene expression is crucial in order to obtain transformed organisms with expected desirable traits. A broad range of transgenic plants use the constitutive cauliflower mosaic virus (CaMV) 35S promoter to drive expression of selectable marker genes. Due to its strong enhancer function, this promoter can disturb the specificity of nearby eukaryotic promoters. When inserted immediately downstream of the 35S promoter in transformation vectors, special DNA sequences called insulators can prevent the influence of the CaMV35S promoter/enhancer on adjacent tissue-specific promoters for the transgene. Insulators occur naturally in organisms such as yeasts and animals but few insulators have been found in plants. Therefore, the goal of this study is to identify DNA sequences with insulator activity in Arabidopsis thaliana. A random oligonucleotide library was designed as an initial step to obtain potential insulators capable of blocking enhancer-promoter interactions in transgenic plants. Fragments from this library with insulator activity were identified and re-cloned into pB31, in order to confirm their activity. To date, one insulator sequence (CLO I-3) has been identified as likely possessing enhancer-blocking activity. Also, two other oligonucleotide sequences (CLO II-10 and CLO III-78) may possess insulator activity but more sampling is needed to confirm their activity. Further studies are needed to validate the function of plant insulator(s) and characterize their associated proteins.

by Tam Fung-ping. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 254-264). / Chapter I. --- Title page --- p.I / Chapter II. --- Abstract --- p.II / Chapter III. --- Acknowlegements --- p.III / Chapter IV. --- Table of contents --- p.IV / Chapter V. --- List of tables --- p.V / Chapter VI. --- List of figures --- p.VI / Chapter VII. --- Abbreviations --- p.VII / Chapter Chapter 1. --- Literature Reviews / Chapter 1.1 --- Modes of energy generation in facultative bacteria --- p.1 / Chapter 1.1.1 --- Difference in energy generation mechanism between respiratory and fermentative pathways --- p.2 / Chapter 1.1.2 --- Difference in carbon metabolism during anaerobiosis --- p.6 / Chapter 1.2 --- Repression and derepression of genes during anaerobiosis --- p.8 / Chapter 1.3 --- Global regulatory network for respiratory control --- p.8 / Chapter 1.3.1 --- Fnr-regulated gene expression --- p.10 / Chapter 1.3.2 --- NarL-regulated gene expression --- p.11 / Chapter 1.3.3 --- Crp-regulated gene expression --- p.12 / Chapter 1.3.4 --- ArcA-regulated gene expression --- p.13 / Chapter 1.3.5 --- Overlapping control of gene expression --- p.14 / Chapter 1.3.6 --- Regulatory mechanism of respiratory control --- p.16 / Chapter 1.4 --- Other regulatory systems in respiratory control --- p.19 / Chapter 1.5 --- The puzzle of regulatory network in anaerobiosis --- p.22 / Chapter 1.6 --- ArcA-ArcB system in Escherichia coli --- p.24 / Chapter 1.6.1 --- Arc A and ArcB for aerobic respiratory control --- p.24 / Chapter 1.6.2 --- arcA/dye/msp/fex/sfrA/cpxC gene are on identical genetic locus --- p.26 / Chapter 1.6.3 --- Arc function and Sfr function of Arc A protein are separately regulated --- p.28 / Chapter 1.6.4 --- ArcB-ArcA as sensor regulator in two component system for respiratory control --- p.29 / Chapter 1.7 --- Objectives and strategies of present study --- p.37 / Chapter Chapter 2. --- Materials / Chapter 2.1 --- Bacterial strains --- p.41 / Chapter 2.2 --- Culture mediums --- p.44 / Chapter 2.3 --- "Buffers, chemicals and antibiotics" --- p.46 / Chapter 2.4 --- DNA primers --- p.53 / Chapter Chapter 3. --- Primer extension analysis for locating the transcriptional start point of anaerobic inducible arcA in pFS --- p.34 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Methods --- p.57 / Chapter 3.2.1 --- Preparation of total RNA --- p.59 / Chapter 3.2.2 --- Formaldeyde agarose gel electrophoresis of RNA --- p.60 / Chapter 3.2.3 --- Spectrometric estimation of RNA --- p.61 / Chapter 3.2.4 --- End-labelling of arcAusp primer with 32P --- p.62 / Chapter 3.2.5 --- Precipitation of arcAusp primer with samples RNA --- p.63 / Chapter 3.2.6 --- Primer extension reaction --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Preparation of RNA --- p.67 / Chapter 3.3.2 --- Determination of transcription start site by primer extension --- p.67 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Selective activations of aerobic and anaerobic transcripts in response to oxygen level --- p.76 / Chapter 3.4.2 --- The arcA promoter is a sigma-70 dependent promoter --- p.77 / Chapter 3.4.3 --- Experimental design --- p.77 / Chapter Chapter 4. --- In vitro chemical mutagensis for finding some important regulatory elements of arcA in pFS --- p.34 / Chapter 4.1 --- Introduction / Chapter 4.2 --- Methods --- p.84 / Chapter 4.2.1 --- Large scale preparation of pFS34 plasmid --- p.84 / Chapter 4.2.2 --- PCR-mediated chemical mutagenesis of pFS34 --- p.86 / Chapter 4.2.3 --- Restriction enzyme digestion of PCR-amplified arcA insert after phenol extraction --- p.90 / Chapter 4.2.4 --- Large scale preparation of vector pFZYl and restriction enzyme digestion --- p.91 / Chapter 4.2.5 --- Ligation of EcoRI-SalI digested pFS34 fragment and vector pFZYl --- p.91 / Chapter 4.2.6 --- Preparation of electrotcompetent cell Salmonella typhymurium JR502 and electro-transformation --- p.92 / Chapter 4.2.7 --- Screening of transformed clones by LB-amp50-xgal plates --- p.93 / Chapter 4.2.8 --- Screening of recombinants colonies by Polymerase chain reaction (PCR) --- p.94 / Chapter 4.2.9 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.96 / Chapter 4.2.10 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.98 / Chapter 4.2.11 --- Sequencing of mutated clones --- p.101 / Chapter 4.2.11.1 --- Recombinant M13 single-stranded sequencing of the mutated clones --- p.101 / Chapter 4.2.11.2 --- pUC18 double-stranded DNA sequencing of mutated clones --- p.105 / Chapter 4.3 --- Results --- p.108 / Chapter 4.3. --- l PCR-mediated chemical mutagenesis of pFS34 --- p.108 / Chapter 4.3.2 --- Screening of transformed clones by LB-amp50-xgal plate --- p.112 / Chapter 4.3.3 --- Screening of recombinants colonies by polymerase chain reaction (PCR) --- p.112 / Chapter 4.3.4 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.114 / Chapter 4.3.5 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.117 / Chapter 4.3.6 --- Sequencing of mutated clones --- p.123 / Chapter 4.4 --- Discussions --- p.135 / Chapter 4.4.1 --- The possible mechanisms in anaerobic transcription --- p.135 / Chapter 4.4.2 --- The possible mechanisms in aerobic transcription --- p.143 / Chapter 4.4.3 --- Experimental design --- p.146 / Chapter Chapter 5 --- Investigation of the effect of integration host factor (IHF) and autoregulation on the expression of pFS34 / Chapter 5.1 --- Introduction --- p.152 / Chapter 5.2 --- Methods --- p.154 / Chapter 5.2.1 --- Construction of Escherichia coli mutant --- p.155 / Chapter 5.2.2 --- PCR check of mutant for the presence of pFS34 and pFZYl plasmid --- p.157 / Chapter 5.2.3 --- β-galactosidase assay of aerobic and anaerobic activities change of pFS34 --- p.157 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Effect of integration factor (IHF) on pFS34 --- p.158 / Chapter 5.3.1.1 --- PCR analysis of E. coli. himA and himD mutant for the presence of pFS34 and pFZYl plasmid --- p.158 / Chapter 5.3.1.2 --- β-galatosidase assay of aerobic and anaerobic activities of pFS34 in E. coli. himA and himD mutant --- p.158 / Chapter 5.3.2 --- Autoregultion on expression of pFS34 --- p.162 / Chapter 5.3.2.1 --- PCR analysis of E. coli. arcA mutant for the presence of pFS34 plasmid --- p.162 / Chapter 5.3.2.2 --- β-galctosidase assay of aerobic and anaerobic activities of pFS34 (arcA-lacZ) in E. coli. arcA mutant --- p.162 / Chapter 5.4 --- Discussions --- p.167 / Chapter 5.4.1 --- Effect of IHF on aerobic and anaerobic expression of arcA --- p.167 / Chapter 5.4.1.1 --- Possible regulatory mechanism of IHF on aerobic transcription --- p.167 / Chapter 5.4.1.2 --- Possible regulatory mechanism of IHF on anaerobic transcription --- p.170 / Chapter 5.4.1.3 --- Affinity binding of IHF depends on topological state of arcA --- p.172 / Chapter 5.4.1.4 --- Possible role of IHF in global regulation of anaerobiosis --- p.173 / Chapter 5.4.1.5 --- Experimental design --- p.174 / Chapter 5.4.2 --- Autoregulatory expression of arcA in pFS34 --- p.176 / Chapter Chapter 6. --- PCR walking of arcA from Salmonella typhimurium LT2 / Chapter 6.1 --- Introduction --- p.177 / Chapter 6.2 --- Methods --- p.186 / Chapter 6.2.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.186 / Chapter 6.2.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.187 / Chapter 6.2.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R & delC-34R) --- p.188 / Chapter 6.2.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.189 / Chapter 6.2.5 --- High stringency PCR amplification with arcAusp2 and delC-23 primer --- p.190 / Chapter 6.2.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer" --- p.191 / Chapter 6.2.7 --- High stringency PCR amplification with arcAusp2 only and Sau3A restriction enzyme digestion of PCR products --- p.192 / Chapter 6.2.8 --- Cloning of PCR walking products into pUC18 and heat shock transforming into E.coli. JM83 --- p.193 / Chapter 6.2.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.194 / Chapter 6.2.10 --- Dideoxy sequencing of PCR walking arcA fragments in pUC18 --- p.194 / Chapter 6.2.11 --- Subcloning of arcA fragment into pFZYl and PCR analysis for insertion of one insert with proper orientation --- p.195 / Chapter 6.2.12 --- arcA-galactosiadase assay of PCR walking arcA fragment-lacZ fusion --- p.196 / Chapter 6.3 --- Results --- p.198 / Chapter 6.3.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.198 / Chapter 6.3.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.198 / Chapter 6.3.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R and delC-34R) --- p.200 / Chapter 6.3.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.200 / Chapter 6.3.5 --- High stringency PCR amplification with arcAusp2 、 primer and delC-23 prime --- p.203 / Chapter 6.3.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer to check for flanking ends of bands" --- p.205 / Chapter 6.3.7 --- High stringency PCR amplification with arcAusp2 primer and Sau3A restriction enzyme digestion of PCR products --- p.207 / Chapter 6.3.8 --- Cloning of PCR walking products into pUC18 and heat-shock transforming into E. coli. JM83 --- p.210 / Chapter 6.3.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.210 / Chapter 6.3.10 --- Dideoxy sequencing of arc A PCR walking fragment: :pUC18 --- p.210 / Chapter 6.3.11 --- Subcloning of arcA fragment into pFZYl and PCR check for right insertion of single insert with proper orientation --- p.226 / Chapter 6.3.12 --- β-galactosidase assay --- p.232 / Chapter 6.4 --- Discussions --- p.227 / Chapter 6.4.1 --- PCR based gene walking strategy --- p.227 / Chapter 6.4.2 --- Confirmation of cloned arcA gene in pFS34 was a geniune arcA gene of S. typhimurium --- p.240 / Chapter 6.4.3 --- Promoter activity of further upstream arcA clones - AU87::pFZYl --- p.241 / Chapter Chapter 7. --- Overall Discussion --- p.244 / Chapter 7.1 --- Summary --- p.244 / Chapter 7.2 --- Proposed Model of regulation of arcA in Salmonella typhimurium --- p.249 / Chapter 7.3 --- Further Studies --- p.251 / References --- p.254

by Cheung, Man Wai William. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 113-123). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.ii / DEDICATION --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- General Introduction --- p.1 / Chapter 1.2. --- Purpose of Study --- p.3 / Chapter 2. --- Literature Review --- p.7 / Chapter 2.1. --- Central Pathways of Aerobic and Anaerobic Carbon Catabolism --- p.7 / Chapter 2.2. --- Global Regulation of Gene Expression by Oxygen --- p.10 / Chapter 2.2.1. --- Two approaches for the studies --- p.10 / Chapter 2.2.2. --- FNR regulation --- p.12 / Chapter 2.2.3. --- ArcAB regulation --- p.19 / Chapter 2.2.3.1. --- arcA --- p.19 / Chapter 2.2.3.2. --- arcB --- p.20 / Chapter 2.2.3.3. --- A member of the Two- Components regulatory systems --- p.21 / Chapter 2.3. --- Molecular Analysis of Promoters --- p.26 / Chapter 2.3.1. --- S1 mapping --- p.29 / Chapter 2.3.2. --- Primer extension --- p.29 / Chapter 2.3.3. --- DNaseI footprinting --- p.30 / Chapter 2.3.4. --- Mutational analysis of promoters --- p.32 / Chapter 3. --- Materials and Methods --- p.35 / Chapter 3.1. --- Bacterial strains and Plasmids --- p.35 / Chapter 3.2. --- Media --- p.35 / Chapter 3.3. --- Solutions --- p.38 / Chapter 3.4. --- Small Scale Preparation of Plasmid DNA --- p.40 / Chapter 3.5. --- Large Scale Preparation of Plasmid DNA --- p.41 / Chapter 3.5.1. --- Growth of bacterial culture --- p.41 / Chapter 3.5.2. --- Lysis by alkali --- p.43 / Chapter 3.5.3. --- Purification of closed circular DNA by cesium chloride gradient equilibrium centrifugation --- p.44 / Chapter 3.5.4. --- Digestion of DNA with restriction endonucleases --- p.45 / Chapter 3.6. --- Analysis of DNA Samples with Agarose Gel Electrophoresis --- p.45 / Chapter 3.7. --- Cloning of DNA Fragments from Nest-deleted M13mpl8 Clones to pFZYl --- p.47 / Chapter 3.8. --- Introduction of Plasmids into Cells --- p.48 / Chapter 3.8.1. --- Heat shock transformation --- p.48 / Chapter 3.8.1.1. --- Preparation of competent cells (I) --- p.48 / Chapter 3.8.1.2. --- Preparation of competent cells (II) --- p.49 / Chapter 3.8.2. --- High efficiency transformation by electroporation --- p.50 / Chapter 3.8.2.1. --- Preparation of electro- competent cells --- p.50 / Chapter 3.8.2.2. --- Electro-transformation --- p.51 / Chapter 3.9. --- DNA Sequencing by Chain Termination Method --- p.51 / Chapter 3.9.1. --- Preparation of single-stranded M13 templates for sequencing reaction --- p.51 / Chapter 3.9.2. --- Sequencing reactions using single- stranded templates --- p.53 / Chapter 3.9.3. --- Preparation of polyacrylamide gel for sequencing --- p.54 / Chapter 3.9.4. --- Electrophoresis of the DNA samples --- p.55 / Chapter 3.10. --- Construction of Nested Clones by Exonuclease III Unidirectional Deletions --- p.55 / Chapter 3.10.1. --- Unidirectional nested deletion of M13mpl8 clones --- p.55 / Chapter 3.10.2. --- Screening of nested clones by Direct gel electrophoresis --- p.56 / Chapter 3.10.3. --- Screening of nested clones of M13mpl8 and pFZYl by Polymerase Chain Reaction --- p.57 / Chapter 3.11. --- β-galactosidase Assay --- p.59 / Chapter 3.12. --- Primer Extension --- p.60 / Chapter 3.12.1. --- Preparation of total RNA from Gram- negative bacteria --- p.60 / Chapter 3.12.2. --- Labelling the 5' end of the oligonucleotides --- p.61 / Chapter 3.12.3. --- Hybridization and primer extension --- p.62 / Chapter 4. --- Result --- p.63 / Chapter 4.1. --- Subcloning of arcA promoter into M13mpl8/19 --- p.63 / Chapter 4.2. --- Sequencing of p34一18i and p3419i using M13 Sequencing primers (-47) and ArcA-cds Primers --- p.63 / Chapter 4.3. --- Unidirectional Nested Deletion of p3418i using Exonuclease III --- p.65 / Chapter 4.3.1. --- Large scale preparation of p3A18i DNA for Exonuclease III unidirectional nested deletion --- p.65 / Chapter 4.3.2. --- Construction of 3' and 5' overhangs --- p.65 / Chapter 4.3.3. --- Exonuclease III digestion --- p.67 / Chapter 4.3.4. --- Repairing of the 3' and 5' overhangs to generate blunt ends --- p.67 / Chapter 4.3.5. --- Blunt-end ligation of the nested deletion M13mpl8 subclones p3418i --- p.67 / Chapter 4.3.6. --- Transformation --- p.69 / Chapter 4.3.7. --- Screening of nest-deleted p3418i clones by Direct Gel --- p.71 / Chapter 4.3.8. --- Screening of nested deletion p3418i clones by PCR Screening --- p.73 / Chapter 4.3.9. --- Sequencing of the nested deletion p3418i clones --- p.76 / Chapter 4.4. --- Cloning of Nested Deletion DNA Fragments from M13mpl8 into pFZYl --- p.80 / Chapter 4.4.1. --- Screening of pFZYl clones using PCR Screening --- p.80 / Chapter 4.5. --- Expression of Nest-Deleted arcA Promoter Clones in E. coli MC1061-5 --- p.87 / Chapter 4.6. --- Expression of Nest-Deleted arcA Promoter Clones in S. typhimurium JR501 --- p.89 / Chapter 4.7. --- Primer Extension --- p.89 / Chapter 5. --- Discussion --- p.93 / Chapter 5.1. --- Sequencing of arcA Promoter --- p.93 / Chapter 5.2. --- Unidirectional Nested Deletion of p3A18i using Exonuclease III --- p.94 / Chapter 5.3. --- Screening of Nest-deletion p3418i Subclones --- p.95 / Chapter 5.4 --- Cloning of Nest-deleted DNA Fragments from M13mpl8 Subclones into pFZYl --- p.99 / Chapter 5.5. --- Screening of Nest-deleted pFZYl Subclones of p3418i --- p.101 / Chapter 5.6. --- The Effect of 5' Unidirectional Nested Deletion on the Expression of the Cloned arcA promoter in E. coli M1061-5 and S typhimurium JR501 --- p.102 / Chapter 5.7. --- Primer Extension --- p.102 / Chapter 5.8. --- Sequence Analysis of the Cloned arcA Promoter --- p.104 / Chapter 6. --- Conclusion and Further Studies --- p.111 / Chapter 7. --- Reference Cited --- p.113

Transcriptional regulation is a determinant of stability of recombinant protein production in CHO cells. Fundamental studies of recombinant gene transcription in relation to chromatin environment and promoter regulation are important for CHO cell line development and selection. This study has developed a methodology based on a cell/vector system to study recombinant transcription and expression stability of different promoters and/or proteins in the similar genomic environment. The CHO-FRT mini-pools developed in this project were mini-pools of CHO-S cell lines containing Flp Recombination Target (FRT) sites with ß-galactosidase gene, under the influence of a SV40 promoter. Continuous culture of these mini-pools for 8 weeks using a robotic system demonstrated that 20% of the mini-pools studied revealed an unstable profile (with 30% loss of protein expression). Two of these mini-pools with different characteristics, CHO-FRT 1 (low producer/unstable) and CHO-FRT 108 (high producer/stable), were selected to be used on the study of influence of SV40 and CMV promoters in long-term recombinant expression. Genes encoding fluorescent proteins were integrated in a site-directed manner under the influence of SV40 or CMV promoters. A sub-clonal population of the top 10% yellow fluorescent protein (YFP) expressing cells of each mini-pool/promoter combination was selected by cell sorting and cultured for 4 weeks. During this period protein expression was monitored by flow cytometry and compared between both promoters. The results revealed that both SV40 and CMV promoters had an unstable expression with different degrees of instability and long-term expressing behaviours. For CMV, instability was considerably high displaying a long-term logarithmic loss of 50-80% of productivity while for SV40 the loss of productivity observed was only 40-45% with a linear behaviour during long-term culture. The vector system generated contained an MS2-RNA tag sequence cloned 3'- of the recombinant gene to track the recombinant mRNA by using the MS2/MCP-GFP system. This study showed the development of a protocol to measure the transcriptional output of recombinant promoters in CHO cells. The results showed background signal in CHO cells that requires further optimisation studies to allow the direct live cell image quantification of the transcriptional activity of recombinant promoters. Although not yet optimised, the successful combination of site-directed integration with recombinant mRNA tagging method has the potential to become a valuable tool to study the mechanisms of transcriptional activity and stability of transcription driven by different promoters in CHO cells.

Enhancers are non-coding DNA sequences which are able to activate the expression of a gene in a specific tissue manner and at a precise stage during embryonic development. First identified almost 40 years ago, our growing understanding of enhancers has transformed the concept of gene regulation to recognise the key role of these sequences in the expression of many genes. Moreover, the identification of human diseases caused by genetic variation in non-coding enhancer elements highlights the importance of characterising enhancers in order to understand human disease. However, enhancers are often located far from the promoter they influence and the mechanisms through which enhancers govern gene expression remain unclear. The most widely accepted model for the action of distal enhancers involves the formation of a chromatin loop, in which the enhancer and promoter physically interact at the loop base. The kinetics or molecular basis for the formation of enhancer/promoter loops is unknown and it remains unclear whether this mechanism of enhancer communication is universal, or indeed whether it is the most pervasive. The aim of my PhD is to investigate further the mechanism of action of distal enhancers in the regulation of developmental genes. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells in order to see which Shh enhancer is active in neural progenitor cells (NPCs), I report the identification of a novel long-range enhancer for Shh - Shh-Brain- Enhancer-6 (SBE6) – that is located 100kb upstream of Shh and that is required for the proper induction of Shh expression during a neural differentiation programme. SBE6 enhances Shh expression during the differentiation of neural progenitor cells (NPCs) and is active in the brain of developing zebrafish and mouse embryos. Next, using a super-resolution 3D-FISH based approach to study the enhancer-driven activation of the Sonic hedgehog gene (Shh) I have identified a novel mechanism of longrange enhancer regulation that is incompatible with the looping model. Instead, gene activation is associated with an increase in nuclear distance between Shh and Shh-Brain- Enhancers. Using a synthetic biology approach I have determined that the chromatin unfolding is regulated specifically by the Shh-Brain-Enhancer and is mediated by the recruitment of transcription factor SIX3 and Poly (ADP-Ribose) Polymerase 1. Chromatin decondensation upon gene activation has been observed previously in Drosophila polytene chromosomes. I suggest an analogous decompaction is driven by Shh-Brain-Enhancer to promote the activation of Shh in mouse neural progenitor cells. This ‘chromatin unfolding’ model represents a new mechanism of long-range enhancer-promoter communication in addition to the looping and tracking models.

Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.

In eukaryotes, the organization of DNA into chromatin is a primary determinant of gene expression. Positioned nucleosomes in promoter regions are frequently found to regulate gene expression by obstructing the accessibility of cis-regulatory elements in DNA to trans-factors. This dissertation focuses on the chromatin structure and remodeling program at the S. cerevisiae PHO5 promoter, extending the use of DNA methyltransferases as in vivo probes of chromatin structure. Our studies address the diversity of histone-DNA interactions in vivo by examining nucleosome conformational stabilities at the PHO5 promoter. We present high-resolution chromatin structural mapping of the promoter, required to relate in vivo site accessibility to nucleosome stability and show that the PHO5 promoter nucleosomes have different accessibilities. We show a correlation between DNA curvature and nucleosome positioning, which is consistent with the observed differences in accessibility/stability. Kinetic analyses of the chromatin remodeling program at PHO5 show that nucleosomes proximal to the enhancer are disrupted preferentially and prior to those more distal, demonstrating bidirectional and finite propagation of chromatin remodeling from bound activators and providing a novel mechanism by which transactivation at a distance occurs.