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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Joseph, Maxim B.
Languages: English
Types: Doctoral thesis
Subjects: QD, QH426
The complexity and size of the higher animal genome and relative scarcity of DNA-binding\ud factors with which to regulate it imply a complex and pleiotropic regulatory system. Cisregulatory\ud modules (CRMs) are vitally important regulators of gene expression in higher\ud animal cells, integrating external and internal information to determine an appropriate\ud response in terms of gene expression by means of direct and indirect interactions with the\ud transcriptional machinery. The interaction space available within systems of multiple CRMs,\ud each containing several sites where one or more factors could be bound is huge. Current\ud methods of investigation involve the removal of individual sites or factors and measuring\ud the resulting effect on gene expression. The effects of investigations of this type may be\ud masked by the functional redundancy present in some of these regulatory systems as a\ud result of their evolutionary development. The investigation of CRM function is limited by a\ud lack of technology to generate and analyse combinatorial mutation libraries of CRMs,\ud where putative transcription factor binding sites are mutated in various combinations to\ud achieve a holistic view of how the factors binding to those sites cooperate to bring about\ud CRM function. The principle work of this thesis is the generation of such a library.\ud This thesis presents the development of microstereolithography as a method for\ud making microfluidic devices, both directly and indirectly. A microfluidic device was\ud fabricated that was used to generate oligonucleotide mixtures necessary to synthesise\ud combinatorial mutants of a CRM sequence from the muscle regulatory factor MyoD. In\ud addition, this thesis presents the development of the optimisation algorithms and assembly\ud processes necessary for successful sequence assembly. Furthermore, it was found that the\ud CRM, in combination with other CRMs, is able to synergistically regulate gene expression in\ud a position and orientation independent manner in three separate contexts. Finally, by\ud testing a small portion of the available combinatorial mutant library it was shown that\ud mutation of individual binding sites within of the CRM is not sufficient to show a significant\ud change in the level of reporter gene expression.
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    • Dissolution Kinetics of Polycrystalline Calcium Sulfate-Based Materials: Influence of Chemical Modification...................................................................................358 Ultrasensitive Detection of Dopamine Using a Carbon Nanotube Network Microfluidic Flow Electrode ...............................................................................359
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