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The aims of this work were two-fold - to enhance the 'latent functionality' of a silicon centre by expanding the range of functionalisation reactions available to such compounds, and to develop novel silicon-tethered transformations in order to increase the utility of this attractive synthetic strategy. 1. Aryle Silane Oxidation. Building upon the earlier work of Tamao and co-workers, we have developed a mild, functional group-tolerant oxidation of arylsilanes, allowing a wide range of phenols to be readily accessed. One key insight uncovered during this work was the observation that this oxidation could be acheived with sub-stoichiometric quantities of a fluoride promoter, thus allowing several TBS-protected substrates to be oxidised without any concomitant loss of the protecting group. 2. Silicon-Tethering Methodology. In order to utilise our recently acquired expertise in the field of alkoxy arylsilane synthesis, we sought to develop a novel silicon-tethered iron-catalysed biaryl coupling. Unfortunately, despite our considerable efforts, this methodology was found to suffer from reproducibility issues, and thus our attentions subsequently turned to silicon-tethered palladium- and platinum-catalysed processes. These investigations proved to be more fruitful, with the palladium-catalysed methodology affording a small range of silicon-tethered products. Finally, a novel platinum-catalysed hydro-silylation/electrocyclisation cascade was also developed, allowing a substituted arene to be accessed from a dienyne precursor.

The 2 micron plasmid of Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number of 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-encoded partitioning system and an amplification system to ensure its stable, high-copy propagation. The partitioning system consists of the plasmid encoded proteins, Rep1 and Rep2 and a cis-acting partitioning locus STB. The Rep proteins, together with several host factors, assembled at STB couple plasmid segregation to chromosome segregation. A plasmid lacking an active partitioning system is subject to a ‘diffusion barrier’, which causes it to be retained in the mother cell with a strong bias (mother bias). Currently available evidence favors the hitchhiking model for plasmid segregation, in which the tethering of plasmids to chromosome provides the basis for faithful plasmid partitioning. However, direct evidence to support this hypothesis has been difficult to obtain because of the small size of the budding yeast nucleus and the poor resolution of chromosomes in live cells or in chromosome spreads. In this study, we have attempted to verify the hitchhiking model using single copy derivatives of the 2 micron plasmid as reporters. We demonstrate, using two single copy reporters present in the same nucleus, that plasmid association with chromosome spreads is authentic, and is dependent on the partitioning system. By using a strategy that forces all chromosomes to stay in either the mother or the daughter compartment, we show that plasmid segregation can be uncoupled from nuclear envelope segregation. However, plasmid segregation cannot be uncoupled from chromosome segregation under this condition. This tight coupling between plasmid and chromosome segregation is consistent with the hitchhiking model for plasmid segregation. The plasmid partitioning complex is assembled de novo at STB during each cell cycle during the G1-S window. Plasmid replication or cell cycle cues that signal cellular DNA replication appear to trigger this assembly. Furthermore, there is an apparent temporal hierarchy in the association and dissociation of protein factors at STB. When DNA replication is delayed or blocked, the dissociation of factors from STB from the previous portioning cycle and the association of factors for the new partitioning cycle are delayed or blocked, respectively. The precise role of replication in plasmid segregation has not been elucidated. We have addressed this question by blocking either plasmid replication or all cellular DNA replication. We find that replication is not required for plasmid to overcome mother bias. However, replication is critical for the equal segregation of sister plasmid copies. These results provide a refinement of the hitchhiking model by suggesting that sister plasmids tether to sister chromatids in a replication-dependent manner and hitchhike on them during chromosome segregation. Finally, we have attempted to reconstitute the 2 micron plasmid partitioning system in mammalian cells with the goal of exploiting their larger nuclear size and the considerably higher chromosome resolution they provide. In experiments completed so far, we show that Rep2 expressed in COS7 cells localizes to chromosomes, and Rep1 does so in the presence of Rep2. Furthermore, they show co-localization on sister chromatids in a symmetric fashion, implying that plasmids associated with them are likely to follow suit. These observations suggest, by extrapolation, the Rep1-Rep2 assisted association of sister plasmids with sister chromatids in yeast as well, and are consistent with the refined hitchhiking model for plasmid segregation. / text

In many DNA processes, action at a distance is required for signaling across long distances on DNA. These pathways, generally have an initiation site (site 1) that signals an event at a second location (site 2). Such a paradigm is found in processes such as transcription, replication, and DNA repair. To overcome long distances on DNA, proteins can utilize translocation, oligomerization, and DNA looping to bridge the distance between the initiating signal at site 1 and the site of action at site 2. The utilization of these mechanisms for action at a distance is crucial in eukaryotic mismatch repair. In this pathway, MutS homologs scan DNA and recognize mis-paired bases. The MutS protein then recruits the endonuclease MutLα, which nicks the nascent strand of DNA containing a mis-incorporated DNA base. The MutLα-generated nick leads to downstream mis-pair removal through excision by an exonuclease or strand displacement activities of a DNA polymerase working together with a flap endonuclease. Although, previous models have suggested that MutL homolog endonucleases can form oligomeric complexes on DNA, the role of a MutLα oligomeric complex and how it might facilitate action at a distance has been unclear. Here, I present evidence that the mismatch repair MutLα endonuclease is activated by DNA-DNA associations, and it can use this activity to overcome DNA torsional barriers. Using DNA ligation and pull-down experiments, I determined that a MutLα oligomer associates two DNA duplexes and that this activity can stimulate MutLα’s endonuclease function. I also show evidence that MutLα enhances a topoisomerase without nicking the DNA itself. These behaviors of MutLα could localize nicking on DNA near a mismatch and help overcome barriers that could inhibit additional repair proteins from activating MutLα and facilitating efficient DNA repair. The endonuclease activity of MutLα is critical for efficient mismatch repair, but in addition to this activity, MutLα is also an ATPase, although the crosstalk between the two enzymatic functions has been largely unexplored. It has been shown previously that the ATPase activity of MutLα allows the protein to undergo conformational changes and in vivo is necessary for efficient mismatch repair. Mechanistically, how this activity supports MutLα’s functions in the mismatch repair pathway remains unclear. Using DNA binding and photo-crosslinking experiments, I provide evidence that MutLα recognizes and localizes itself to a nick. Additionally, through DNA protection assays and photo-crosslinking I provide evidence of a signaling mechanism initiated at the nick for a MutLα oligomer to undergo its ATP cycle. These data provide insight into how MutLα uses ATP to signal events for mismatch removal. These data also provide a mechanistic explanation for how MutL proteins interact with DNA during mismatch repair and send signals for additional repair processes after the protein nicks DNA that help explain new models for action at a distance. / Chemistry

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In social situations, people who use a powered wheelchair must divide their attention between navigating the chair and conversing with people. As a solution that maintains a good conversation distance between the wheelchair and the accompanying person, a wheelchair control system was introduced to provide automated side-by-side following by wirelessly tethering the wheelchair to the person. This thesis designed, developed, and evaluated a wireless tethering system using ultrasonic sensors. Two ping sensors and three piezoelectric ultrasonic transducers were used to identify the accompanying person and determine their pose. A trajectory algorithm determined the person’s direction of motion and a drive control algorithm determined the wheelchair’s required direction by maintaining a comfortable conversation distance between the person and the wheelchair user. A plug-and-play prototype was developed using commercially available components and the firmware was implemented using an open-source platform. The prototype developed in this thesis was mounted to a Permobil F3 Corpus powered wheelchair with a modified Eightfold Technologies SmartChair Remote, which controlled the wheelchair direction. Results demonstrated that the system can navigate a wheelchair beside an accompanying person and maintain a comfortable conversation distance, which is advantageous for users who require hands-free wheelchair control during social activities.

Polyadenylation is an essential post-transcriptional modification resulting in a mature mRNA in eukaryotes. Three cis-elements the Far Upstream Element (FUE), Near Upstream Element (NUE), and Cleavage Site (CS) - guide the process of cleavage and polyadenylation with the help of multi-subunit protein complexes cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF) along with cleavage factors and poly(A) polymerase. Protein-protein interactions play an important role in the cleavage and polyadenylation process. WD repeat proteins play an important role in protein-protein interactions and have diverse functions in plant system. In the present study WD repeat proteins AtCstF50 and AtFY were studied for their role in polyadenylation process. Mammalian CstF50 is a WD repeat protein that is one of the subunit of CstF that aids in the cleavage step by associating with CPSF and cleavage factors. AtCstF50 was functionally characterized using T-DNA knock-out lines and by identifying the proteins that interacts with it in the process. Results shows that AtCstF50 is essential and was identified as part of CPSF complex, which is different from its mammalian counter part. CPSF was known to interact with Fip (factor interacting with PAP), Poly(A) polymerase and Poly(A) binding protein and AtCstF50 also interacts with these complexes. AtFY is a 3’ end processing factor which contains WD repeats is one of the subunits of the CPSF complex in Arabidopsis polyadenylation machinery. The AtFY interacts with FCA and promotes the alternative polyadenylation and also plays a role in polyadenylation site choice of FCA mRNA. We characterized the FY expression and localization of FY in the cell by fusing with RFP reporter. Results show that FY accumulates in the nucleus while FY with deleted calmodulin binding domain localizes both to the nucleus and outside the nucleus. The individual N-terminal and C-terminal domains also localized in the nucleus suggesting that they are multiple nuclear localization signals in FY and calmodulin might play a direct or indirect role in FY localization. Using a tethering assay we proved that AtFY is able to recruit the 3’ end processing complex in the proximal polyadenylation site choice of the reporter mRNA.

The 2 micron plasmid of the budding yeast Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number around 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-coded partitioning system and an amplification system to exhibit nearly chromosome-like stability in its host. Plasmid behavior under conditions that missegregate chromosomes suggest that the partitioning system couples plasmid segregation to chromosome segregation. However, the mechanism of this coupling has not been elucidated. A plausible model, consistent with current evidence, is the hitchhiking model, in which plasmid-chromosome tethering provides the basis for faithful plasmid partitioning. Testing this hypothesis unequivocally has been difficult, primarily because of the technical limitations posed by the small size of the budding yeast nucleus and poor resolution of chromosomes. As a result, cell biological assays based on fluorescence microscopy have had only modest success in addressing this problem. In the present study, I devised an experimental verification of the hitchhiking model using a single copy derivative of the 2 micron plasmid as a reporter. The rationale was to establish various conditions that force sister chromatids to co-segregate during mitosis in a bias-free manner or with a bias towards the daughter. The segregation patterns of plasmid sisters were followed under these conditions. The sum of the results from this analysis is accommodated by the hitchhiking model, with sister plasmids associating with sister chromatids in a one-to-one fashion. Episomes of mammalian viruses belonging to the gamma-herpes and papilloma families utilize a hitchhiking mechanism to persist in cells during the latent phase of their infection. Two of the viral partitioning systems have been reconstituted in S. cerevisiae. We wished to exploit these systems to characterize the efficiency of non-native chromosome tethering systems in promoting equal segregation of viral plasmids in S. cerevisiae. We find that the 2 micron plasmid partitioning system is considerably superior to the viral systems. This could be due to the higher efficiency of plasmid-chromosome association and/or due to the ability of plasmid sisters to tether to sister chromatids. / text

The exocyst is thought to tether secretory vesicles to specific sites on the plasma membrane. As a member of the exocyst, Sec5 is implicated in cell survival and membrane growth in Drosophila. Little is known of the exocyst function in mammals, with previous work suggesting involvement of exocyst in GTP-dependent exocytosis. Using RNA interference, we stably down-regulated Sec5 in PC12 cells. We found that these knockdown cells exhibit decreased GTP- and Ca2+-dependent exocytosis of dense-core vesicles (DCVs), and contain less proportion of docked vesicles. Expression of Sec6/8 is also slightly reduced in Sec5 knockdown cells. Our results suggest that Sec5 is involved in both GTP- and Ca2+-dependent exocytosis, possibly through the regulation of DCV docking. We also established doxycycline-inducible knockdown system for Sec5 in PC12 cells which may be more appropriate to study development-related proteins. Efforts were also made to re-introduce Sec5 into the Sec5 knockdown cells for rescue purposes.