Interactive visualisation of oligomer frequency in DNA

Since 1990, bioinformaticians have been exploring applications of the Chaos Game Representation (CGR) for visualisation, statistical characterisation and comparison of DNA sequences. We focus on the development of a new computational algorithm and description of new software tool that enables CGR visualisation of frequencies of K-mers (oligomers) in a flexible way such that it is possible to visualise the whole genome or any of its parts (like genes), and parallel comparison of several sequences, all in real time. User can interactively specify the size and position of visualised region of the DNA sequence, zoom in or out, and change parameters of visualisation. The tool has been written in JAVATM language and is freely available to public

The role of the C-terminal extension of αB-crystallin upon structure and function and the relationship with disease

The eye lens protein αB-crystallin plays an important role in maintaining the refractive index of the lens, however is also found in many non-lenticular tissues suggest that it has general cellular functions over and above its role in light refraction. The role of the C-terminal extension of αB -cryStellin upon solubility, structure and function and the relationship with disease has not been investigated. In this study, the systematic analysis of the role of the C-terminal extension of αB -crystallin aimed to investigate the hypothesis that the C-terminal region in αB -crystallm is important for solubility, structure and function of the protein. This study also aimed to find a link between the role of the C-terminal and the symptoms of disease, thus providing a potential explanation of whether the three congenital mutations (450delA, Q151X and 464delCT), which alter the C-terminal extension of αB -crystallin, cause the various diseases via the loss of chaperone function or perhaps by a different mechanism. Further more this study aimed to elucidate the mechanism of phenotypic heterogeneity associated with these αB -crystallin mutations.I compared the three C-terminal αB -crystallin mutants (450delA, Q151X and 464delCT) to a series of C-terminal truncations (E164X, E165X, K174X and A171X) and found that the C-terminal extension was essential for oligomerisation but not chaperone function, infact the removal of the entire C-terminal extension actually enhanced chaperone activity, however significantly destabilized αB -crystallin causing it to self-aggregate. This instability was supported by refolding analysis, where the 450delA and 464delCT mutants could only be refolded and assayed as a complex with wild type αB -crystallin, however the Q151X αB -crystallin could be refolded alone. From these studies, I conclude that all three disease-causing mutations (450delA, 464delCT and Q151X) in the C-terminal extension destabilise αB -crystฝlin and increase its tendency to self-aggregate. I propose that it is this, rather than a catastrophic loss of chaperone activity, that is a major factor in disease development

Understanding the Molecular Mechanism of Single-Strand Annealing Homologous DNA Recombination in Viruses, by Cryo-Electron Microscopy

The single-strand annealing homologous recombination (SSA) is one of the dsDNA break repair pathways, and albeit its importance from bacteria to bacteriophages, its molecular function is still unknown. The SSA reaction is catalysed by the enzyme complexes known as Exonuclease Annealase Two-component Recombinase (EATRs). The RecT and ORF6 proteins are single-stranded DNA-binding and annealing proteins expressed in E. coli and Kaposi’s sarcoma-associated herpesvirus (KSHV), respectively. RecT has already been shown to catalyse the SSA reaction. Although ORF6 has been shown to bind to ssDNA, further experimental evidence is needed to solidify its annealase activity. Since structure can dictate the function, this thesis aimed to determine the structure of the annealases RecT and ORF6 using a state-in-art cryo-electron microscopy technique. Furthermore, the shadow-casting EM technique has been established by optimising it for the equipment available at UOW, which is helpful for imaging the substrate DNA intermediates and the nucleoprotein complexes formed during SSA to better understand the molecular mechanistic details of this reaction. This thesis includes the details about RecT and ORF6 proteins’ cloning, expression, and purification, which were further optimised for purity and homogeneity for cryo-electron microscopy with the help of negative staining electron microscopy (NSEM). Additionally, based on several NSEM analyses, the C-terminal His-tag containing RecT (RecTCH) oligomerisation on ssDNA was studied, and a general mechanism of its oligomerisation is described. Unfortunately, during the RecTCH protein’s cryo-EM sample optimisation, the LiRecT structure was published by another group. Therefore, work on that project was ceased at that point. Several novel findings on ORF6 are reported in this thesis. Primarily, the concentration of the purified protein was increased 3 times more than the reports in the literature. Based on the NSEM and preliminary cryo-EM map of ORF6, it is shown that the ORF6 structure overall resembles the HSV1-ICP8 protein. Further, based on the steady-state and time-resolved fluorescence resonance energy transfer (FRET) experiments, a model for the ORF6 annealing mechanism is suggested. Towards generating a high-resolution structure, ORF6 monomers and filaments were optimised and imaged by using cryo-EM. Processing a data set obtained from a monomeric ORF6 sample showed the presence of conformational heterogeneity in the particles, which was expected as the ORF6 AlphaFold model shows that the N-terminal and C-terminal domains are connected by an 18 amino acids long loop, allowing C-terminal domain to be relatively flexible to move around. Processing of another data set obtained from a sample containing ORF6 filaments generated 2-dimensional averages that look promising for generating a high-resolution structure. This thesis also shows the details related to the installation and optimisation of the shadowing technique using a modern material, graphene oxide (GO), as a support film. This technique involves optimising both sample preparation and instrumentation for metal evaporation and deposition. For sample preparation, GO was deposited on cryo-EM holey grids, on which the sample was mounted. For instrumentation optimisation, a DENTON brand evaporator was used. The grid stage was re-engineered using AutoCAD to achieve the finest metal evaporation, and parameters such as amperage, vacuum, metal thickness, and angles were optimised. The optimised parameters were used to shadow-cast different lengths of DNA and their complexes with proteins, and good contrast images were acquired for qualitative and quantitative analyses. Overall, this thesis presents two main novel findings. First, RecTCH monomers oligomerise into an open ring-shaped structure, which stacks together to generate short filaments. Second, to anneal two complementary ssDNA strands, ORF6 first forms filaments with both ssDNA, which then come in contact with each other rapidly to anneal the complementary strands. Once the annealing finishes, the annealed dsDNA is released from the filaments as the filaments fall apart into monomers. We also found that ORF6 monomers oligomerise to form the helical and non-helical filaments in the presence of DTT+Mg2+ and DTT-containing buffer, respectively

Nucleases and histone acetyltransferases in DNA repair and immune diversity

DNA repair mechanisms are essential for genome maintenance and adaptive immunity. A careful balance must be achieved whereby highly accurate and efficient canonical repair protects the genome from accumulating mutations that lead to aging and cancer, and yet mutation and error-prone non-canonical repair is required for generating immune diversity. Immune diversity is achieved within a tightly regulated environment in which mutator proteins are directed to the antibody locus to introduce a swathe of DNA damage. This produces high affinity antibodies that recognise an infinite number of invading pathogens. This process of secondary antibody diversification is dependent on both active transcription and DNA repair. Downstream of histone signalling, DNA repair nucleases are recruited to remove the damaged bases. The structure of damaged regions in the DNA can have very different conformations depending on whether the source of the damage is endogenous or exogenous. Specific DNA nucleases recognise particular DNA substrates and generate DNA intermediates that are repaired in conjunction with polymerases and ligases. Despite their multitude and importance to DNA repair, very few nucleases have been characterised, while the activities of some studied nucleases remain controversial. Conventional techniques for studying DNA nucleases have several disadvantages; they are hazardous, laborious, time-consuming, and capture nuclease activity in a discontinuous manner. Recognising a need for a safer, faster alternative, a fluorescence-based method has been developed for the study of DNA nucleases, nickases and polymerases. Key histone modifications that are known to orchestrate canonical DNA repair have since been discovered to regulate non-canonical repair at the antibody locus. The Kat5 histone lysine acetyltransferase functions highly upstream of DNA repair and promotes active transcription, yet a role for Kat5 in secondary antibody diversification has not yet been established. Using chemical inhibitors to prevent the catalytic activities of Kat5, and the genetic method of an inducible degron system for rapid and reversible downregulation of Kat5, a role for Kat5 in secondary antibody diversification is recognised, and the research contributes to our current understanding of the DNA repair signal transduction pathway

The polymorphic nature of amyloid assembly: Exploring fibril morphology and the structural relationship towards mechanical stability

The polymorphic nature of amyloid fibrils is important in the understanding of structural based relationships, such as a morphology influence on cytotoxicity and disease progression. The work reported here uses Atomic force microscopy (AFM) to enhance the understanding of fibril morphology in addition to the relationship between structure and stability towards breakage. A novel quantitative cluster analysis was developed here to identify the vast range of fibril morphologies present within a population. Using fibrils formed from three peptide sequences identified by the WALTZ algorithm, we have characterised the polymorphism displayed by each fibril population and provided structural models to predict the likely filament arrangements accessible to each. The range of fibril polymorphism also conveys mechanical differences, defined here by persistence length values for each respective population. These mechanical differences subsequently affect fibrils stability towards breakage, quantified here using AFM and subsequent image analysis. Additionally, using AFM, a structural comparison was performed between Sup35NM amyloid fibrils formed in vitro and those formed in situ using a synthetic biology approach with the Curli-dependent amyloid generator (C-DAG) in E. Coli. Structural similarities between fibrils formed using this system and those formed in vitro is of great value given the importance of a sequence-structure relationship. The work in this thesis expands on possible fibril morphologies and the related mechanical properties, which has implications in the understanding of disease enhancing structural motifs and the utilisation of amyloid fibrils in a biotechnology role

Super-resolution mapping of receptor engagement during HIV entry

The plasma membrane (PM) serves as a major interface between the cell and extracellular stimuli. Studies indicate that the spatial organisation and dynamics of receptors correlate with the regulation of cellular responses. However, the nanoscale spatial organisation of specific receptor molecules on the surface of cells is not well understood primarily because these spatial events are beyond the resolving power of available tools. With the development in super-resolution microscopy and quantitative analysis approaches, it optimally poises me to address some of these questions. The human immunodeficiency virus type-1 (HIV-1) entry process is an ideal model for studying the functional correlation of the spatial organisation of receptors. The molecular interactions between HIV envelope glycoprotein (Env) and key receptors, CD4 and co-receptor CCR5/CXCR4, on the PM of target cells have been well characterised. However, the spatial organisation that receptors undergo upon HIV-1 binding remains unclear. In this project, I established a Single Molecule Localisation Microscopy (SMLM) based visualisation and quantitative analysis pipeline to characterise CD4 membrane organisation in CD4+ T cells, the main host cell target for HIV-1 infection. I found that prior to HIV engagement, CD4 and CCR5 molecules are organised in small distinct clusters across the PM. Upon HIV-1 engagement, I observed dynamic congregation and subsequent dispersal of virus-associated CD4 clusters within 10min. I further incorporated statistical modelling to show that this reorganisation is not random. This thesis provides one of the first nanoscale imaging and quantitative pipelines for visualising and quantifying membrane receptors. I showed that this quantitative approach provides a robust methodology for understanding the recruitment of HIV-1 receptors before the formation of a fusion pore. This methodology can be applied to the analyses of the nanoscale organisation of PM receptors to link the spatial organisation to function

Characterizing the role of p21-Activated Kinase 3 (PAK3) in AP-1-induced transformation

Includes bibliographical references.Previous studies identified p21-Activated Kinase 3 (PAK3), a serine/threonine kinase, as a potential AP-1 target gene. PAK3 has been implicated in a variety of pathological disorders and over-expression of other PAK-family members has been linked to cancer. In this study, we investigated AP-1 regulation of PAK3 expression and the role of PAK3 in cJun/AP-1-associated cellular transformation. Our results showed elevated PAK3 expression at both the mRNA and protein level in cJun-over-expressing Rat1a fibroblasts, as well as in transformed human fibroblasts. Elevated PAK3 protein levels were also seen in cervical, ovarian, oesophageal and breast cancer cells lines, while poor survival tracked with high PAK3 expression in ovarian cancer patient material. Elevated PAK3 levels appear to play no role in the proliferation of transformed or cancerous cells, however appears vital for the transformed morphology and actin distribution. These cytoskeletal changes seem to be the underlying force governing cellular migration, as inhibition of PAK3 significantly reduced the motility of both transformed fibroblasts and cancer cell lines. Our data shows that elevated PAK3 expression in response to AP-1 over-expression is regulated through the transcriptional activation of the PAK3 promoter by AP-1 binding directly to a single site in the promoter. We also show that constitutive activation of PAK3 results in changes in cJun phosphorylation and an increase in AP-1 activity, which can be inhibited by a serine/threonine kinase inhibitor. PAK3 and AP-1 proteins were also shown to directly interact with each other. Our study is a first to describe a role for AP-1 in regulating PAK3 expression, and PAK3 in regulating AP-1 activity, identifying a potential feedback loop in which PAK3 is an AP-1 target required for cytoskeletal reorganization and migration observed in transformed cells

Bioprocess Monitoring and Control

Process monitoring and control are fundamental to all processes; this holds especially for bioprocesses, due to their complex nature. Usually, bioprocesses deal with living cells, which have their own regulatory systems. It helps to adjust the cell to its environmental condition. This must not be the optimal condition that the cell needs to produce whatever is desired. Therefore, a close monitoring of the cell and its environment is essential to provide optimal conditions for production. Without measurement, no information of the current process state is obtained. In this book, methods and techniques are provided for the monitoring and control of bioprocesses. From new developments for sensors, the application of spectroscopy and modelling approaches, the estimation and observer implementation for ethanol production and the development and scale-up of various bioprocesses and their closed loop control information are presented. The processes discussed here are very diverse. The major applications are cultivation processes, where microorganisms were grown, but also an incubation process of bird’s eggs, as well as an indoor climate control for humans, will be discussed. Altogether, in 12 chapters, nine original research papers and three reviews are presented