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    The development of optical nanomachines for studying molecules : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Mechatronics Engineering at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Andrew, Philippa-Kate
    Optical tweezers have been used for a number of applications since their invention by Arthur Ashkin in 1986, and are particularly useful for biological and biophysical studies due to their exceptionally high spatial and force-based resolution. The same intense laser focus that allows light to be used as a tool for micro-nanoscale manipulation also has the potential to damage the objects being studied, and the extremely high force resolution is coupled with the limitation of very low forces. There is potential to overcome these drawbacks of optical manipulation through making use of another laser based technique: two-photon absorption polymerisation (TPAP). This thesis has brought these together to demonstrate the uses of optical nanomachines as helpful tools for optical tweezer studies. The project was highly interdisciplinary, concerning the intersection of optical trapping, 3D micromachine design and development, and DNA stretching. The thesis was based around the strategy of first developing microrobots and demonstrating their manipulation using optical tweezers, then adjusting the design for specific applications. Microlevers were developed for lever-assisted DNA stretching and amplification of optical forces. The influence of design features and TPAP parameters on microlever functionality was investigated; particularly the influence of overlapping area and presence of supports, and the effects of differently shaped "trapping handles". These features were important as lever functionality was tested in solutions of different ionic strength, and stable trapping of the levers was required for force amplification. DNA stretching was chosen as a target application for distanced-application of optical forces due to its status as a well-known and characterised example of single-molecule studies with optical tweezers. Amplification of optical forces was also seen as an application that could demonstrate the utility of optical micromachines, and microlevers with a 2:1 lever arm ratio were developed to produce consistent, two-fold amplification of optical forces, in a first for unsupported, pin-jointed optical microrobotics. It is hoped that in the future fully-remote, micromachine-assisted studies will extend optical tweezer studies of laser-sensitive subjects, as well as increasing the forces that can be applied, and the results obtained in this thesis are encouraging. All in all, the thesis confirms the potential of optical micromachines for aiding studies using optical tweezers, and demonstrates concrete progress in both design and application.
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    Capture-seq and small RNA-seq to identify noncoding RNAs in the mouse ribosomal RNA gene repeat intergenic spacer : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University (Albany), New Zealand
    (Massey University, 2018) Fitch, Jessica Leigh
    Cancer is a leading cause of mortality in developed countries. Due to the genetic and epigenetic heterogeneity of this disease, we still don’t have effective long-term therapies for many cancers. A characteristic of many cancer cells is an alteration in the structure of the nucleolus - the primary location of the ribosomal DNA (rDNA). The rDNA encodes ribosomal RNA, which is the major structural and catalytic component of ribosomes – the cellular machinery responsible for protein biosynthesis. Accordingly, the rDNA and its transcription is a key regulator of cell proliferation. Despite this critical role, the highly repetitive nature of the rDNA has made it difficult to study, thus it remains an attractive target for anti-cancer therapies. Indeed, the promising anti-cancer drug, CX-5461, developed by our collaborators, targets the rDNA through the inhibition of the rDNA dedicated RNA polymerase I (currently in clinical trials). In preliminary experimentation, there is a dramatic change in expression of non-coding RNAs (ncRNAs) from the rDNA during the transition to malignancy. Although the function of rDNA ncRNAs is almost entirely unknown, ncRNAs from other regions of the genome have a multitude of regulatory functions, including involvement in cancer. We hypothesise that these transcripts play a role in malignancy and CX-5461 sensitivity. Utilising a mouse B-lymphoma model (Eμ-myc), we first applied a high throughput hybridisation-based RNA-sequencing approach (capture-seq), to enrich for rDNA intergenic spacer (IGS) ncRNA transcripts within 11 cDNA sequencing libraries. Regions of transcription throughout the IGS were identified using several bioinformatic tools, and qPCR was performed to validate transcription status as well as assess for CX-5461-dependent transcriptional changes. We also utilised other bioinformatics tools, to predict small RNAs arising from the IGS and other regions of the Eμ-myc genome, and briefly assessed their response to CX-5461 treatment. miRNAs of interest were assessed for potential pathway targets using several bioinformatic targets. Lastly, we aimed to further characterise the Eμ-myc model. With this, we assessed efficacy of methods that could be used for downstream knockdown/over expression analysis. Overall, using the capture-seq method we identified 8 major clusters of exons (known as exon cluster groups), that were consistently predicted between RNA library preparations. These were confirmed to be transcriptionally active by qPCR, with one of these clusters. Additionally, we identified several sites in the mouse rDNA IGS that may express small RNAs, with small RNA reads aligning to these sites with some consistency between library preparations. Some of these, due to presence and absence patterns in either CX-5461 treated or control libraries, may show some signs of treatment-dependent differential expression. We also identified miRNAs from other regions of the genome which show similar patterns. We assessed potential small RNAs for gene target enrichment. No pathways/cellular components appeared to be biologically significant. We assessed a method of viral-mediated gene knockdown in a number of cell lines, which did not show efficacy in the mouse lines we had available. In conclusion, if these exons produce ncRNAs that contribute to malignancy, the ncRNAs will form attractive new targets for therapy, independently or in combination with CX-5461, and could be used as diagnostic and prognostic markers of cancer. The future trajectories of this project include selecting promising IGS transcripts, particularly those differentially expressed, to confirm their size by northern blot. Then, to assess their role in malignant cells, to perform knockdown/overexpression assays and assess cellular response. Further, we would target the rDNA ncRNAs in several cancer and non-cancer cell lines, to broaden our understanding of anti-cancer application.
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    16S ribosomal DNA probes for the detection and enumeration of proteolytic rumen bacteria :|ba thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology at Massey University
    (Massey University, 1999) Reilly, Kerri
    Bacterial degradation of protein causes inefficient nitrogen retention in New Zealand ruminants. The 16S rRNA genes of a Butyrivibrio fibrisolvens-like strain and three Streptococcus bovis strains, isolated from New Zealand cattle were sequenced to further characterise these isolates. Based on 16S rDNA analysis the B. fibrisolvens-like isolate was classified as Clostridium proteoclasticum, while the three S. bovis isolates were confirmed as S. bovis strains. In the absence of selective media for enumeration of these bacteria, a competitive PCR (cPCR) approach was developed for enumeration of these bacteria from rumen samples. PCR primers were designed to variable regions within the 16S ribosomal RNA genes of both S. bovis and C. proteoclasticum. These primers were used in conjunction with the universal forward primer fD1*, to allow amplification of 16S rDNA fragments from these organisms. DNA database searches revealed that the B316 830 primer sequence was present in four B. fibrisolvens strains. Analysis of 16S rDNA sequences indicated that these B. fibrisolvens strains are closely related to C. proteoclasticum and that the B316 830 primer circumscribes these five strains.. The B315 454 primer sequence was found in the 16S rDNA of 10 Streptococcus species. Primer specificity was tested in amplification reactions with DNA extracted from 85 bacterial isolates, mainly of rumen origin. The C. proteoclasticum primer B316 830 and fD1* produced a specific PCR product from C. proteoclasticum DNA only, while the S. bovis primer B315 454 and fD1* gave specific PCR product from DNA of all strains of S. bovis tested but from no other rumen bacterium. An internal control was developed for both S. bovis and C. proteoclasticum to use in cPCR reactions for quantitation. Standard curves were constructed relating the PCR product intensity of target DNA extracted from a known number of cells and the intensity of internal control DNA PCR product. The standard curves were used to quantitate populations of S. bovis and C. proteoclasticum in rumen samples collected from eight dairy cows fed a rotation of four diets. Populations detected ranged from 2 x 106 to 2.8 x 107 for C. proteoclasticum and 1.7 x 107 to 1.3 x 108 for S. bovis. Diet had no significant effect on the populations of either of these proteolytic bacteria.
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    The binding of small volatile molecules by bovine [beta]-lactoglobulin : a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University
    (Massey University, 2008) Hsu, Yu-Ting
    Bovine ß-lactoglobulin (ß-Lg) has been studied extensively but there is no clear identification of its biological function. Hydrophobic molecules have been observed binding into the hydrophobic calyx of ß-Lg. By comparison with other members of lipocalin family, it is probable that ß-Lg plays a role of transport of ligands, as ligands also bind into the central cavity of lipocalins. The structurally similar MUP is a pheromone-binding protein; therefore, it is possible that ß-Lg may also fulfil a similar role. This study has begun to test this hypothesis by investigating the interactions between bovine ß-Lg and several small volatile molecules (2-sec-4,5-dihydrothiazole, 3-methyl-2-butenal, 3-methyl-2-buten-1-ol and phenylacetic acid). The interactions between the volatile molecules and ß-Lg were studied by both two-dimensional NMR spectroscopy and X-ray crystallographic methods. TOCSY spectra were recorded for ß-Lg and the complex between ß-Lg and the ligands. The observed chemical shifts in the HN-Ha region are sensitive to the proximity of ligands, and hence chemical shift changes on ligand binding provide information on possible binding sites. It appears that several amino acids with hydrophobic sidechains are affected by interaction with volatile molecules at pH 2.0. The X-ray crystallographic study at pH 8.5 showed that the potential ligand, 2-sec-4,5-dihydrothiazole, may have decomposed into a linear 2-methyl-butanol. The refined structure (R=0.281, Rfree=0.354 for reflections to 2.6 Å resolution) reveals that the potential ligand may bind to the central cavity in a manner similar to the binding of 12-bromodecanoic acid to ß-Lg.