Kinesin is a molecular motor which walks on microtubule tracks in the eukaryotic cytoskeleton. It transports cargo but is also involved in cytoskeletal organisation. This thesis demonstrates fusing kinesin and DNA to construct a molecular transport system using self-organised tracks and to study the mechanics of the minimal motor unit of kinesin. The programmability of DNA allows for the formation of nanostructures with controllable interactions. Kinesin is conjugated to various DNA nanostructures to accomplish different tasks. Instructions encoded into DNA sequences are used to direct the assembly of a polar array of microtubules, to control the loading, active concentration and unloading of cargo on this track network and to trigger the disassembly of the network. Fluorescence microscopy was used to observe these microtubule arrays and the movement of cargo. It was found that the DNA signals used to control the unloading of cargo and the disassembly of the network had to be actively transported, rather than relying on diffusion, for effective delivery of the signal. This work lead to a first author publication, Wollman et al. (2013). DNA was also used to study kinesin by linking defined numbers of minimal functional motor units, single kinesin heads, into teams of 4-12 heads and observing their movement along microtubules via fluorescent labelling. A minimum of 5 heads were required for sustained movement, in agreement with the predictions of Hancock and Howard (1998). The velocity of teams increased with more heads, up to 8, and then a decrease was observed in teams with more heads.

The emergence of bionanotechnology has allowed the design of novel technological tools for a variety of biomedical applications. Despite the incompatibility of native semiconductor quantum dots with biological environment, this class of nanocrystals was among the first nanomaterials to demonstrate their use in biological labeling and imaging applications. In this thesis, new surface modification chemistry and synthetic strategies were developed to produce high quality biocompatible and bioconjugatable near-infrared emitting quantum dots for deep tissue in vivo imaging and detection applications. By carefully selecting a specific mixture of semiconductor elements to obtain a desired bandgap energy and optimizing the procedure for surface coating, successful synthesis of high quality, watersoluble, near-infrared emitting quantum dots were demonstrated. These developments allows for the use of quantum dots as alternative contrast agents for sophisticated biological imaging applications that are currently unachievable using conventional uorophores. In addition, using metallic nanoparticles, it was found that cells possess the ability to differentiate nanoparticles of various sizes upon their binding with specific membrane receptors. These receptors undergo rapid cellular internalization which altered their trafficking dynamics and down-stream signaling processes. The amplitude of such alteration was highly dependent on the size of the nanoparticle with most efficient internalization occurring at 40 nm - 50 nm size range. These observations raise important questions regarding the mechanisms governing similar processes and cell behaviours documented during viral infections. Whether such biological phenomenon are evolutionarily conserved as natural defense mechanisms to counter foreign invasion, or whether many of the known viruses are naturally selected to breach the primary defense of cells - the plasma membrane, remains to be elucidated. In summary, nanotechnology offers great promises for biological research and medicine. This thesis demonstrates the use of semiconductor and metallic nanostructures for imaging, detecting and administrating therapeutics in cancerous cells, tissues and animal models. Although the results presented in this thesis are preliminary, and the technologies demonstrated are still years away from practical use, these studies nevertheless, pave the way for future experimental researches within the field of nanomedicine, and provide insights into the understanding of the most fundamental yet highly complex processes in cell biology.

Self-assembling materials have gained attention in the field of nanotechnology due to their potential to be used as building blocks for fabricating complex nanoscale devices. The biological world is abundant with examples of functional self-assembling biomolecules. Proteins are one such example, found in a variety of geometries and shapes. This research is focussed on the use of ring-shaped self-assembling proteins, called Lsm proteins, as componentary for applications in bionanotechnology. Lsm proteins were used because of their spontaneous association into stable rings, tolerance to mutations, and affinity to RNA. This thesis primarily focussed on the thermophilic Lsmα (from Methanobacterium. thermoautotrophicum) that assembles as heptameric rings. The oligomeric state of the heptameric protein, and hence the diameter of its central cavity, was manipulated by judiciously altering appropriate residues at the subunit interface. Lsmα presented a complex set of interactions at the interface. Out of the mutations introduced, R65P yielded a protein for which SEC and SAXS data were consistent with a hexameric state. Moreover, key residues, L70 and I71, were identified that contribute to the stability of the toroid structure. Covalent linking of rings provided nanotubular structures. To achieve this, the surface of the Lsmα ring scaffold was modified with Cys residues. This approach led to the formation of novel Lsmα nanotubes approximately 20 nm in length. Importantly, the assembly could be controlled by changing the redox conditions. As an alternative method to manipulate the supramolecular assembly, His6-tags were attached at the termini of the Lsmα sequence. The higher-order organisation of the constructs was influenced by the position of the His6-tag. The N-terminally attached His6-tag version of Lsmα showed a metal-dependent assembly into cage-like structures, approximately 9 nm across. This organisation was highly stable, reproducible, and reversible in nature. The results presented in this thesis aid the understanding of generating complex nanostructures via in vitro self-assembly. The Lsmα rings were assembled into higher-order architectures at the quaternary level by employing protein engineering strategies. Future work is necessary to functionalise these supramolecular structures; however, this study confirms the potential role of Lsmα proteins as a molecular building block in bionanotechnology.

The emergence of bionanotechnology has allowed the design of novel technological tools for a variety of biomedical applications. Despite the incompatibility of native semiconductor quantum dots with biological environment, this class of nanocrystals was among the first nanomaterials to demonstrate their use in biological labeling and imaging applications. In this thesis, new surface modification chemistry and synthetic strategies were developed to produce high quality biocompatible and bioconjugatable near-infrared emitting quantum dots for deep tissue in vivo imaging and detection applications. By carefully selecting a specific mixture of semiconductor elements to obtain a desired bandgap energy and optimizing the procedure for surface coating, successful synthesis of high quality, watersoluble, near-infrared emitting quantum dots were demonstrated. These developments allows for the use of quantum dots as alternative contrast agents for sophisticated biological imaging applications that are currently unachievable using conventional uorophores. In addition, using metallic nanoparticles, it was found that cells possess the ability to differentiate nanoparticles of various sizes upon their binding with specific membrane receptors. These receptors undergo rapid cellular internalization which altered their trafficking dynamics and down-stream signaling processes. The amplitude of such alteration was highly dependent on the size of the nanoparticle with most efficient internalization occurring at 40 nm - 50 nm size range. These observations raise important questions regarding the mechanisms governing similar processes and cell behaviours documented during viral infections. Whether such biological phenomenon are evolutionarily conserved as natural defense mechanisms to counter foreign invasion, or whether many of the known viruses are naturally selected to breach the primary defense of cells - the plasma membrane, remains to be elucidated. In summary, nanotechnology offers great promises for biological research and medicine. This thesis demonstrates the use of semiconductor and metallic nanostructures for imaging, detecting and administrating therapeutics in cancerous cells, tissues and animal models. Although the results presented in this thesis are preliminary, and the technologies demonstrated are still years away from practical use, these studies nevertheless, pave the way for future experimental researches within the field of nanomedicine, and provide insights into the understanding of the most fundamental yet highly complex processes in cell biology.

A cell’s interaction with its extracellular environment is critical to tissue structure and function. This work investigates the effect of substrate topography on selective cell adhesion and morphology. Alterations in cell response to micro- and nanoscale signals and cues can cause changes in downstream functions of proteins and complexes such as invasive and metastatic motility of malignant tumour cells and the differentiation direction of stem cells. Biomaterial surfaces can be modified to provide different chemical and topographical cues and encourage controlled cell-substrate interaction. At the protein level, template substrates have shown and increased affinity for selective binding of the imprinted antigen or antibody. Topography of a cell’s microenvironment may be replicated as a permanent polymer mould by bioimprinting technology, which was developed at University of Canterbury. The resulting high resolution methacrylate polymer samples have been used for imaging and analysis, but have not previously been investigated as cell culture substrates. This work investigates the effect of bioimprint and photolithographic substrate patterning on cell behaviour in culture. Optimisation of a methacrylate co-polymer resulted in a 6:3:1 ethylene glycol dimethacrylate: methacrylic acid: photoinitiator polymer mixture cured by 240 seconds of UV exposure. The polymer was used to replicate cell membrane features into a permanent polymer mould [a bioimprint]. The resulting high resolution methacrylate bioimprints were cleaned and sterilised for use as a secondary cell culture substrate. Ishikawa endometrial cancer cells were cultured on bioimprinted methacrylate polymer substrates. Preliminary results showed preferential cell adhesion to bioimprinted areas over flat areas and also showed three dimensional spheroid growth instead of lateral two dimensional monolayer spreading. At higher seeding densities, preferential adhesion was similarly noted as well as peeling artefacts of shear stresses and cell size variation on flat methacrylate substrate regions. Fluorescent imaging and cell culture stencilling highlighted the association of secondary cells with bioimprint substrate features. To determine whether preferential cell adhesion effects were due to bioimprint features or general topography modification, secondary cancer cells were cultured on comparable photolithographically-defined, geometrically-patterned substrates. Methods for transferring regular pattern arrays into methacrylate polymer substrates were developed. No organisation or preferential adhesion effects were observed in association with pillar and hole patterns between 5-30 µm. However, artefact incidence in methacrylate polymer replication techniques led to development and adaptation of polystyrene patterning techniques. Experimental analysis of substrate-dependent effects on cell culture adhesion and organisation was extended to a non-cancerous cell line model. C2C12 mouse skeletal muscle cells were chosen for these investigations because of their ability to differentiate further, into myocytes or myofibrils. C2C12 myoblasts seeded on common cell culture substrates showed a notable morphology variation and extent of differentiation between cells grown on tissue culture polystyrene [TCPS] and polydimethylsiloxane [PDMS]. Myoblasts were plated on geometrically-patterned polystyrene and PDMS substrates. Significant alignment to grated pattern features was observed on both substrate types, before and after driven differentiation. Peeling artefacts of confluent tissue-like culture from PDMS surfaces which were observed were unreported previously in literature. The results reported in this thesis provide a foundation for potential research and commercial application for surface modification methods. The biomimetic topography provided by bioimprinted substrates can be used to identify and investigate cell activities, including for example the mechanisms of cell adhesion and separation in metastatic and invasive cancer research. Altering the material of the bioimprinted substrates may attune substrate topographies as scaffolds to direct specific stem cell differentiation for regenerative tissue engineering applications.

A successful cancer treatment is a combination of early diagnosis and efficient use of anticancer drugs. There is a chance of approximately 70 - 90% of cancer patients surviving if the diagnosis is conducted early. That means if a diagnosis system is in place which can detect multiple types of cancer at an early stage, a potential cancer therapy is most likely to succeed. However, at present, the available biomedical sensors are unable to detect and differentiate between cancerous cells or tumours. They are also not able to provide continuous real-time monitoring of a patient. Additionally, oral anticancer drugs given during chemotherapy, at the moment, suffer from low bioavailability. Also, a variety of these drugs is not targeted in nature. That means the drug will potentially affect areas of the body which do not need it. The low bioavailability of the drug will not only increase the chemotherapy sessions but also makes the entire process more aggravating for the cancer patient. Therefore, there is an absolute need to have innovative and efficient anticancer drug delivery mechanisms. Finally, current biomedical sensors are primarily made up of silicon (Si) or hard substrates based materials. Even if the biomedical sensor is of a flexible material, the material is either a fragile film or flexible but not stretchable polymers such as polyimide (PI). By having a biomedical sensor which is moderately flexible or not flexible at all, a continuous on-body biomedical sensing is not possible in an efficient manner. That is because hard substrates based biomedical sensors would be difficult to be placed on a body at all times. Furthermore, the flexible biomedical sensors currently suffer from problems such as the electrode on top cracking and damaging after few uses rendering them unusable. Hence, a new fabrication process needs to be devised to solve the issues mentioned above. In this work, an attempt is made to utilise zinc oxide (ZnO) nanostructures for biomedical sensing, drug delivery and biomimetics. ZnO nanostructures are synthesised by using a low-cost wet chemistry process known as hydrothermal growth. Due to the inherent biocompatibility and unique electrical/ piezoelectric properties of ZnO, they acted as prime candidates for the applications outlined above. A high-throughput process is used to synthesise ZnO nanowires (NWs) on Si, polyimide-onsilicon (PI/Si) and directly on PI and polydimethylsiloxane (PDMS) substrates. The work utilises a variety of characterization tools. ZnO nanostructures' morphology is characterised by using a Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM) and Atomic Force Microscope (AFM). X-ray diffraction (XRD) was used to calculate the crystallite size and the crystalline orientation of the nanostructures. A novel fabrication process is developed to allow direct synthesis and direct patterning of metal electrodes on fully flexible, stretchable and bendable PDMS substrates by using standard photolithography. This novel fabrication process makes the PDMS substrates not expand when exposed to temperatures up to 110 °C. Also; the new fabrication process does not cause the PDMS to swell when exposed to various chemicals such as isopropyl alcohol (IPA) or acetone. The fabrication process has created a new paradigm shift in the field of patterning and producing devices directly on flexible and stretchable substrates. The PDMS substrate is further utilised as a sensitive bovine serum albumin (BSA) protein sensor which is capable of detecting up to femtomolar concentrations in just under 5 min of incubation time. Protein biosensing tests were carried out by measuring the change in resistance at 1V bias voltage. The PDMS based biosensor is tested as a protein sensor because proteins are important biomarkers in cancer diagnosis. Also, protein sensors are immensely useful in the detection of bacteria and viruses thereby allowing further expansion to the technology developed herewith. For the first time, ZnO NWs are used to deliver hydrophobic organic dye, Nile red, in a human body like environment. The Nile red simulates an anticancer drug as they share similar surface chemistry. There is an approximately 80% release of Nile red which shows that ZnO NWs can be used as an efficient anticancer drug delivery system with high bioavailability. For the drug delivery experiments, the dynamic dialysis based release of Nile red (Nr) from the ZnO nanowires is carried out by using UV-Visible (UV-Vis) spectroscopy. Fourier Transform Infrared (FTIR) was used to determine the coordination of Nr across the ZnO nanowires. Finally, a novel synthesis process is used to produce individual ZnO NWs on a single ZnO nanoplate (NP) which are named as ZNWNP nanostructures. ZNWNP nanostructures have high hydrophobicity without the need of any functionalization. The hydrophobicity of the hybrid ZnO nanowires on ZnO nanoplate nanostructures (ZNWNP) is characterised by using contact angle goniometry (CAG). Various contact angle theories have been used to calculate the surface free energy (SFE) of the ZNWNP nanostructures. The high hydrophobicity allows these nanostructures to be used for biomimetic applications such self-cleaning, bioinspired sensors and multimodal biosensing. Additionally, ZNWNP nanostructures can be used in biomedical sensors to create multimodal analysis. The multimodal analysis is immensely useful in cancer detection as at least three or more cancer biomarkers can be used to triangulate the diagnosis. The work presented in the thesis aims to utilise ZnO nanostructures for a variety of biomedical applications. The new fabrication process mentioned above has applications not only in biomedicine but also in the flexible electronics industry. The biomimetic nanostructures combined with the biomedical sensor gives rise to a robust multimodal analysis system which can change the course of the cancer diagnosis. That coupled with the usage of ZnO NWs as an effective anticancer drug delivery system gives an immense promise in advancing cancer therapy as a whole and making the entire treatment process less aggravating and less painful for cancer patients.

One of the most crucial steps towards nanopore-based nucleic acid analysis is extending the dwell time of DNA molecules within the sensing region of the nanopore. I address this issue by interfacing solid-state nanopores with gel media, which sterically hinders translocating DNA molecules, increasing dwell times. Specifically, my experimental results focus on two reptation regimes: when the DNA molecule is flexible on the length scale of the gel pore, and when the DNA molecule is inflexible on the length scale of the gel pore. The first regime is achieved through the use of agarose gel and 5 kbp dsDNA fragments, and produces a wide distribution of translocation times, spanning roughly three orders of magnitude. The second regime is achieved through the use of polyacrylamide gel and 100 bp dsDNA fragments, and displays a shift in translocation times by an order of magnitude while maintaining a tight distribution.

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No description available.

The oligomerisation of protein subunits is an area of much research interest, in particular the relationship to protein function. In the last decade, the potential to control the interactions involved in order to design constructs with tuneable oligomeric properties in vitro has been pursued. The subject of this thesis is the quaternary structure of members of the peroxiredoxin family, which have been seen to assume an intriguing array of organisations. Human Peroxiredoxin 3 (HsPrx3) and Mycobacterium tuberculosis alkyl hydroperoxide reductase (MtAhpE) catalyse the detoxification of reactive species, preferentially hydrogen peroxide and peroxynitrite respectively, and form an essential part of the antioxidant defence system. As well as their biomedical interest, the ability of these proteins to form organised supramolecular assemblies makes them of interest in protein nanotechnology. The work described focusses on the elucidation of the quaternary structure of both proteins, resolving previous debates about their oligomeric state. The factors influencing oligomerisation were examined through biophysical characterisation in different conditions, using solution techniques including chromatography, light and X-ray scattering, and electron microscopy. The insight gained, along with analysis of the protein-protein interfaces, was used to alter the quaternary structure through site-directed mutagenesis. This resulted in a level of control over the protein’s oligomeric state to be achieved, and novel structures with potential applications in nanotechnology to be generated. The activity of the non-native structures was also assessed, to begin to unravel the relationship between peroxiredoxin quaternary structure to enzyme activity. The formation and structure of very high molecular weight complexes of HsPrx3 were explored using electron microscopy. The first high resolution structural data for such a complex is presented, analysis of which allowed the theory of an assembly mechanism to be proposed.