Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle.

<p>Various aspects of and approaches towards the steric stabilisation of liposomes have been investigated, mainly by use of fluorescence techniques and cryo-transmission electron microscopy (cryo-TEM). It is shown that PEG(2000)-lipids can be incorporated in the liposome membrane up to a critical concentration of 8-10 mol% without any observable structural perturbations. Above 10 mol%, a breakdown of the liposome structure into flat lamellar discs was observed. The sterically stabilised liposomes displayed similar, or even reduced, membrane permeability as compared with conventional liposomes. The presence of PEG-lipids in the EPC membrane was shown to affect the liposome-to-micelle transition in mixtures containing OG. Little or no effects of the PEG-lipids were found on the transition in mixtures containing C₁₂E₈.</p><p>The interactions between a number of PEO-PPO-PEO triblock copolymers and PC or PC/Chol liposomes have been investigated. It is shown that these polymers adsorb rapidly onto the liposome surface and induce a substantial increase in membrane permeability as well as structural perturbations. No evidence of an effective steric stabilisation due to the presence of the polymers at the membrane surface was found. This was shown, by the use of a QCM-technique, to be a consequence of the weak interaction between the polymers and the lipid membrane. </p><p>Dispersions of reversed lipid phases in mixtures of DOPE and PEG-lipids were characterised using cryo-TEM. Dispersions displaying reasonable colloidal stability were obtained and particles exhibiting a periodic dense inner structure were observed.</p><p>PEG-lipid micelles were characterised mainly using light scattering techniques. Micelle aggregation numbers and hydrodynamic radii were determined as a function of temperature. It is shown that the inter-micellar interactions are dominated by the steric repulsion.</p><p>PEG-lipid stabilised liposomes loaded with boronated drugs intended for BNCT have been characterised. The drugs were efficiently encapsulated into the liposomes, resulting in a drug precipitation in the water core of the liposomes.</p>

Various aspects of and approaches towards the steric stabilisation of liposomes have been investigated, mainly by use of fluorescence techniques and cryo-transmission electron microscopy (cryo-TEM). It is shown that PEG(2000)-lipids can be incorporated in the liposome membrane up to a critical concentration of 8-10 mol% without any observable structural perturbations. Above 10 mol%, a breakdown of the liposome structure into flat lamellar discs was observed. The sterically stabilised liposomes displayed similar, or even reduced, membrane permeability as compared with conventional liposomes. The presence of PEG-lipids in the EPC membrane was shown to affect the liposome-to-micelle transition in mixtures containing OG. Little or no effects of the PEG-lipids were found on the transition in mixtures containing C12E8. The interactions between a number of PEO-PPO-PEO triblock copolymers and PC or PC/Chol liposomes have been investigated. It is shown that these polymers adsorb rapidly onto the liposome surface and induce a substantial increase in membrane permeability as well as structural perturbations. No evidence of an effective steric stabilisation due to the presence of the polymers at the membrane surface was found. This was shown, by the use of a QCM-technique, to be a consequence of the weak interaction between the polymers and the lipid membrane. Dispersions of reversed lipid phases in mixtures of DOPE and PEG-lipids were characterised using cryo-TEM. Dispersions displaying reasonable colloidal stability were obtained and particles exhibiting a periodic dense inner structure were observed. PEG-lipid micelles were characterised mainly using light scattering techniques. Micelle aggregation numbers and hydrodynamic radii were determined as a function of temperature. It is shown that the inter-micellar interactions are dominated by the steric repulsion. PEG-lipid stabilised liposomes loaded with boronated drugs intended for BNCT have been characterised. The drugs were efficiently encapsulated into the liposomes, resulting in a drug precipitation in the water core of the liposomes.

The bilayer disc is a flat, lipid aggregate structure in the nanometre regime. It is composed of a bilayer of amphiphilic molecules with micelle-forming amphiphilic molecules supporting the rim, which prevent disc fusion and self-closure. Stable discs have been found in lipid mixtures containing polyethylene glycol (PEG)-lipids as a rim-stabilizing component. One of the aims of the work described in this thesis was to increase the fundamental knowledge and understanding of the systems in which these discs are formed. Other micelle-forming surfactants apart from PEG-lipids were also explored to see if they could be used to stabilize the disc aggregate structure. Due to the similarities of these lipid discs with natural membranes it was hypothesized that they could be used as models for biological membranes. It was demonstrated that discs are formed in PEG-lipid/lipid systems when the lipid mixture contains components that reduce the spontaneous curvature and increase the monolayer bending rigidity. Discoidal structures are furthermore preferred when the lipids are in the gel phase, probably due to a combination of high bending rigidity and reduced PEG-lipid/lipid miscibility. The disc size could be varied by changing the PEG-lipid concentration. The size and size homogeneity of the discs could also be varied by changing the preparation path. Generally, the preferences of certain lipid systems to form discs remained when the PEG-lipid was replaced by more conventional surfactants. However, discs prepared in PEG-lipid/lipid systems are more useful as model membranes because of their relatively large size and good temperature, dilution and long-term stability. Data obtained with isothermal titration calorimetry and drug partition chromatography indicate that these bilayer discs may serve as an attractive and sometimes superior alternative to liposomes in studies of drug-membrane interactions.

When increasing magnetic resonance imaging (MRI) resolution into the micron scale, image signal-to-noise ratio (SNR) can be maintained by using small radiofrequency (RF) coils in close proximity to the sample being imaged. Micro-scale RF coils (microcoils) can be easily fabricated on chip and placed adjacent to a sample under test. However, the high series resistance of microcoils limits the SNR due to the thermal noise generated in the copper. Cryo-cooling is a potential technique to reduce thermal noise in microcoils, thereby recovering SNR. In this research, copper microcoils of two different geometries have been cryo-cooled using liquid nitrogen. Quality-factor (Q) measurements have been taken to quantify the reduction in resistance due to cryo-cooling. Image SNR has been compared between identical coils at room temperature and liquid nitrogen temperature. The relationship between the drop in series resistance and the increase in image SNR has been analyzed, and these measurements compared to theory. While cryo-cooling can bring about dramatic increases in SNR, the extremely low temperature of liquid nitrogen is incompatible with living tissue. In general, the useful imaging region of a coil is approximately as deep as the coil diameter, thus cryo-cooling of coils has been limited in the past to larger coils, such that the thickness of a conventional cryostat does not put the sample outside of the optimal imaging region. This research utilizes a scheme of microfluidic cooling (developed in the Texas A&M NanoBio Systems Lab), which greatly reduces the volume of liquid nitrogen required to cryo-cool the coil. Along with a small gas phase nitrogen gap, this eliminates the need for a bulky cryostat. This thesis includes a review of the existing literature on cryo-cooled coils for MRI, as well as a review of planar pair coils and spiral microcoils in MR applications. Our methods of fabricating and testing these coils are described, and the results explained and analyzed. An image SNR improvement factor of 1.47 was achieved after cryo-cooling of a single planar pair coil, and an improvement factor of 4 was achieved with spiral microcoils.

AEGIS (Antimatter Experiment, Gravity, Interferometry and Spectroscopy) isan experiment under development at CERN which will measure earth's gravitationalforce on antimatter. This will be done by creating a horizontal pulsedbeam of low energy antihydrogen, an atom consisting of an antiproton anda positron. The experiment will measure the vertical de ection of the beamthrough which it is possible to calculate the gravitational constant for antimatter.To characterise the production process in the current state of the experimentit is necessary to develop an imaging detector for single excited hydrogenatoms. This thesis covers the design phase of that detector and includes studiesand tests of detector components. Following literature studies, tests and havingdiscarded several potential designs, a baseline design was chosen. The suggesteddetector will contain a set of ionising rings followed by an electron multiplyingmicrochannel plate, a light emitting phosphor screen, a lens system and nallya CCD camera for readout. The detector will be able to detect single hydrogenatoms, measure their time of ight as well as being able to image electronplasmas and measure the time of ight of the initial particles in such a plasma.Tests were made to determine the behaviour of microchannel plates at the lowtemperatures used in the experiment. Especially, the resistance and multiplicationfactor of the microchannel plates have been measured at temperaturesdown to 14 K. / AEGIS

Eukaryotic cells face a considerable challenge organizing a complicated interior with spatial and temporal precision. They do so, in part, through the deployment of the microtubule- based molecular motors kinesin and dynein, which translate chemo-mechanical force production into the movement of diverse cargo. Many aspects of kinesin’s motility mechanism are now known in detail, whereas fundamental aspects of dynein’s motility mechanism remain unclear. An important unresolved question is how dynein couples rounds of ATP binding and hydrolysis to changes in affinity for its track, a requisite for a protein that takes steps. Here we report a sub- nanometer cryo-EM reconstruction of the high affinity state of dynein’s microtubule binding domain in complex with the microtubule. Using molecular dynamics flexible fitting, we determined a pseudoatomic model of the high affinity state. When compared to previously reported crystal structure of the free microtubule binding domain, our model revealed the conformational changes underlying changes in affinity. Surprisingly, our simulations suggested that specific residues within the microtubule binding domain may tune dynein’s affinity for the microtubule. We confirmed this observation by directly measuring dynein’s motile properties using in vitro single molecule motility assays, which demonstrated that single point mutations of these residues dramatically enhance dynein’s processivity. We then sought to understand why dynein has been selected to be a restrained motor, and found that dynein-driven nuclear oscillations in budding yeast are defective in the context of highly processive mutants. Together, these results provide a mechanism for the coupling of ATPase activity to microtubule binding and release by dynein, and the degree to which evolution has fine-tuned this mechanism. I conclude with a roadmap of future approaches to gain further insight into dynein’s motility mechanism, and describe our work developing materials and methods towards this goal.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

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.