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Knowledge of protein sequences has exploded, but knowledge of protein function is needed to make use of sequence information, and this lags behind. A protein's function must be understood in context and part of this is the network of interactions between proteins. What are the relationships between protein function and the structure of the interaction network? In the first part of my thesis, I investigate the functional relevance of clusters, or communities, of proteins in the yeast protein interaction network. Communities are candidates for biological modules. The work I present is the first to systematically investigate this structure at multiple scales in such networks. I develop novel tests to assess whether communities are functionally homogeneous, and demonstrate that almost every protein is found in a functionally homogeneous community at some scale. The evolution of protein sequences IS well-studied, but comparatively little is known about the evolution of protein function. Such knowledge is needed to un- derstand when it is appropriate to annotate newly sequenced proteins by transferring functional information from homologs-i.e. evolutionarily related proteins. In the sec- ond part of my thesis, I assess the success of transferring protein-protein interactions across species and use this to estimate the rate at which interactions are lost in evolu- tion. At levels of sequence similarity associated with functional annotation transfer, I demonstrate that protein-protein interaction transfer is unreliable. The relevance of community structure for understanding protein function and the low conservation of individual interactions, suggests a possible role for communities in the evolution of cellular function. I discuss this possibility in my conclusions.

Abstract The small Rho GTPases are a family of signalling proteins that act as molecular switches to regulate a number of key cellular processes, including actin remodelling, cell adhesion, cell polarity and cell migration. Recently, they were also found to play vital roles in nervous system development, immune system development and angiogenesis. The human family of Rho GTPases contains 20 members; however, only three of them; RhoA, Racl and Cdc42, have been studied in detail. Solving the pathways controlled by the other members of this important signalling protein family is clearly essential to our understanding of the actin cytoskeleton. The research within this thesis focused on the characterization of the signalling pathways of one novel and poorly-defined Rho GTPase: Rif. The Rif GTPase is a relatively recent addition to the Rho family; it is found only in chordates and displays a relatively low homology to other family members. Activated Rif was found to be an alternative trigger for the formation of actin stress fibers in epithelial cells through effector mDial. Unlike the classical stress fiber inducer RhoA, Rif does not raise ROCK activity in cells, instead Rif appears to regulate the localization of myosin light chain phosphorylation. This study establishes Rif as a general regulator of Diaphanous-related formins and shows how non-classical Rho family members can access classical Rho pathways to create new signalling interfaces in cytoskeletal regulation. Proteomics were carried out in this thesis to identify potential binding partners of Rif. Through these studies I have shown that Rif interacts with FARPl, which is a RhoA GEF; and plexinA4, which is a developing nervous system guidance cue receptor. PlexinA4, FARPl and Rif form a heterotrimer at the cell membrane. At low local expression levels, the priority for Rif is to bind the C-terminus of FARPl and enhances its RhoGEF activity. In contrast, at higher local expression levels, Rif competes with the FERM domain ofFARPl for plexinA4 binding and forms a feedback loop to prevent overactivation of plexinA4/FARPl signalling pathway. These results suggest that Rif can not only work as a GTPase to remodel actin cytoskeleton, but also act as a regulator to regulate the activity of the other small Rho GTPases.

The endosomal network acts as a master co-ordinator of traffic from both endocytic and bio-synthetic pathways. A vast multitude of cargoes are sorted within this system destined for distinct intra- cellular locations. The targeted and efficient distribution of endosomal cargo is reliant upon interactions with protein containing sorting machineries. Sorting machineries have evolved modular domains that associate with defining composites and morphologies of endosomal membranes that assure for correct steady state localisation and therefore function. The Sorting Nexins (SNXs) are a family of proteins characterised by the presence of a phosphoinositide-binding motif, termed the SNX- PX domain. By virtue of this domain, SNXs associate with membranes of the endocytic network and from here co-ordinate control over endocytosis, endosomal sorting and endosomal signalling. Two sub-families of SNXs are conjugated to additional modular domains, which confer to them diverse functionalities. In this thesis I have examined and characterised the mechanistic details of how SNX8 and SNX15 co-ordinate distinct events in endosomal trafficking. SNX8 utilises co-incidence detection to localise to high-curvature, phosphoinositide-enriched sub-domains of maturing endosomes; from here SNX8 co-ordinates the production of cargo-enriched carriers that are spatially distinct from more other well characterised tubular exit sites. By recruiting additional sorting machineries, SNX8 carriers are able to undergo long-range transport events, culminating in recognition and ultimately fusion with a recipient compartment, the Golgi apparatus, to facilitate cargo delivery. SNX15 associates with recently. endocytosed carriers, throuqh a direct association with clathrin, and facilitates their directional movement away from the cell periphery into the endo-Iysosomal degradative pathway.

The molecular chaperone Hspl04, in conjunction with two other chaperones Hsp70 and Hsp40, provides the yeast Saccharomyces cerevisiae with the necessary machinery to ensure that aggregated proteins that form in stressed cells can be remodelled back to a correctly folded and functio nal form. Hspl04 also plays an essential role in non-stressed cells in the propagation of yeast prions such as [PSt1, the prion form of the translation termination factor Sup3S. To date no functional mammalian orthologues of Hspl04 have been described although TorsinA, a 37 kOa ER located protein shows some degree of amino acid identity to Hspl04 predominantly through its conserved AAA+ ATPase domain. Earlier studies in mammalian cells had suggested that TorsinA may share more than sequence similarity with Hspl04 and function as a chaperone although this activity had not been formally demonstrated in vivo. This thesis describes a study of the chaperone function of mammalian TorsinA expressed in yeast. TorsinA was shown to partially rescue the ability of cells lacking Hspl04, to refold-heat denatured luciferase. This putative protein disaggregation function was impaired by mutations in the single ATPase domain of TorsinA and inhibited in cells grown in the presence of guanidine hydrochloride (GdnHCI), an inhibitor of the ATP-dependent function of Hspl04. These findings are consistent with Torsin A acting as an ATP-driven chaperone. However, mutating t he non-canonical Walker A motif of Torsin A nucleotide binding domain into a canonical sequence did not affect the ability of Torsin A to reactivate misfolded luciferase. Several variants of the [PSf1 prion were identified that were destabilised in cells exp ressing TorsinA, and when endogenous Hspl04 levels were elevated. Constitutive expression of Torsin A also protected yeast cells against various forms of stress in absence of Hspl04 demonstrating that TorsinA recapitulates a number of the important chaperone-related properties of Hspl04. A glutamic acid deletion in the (-terminus of Torsin A (associated with a movement disorder called torsion dystonia) reduced the ability of Torsin A to protect cells against stress and to eliminate [PSt] variants but did not affect its ability to reactivate heat-denatured luciferase. Torsin B belongs to the same gene family and shares a high sequence similarity (89.4% similarity) with TOrsin A but the chaperone function of Torsin B had not been established. Consequently its possible chaperone function was examined in yeast but was found not to protect cells against stress or protein aggregation and was not able to eliminate [PSf] but it inh ibited the growth of both yeast and mammalian cells. The ER targeting signal sequence ofTorsin A was recognized in yeast since a significant proportion of Torsin A was detected in the ER. Mutations in Torsin A that would be expected to impair ATP binding and hydrolysis did not affect its localisation although deletion of the N-terminal ER signal sequence resulted in its accumulation in the cytoplasm. 6N Torsin A mutant maintained the protective functions in yeast but exhibited reduced chaperone abilities. Under heat shock conditions Torsin A was translocated to the cytoplasm where it could process substrates, such as luciferase. Identification of a mammalian orthologue of Hspl04 is important for developing therapeutic strategies for protein aggregation-related disorders, such as Parkinson's disease, and improving recombinant protein production. The data in this thesis paint to Torsin A as a potential target.

Electrochemical impedance spectroscopy (EIS) has been employed to study protein-carbohydrate interaction using concanavalin A as a model protein. A novel biosensor based on Con A has been developed using a mixed self-assembled monolayer (mSAM) format comprising of 16- mercaptohexadecanoic acid (MHDA) and the interchelating phospholipids bearing a biotin group (biotin caproyl-DPPE) on a planar electrode. Biotinylated Con A was then linked to the layer using Neutravidin as crosslinker. Each stage in the biosensor construction and analyte detections were investigated using EIS and cyclic voltammetry (CV). Quartz crystal microbalance (QCM) and total internal reflection ellipsometry (TIRE) analysis were also carried out to follow each step of the biosensor construction. Interactions between Con A and carbohydrates of differing in chain length, ranging from monosaccharides to polysaccharides, and the glycoprotein human choriogonadtrophin (hCG) were investigated. It was found that larger oligomers could produce disruption in the impedance signa!. Data from further investigation using a series of defined oligomers with degree of polymerisation (DP) 1 to 7 (glucose to maltoheptaose respectively) implied that protein aggregation occurred at four units of glucose or more and that this was due to pinhole generation on the biosensor surface. This was indicated by a decrease in the charge-transfer resistance (Rct). However, the signal disruption was not observed when using a base layer of a copolymer of aniline and 2-aminobenzylamine (2-ABA) as the tethering template for lectin immobilisation. This suggests that a thick and rigid tethering template is required for larger glycoconjugates detection by lectins. Human blood group-specific lectins were then utilised in the copolymer system as proof-of- principle for blood typing. The biosensor was highly specific to blood group 0 sample with low detection limit of 100 erythrocytes/ml

Spectrofluorimetry is utilized as a novel real time non-invasive optical assay to investigate whether the exposure of bovine serum albumin (BSA) to extremely low frequency (ELF) and radio frequency (RF) electromagnetic (EM) fields causes conformational changes in the protein distinct from those due to heating alone. Such conformational changes would alter the protein's fluorescence spectrum, and could indicate a potential for EM induced altered biological activity of proteins in-vivo. The development of both the ELF and RF exposure system are described, discussed and evaluated. Since ELF electric fields do not exhibit propagation phenomena within the dimensions of the apparatus, a quasi-static approach is taken and a stainless steel parallel-plate exposure system is designed in a cuvette. While at RF, FDTD software is employed to facilitate a TEM cell based exposure system evaluation and optimization, and also to provide preliminary SAR distributions in the BSA sample as a control for future experimental measurements. The sensitivity of the systemic methodology (including the statistical analysis method) is demonstrated, indicating that the system is capable of detecting fluorescence changes as small as 0.032%. At ELF, the effects of six different high field strength 100Hz EM exposure paradigms at different temperatures on BSA conformational changes are presented and critically discussed. While at RF, four different 430MHz exposure paradigms are applied, with either intermittent or continuous bursts of TETRA or CW signals, at different average SAR levels. Statistical tests are used to analyse the experimental data. Periodical fluorescence oscillations are observed under both ELF and RF exposures, but their origins are considered to be convection currents due to unevenly distributed energy injections into the solution.

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