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Abstract: Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain–containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif–containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.

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Abstract: Neuronal calcium sensors play a crucial role in different pathways of Ca2+-mediated neurotransmission. Among them guanylate cyclase-activating protein 1 (GCAP1) is expressed only in photoreceptors and activates or inhibits retinal guanylate cyclase 1 (retGC1) depending on cellular Ca2+ concentrations during phototransduction. To date, 22 pathogenic mutations responsible for retinal dystrophy have been associated to GCAP1, but a complete picture of the molecular determinants of the disease is still missing. The only crystal structure available so far is the wt Ca2+-bound monomeric homologue from chicken and no cure exists for retinal dystrophy. In this work I report for the first time that the recombinant human GCAP1 is characterized by a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and by the nature of the divalent ion bound. Surprisingly, I discovered that also the chicken protein shows a similar mechanism, suggesting that this property could be potentially functional for GCAP1 activity and conserved among different species. Despite the large number of crystallization trials, no diffracting crystal of the human GCAP1 was obtained, probably due to the flexible C-terminal tail and the intrinsic dynamicity of the protein. To overcome this issue, I produced a construct lacking the 12 C-term residues and stabilized by a disulfide bridge between the N- and C-term domains which was successfully crystallized. We showed that such engineered construct is able to regulate retGC1 as well as the wt protein. By combining SAXS, protein-protein docking and molecular dynamics simulation we propose two novel three-dimensional models of Ca2+-bound GCAP1 dimer which are stabilized by some of the residues involved in the interaction with the retGC1. We used a biophysical and biochemical approach to thoroughly investigate three pathogenic variants (D100G, E155A and E155G) characterized by mutations in residues directly involved in Ca2+-coordination. All the three variants were able to form oligomers in solutions, showing a decreased affinity for Ca2+ and constitutively activating retGC1 at physiological calcium concentrations. Besides local structural effects, the mutations perturb also the oligomeric state of GCAP1 suggesting that the multimeric assembly of the protein could affect its proper biological function. A recombinant baculovirus for the expression of the cytoplasmic domain of retGC1 in insect cells was produced with the aim to get atomic structural information on the GCAP1/retGC1 5 complex. This will facilitate the identification of drug candidates able to recognize the binding region of the pathogenic GCAP1 mutants with the cyclase and to competitively inhibit the constitutive retGC1 activation, restoring the homeostasis of second messengers which is impaired in retinal degenerative diseases. A preliminary molecular docking based on the crystal structure of the chicken protein was performed and I identified four molecules able to bind the wt human GCAP1 in the millimolar/micromolar range. Together these results shed new light on the quaternary assembly of the wt human GCAP1, showing how the structural changes related to the presence of Ca2+ or Mg2+ are reflected in the different measured dimerization constant. Such conformational changes are in turn likely related to the regulatory mechanism of GCAP1 in the modulation of the retGC1 activity. The differences between the oligomerization state of D100G, E155G and E155A variants suggest a correlation between the altered quaternary assembly of GCAP1 and the aberrant activity of the mutants, representing a step forward to dissect the structural bases of the altered regulatory mechanism of GCAP1 in retinal dystrophies.

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Abstract: We investigate how apparent slight changes to the chemical structure of amino acid-functionalised perylene bisimides (PBIs) affect the self-assembled aggregates formed and their resulting physical and optical properties. PBIs functionalised with L-valine (PBI-V), L-leucine (PBI-L) and L-isoleucine (PBI-I) are investigated due to their similarly branched structure and their assemblies in water were studied using UV-vis absorption spectroscopy, cyclic voltammetry (CV), small angle X-ray scattering (SAXS) and viscosity at different pHs. It was seen that each PBI behaved differently. Each of the PBIs were then used to prepare hydrogels, and their properties again assessed, with PBI-I forming different hydrogels than the other PBIs. By understanding how slight changes in chemical structure can affect bulk properties we become a step closer to designing gels with specific physical and electrical properties.

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Abstract: To be effective, a drug must be efficiently delivered in sufficient quantities over a period of time long enough for it to carry out its desired effect. A major challenge in this sense is poor retention and bioavailability of drugs, particularly those that display poor solubility. The number of newly discovered drugs is disproportionate to the number that make it to market because of less than desirable solubility and permeability profiles, but smart drug delivery systems, such as lipid-based systems, are capable of overcoming these challenges. One particular system, the amphiphilic, biocompatible, and biodegradable lipid cubic phase, has shown promise as an effective carrier system for the controlled release of drugs varying in solubility. This work explores the behaviour of a variety of industrially relevant small molecule pharmaceuticals in lipid cubic phases formulated with different host lipids for potential controlled delivery applications. An understanding of the molecular mechanisms underlying the process of dissolution/diffusion from these phases was elucidated to support the field of controlled drug delivery using in silico molecular dynamic modelling and empirical approaches. A comprehensive characterization approach was taken both macroscopically and microscopically using small-angle X-ray scattering and polarized light to ascertain the mesophase accessed upon incorporation of molecules of varying solubilities and size. In the first instance, the influence of environmental conditions on the release profile of four antihistamine molecules was studied to establish in vitro models that might assist in predicting the dissolution behaviour of a given pharmaceutical with known physicochemical properties. Two model first-generation and two model second-generation H1 antagonist antihistamine drugs were selected and formulated in two separate monoacyglycerol-derived matrices. The impact of encapsulating the molecules in the lipid cubic systems on their mucoadhesive properties was demonstrated using multi-parametric surface plasmon resonance (MP-SPR). With a potential application in developing therapies for the treatment of allergic reactions, the ability of the formulations to inhibit mediator release utilizing RBL-2H3 mast cells with the propensity to release histamine upon induction was presented. Lipid cubic formulations can enhance the intestinal solubility and subsequent bioavailability of notoriously hydrophobic drug entities by reducing drug precipitation and facilitating mass transport to the intestinal surface for absorption. In this context, the aims of the second study were twofold: to evaluate an approach to regulate the rate of degradation of lipid cubic phase drug delivery systems by targeting the enzyme interactions responsible for their demise; and to study the subsequent drug release profiles from bulk lipid cubic gels using model drugs of contrasting hydrophobicity. In a novel approach, monoacylglycerol cubic phases were formulated with a potent lipase inhibitor tetrahydrolipstatin displaying controlled degradation with at least a 4-fold longer release compared to the blank systems. Sustained release of a model hydrophobic pharmaceutical (a clofazimine salt) was studied over 30 days to highlight the advantage of incorporating an inhibitor into the cubic network to achieve tunable lipid release systems. The final aspect of this thesis deals with the interplay between the lipolysis rate and the interfacial interaction of porcine pancreatic lipase with lipid cubic substrates encapsulating the THL. In the final chapter, inhibitor-modified monoolein lipid cubic formulations designed to encapsulate and control the release of a model BCS class IV drug paclitaxel (PTX) were examined under simulated lipolysis in the presence of lipase and its cofactors colipase and calcium.

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Abstract: Human immunoglobulin IgG3 possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved glycosylation sites in the Fc region is unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s020,w of 5.82-6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) of both forms of IgG3 showed a maximum length of 25-28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modelling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically-realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each of the two forms of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively-restricted Fab and Fc conformations joined by an extended semi-rigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2 and IgG4.