The Structural and Functional Study of GIT1 Paxillin Binding Domain

The G protein coupled receptor (GPCR)-kinase (GRK) interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of p21-activated kinase (PAK) and PAK-interactive exchange factor (PIX) to focal adhesions, and its activation is regulated by the interaction between its C terminal paxillin-binding domain (PBD) and the LD motifs of paxillin. In this dissertation, we determined the solution structure of rat GIT1 PBD by nuclear magnetic resonance (NMR) spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting (FAT) domain and the vinculin tail (Vt) domain. The PBD is more stable than the FAT domain and there is no evidence of helix 1 swapping. Previous studies showed that GIT1 interacts with paxillin through the LD4 motif. However, studies in this dissertation demonstrated that in addition to the LD4 motif, the GIT1 PBD can also bind to the paxillin LD2 motif; and both LD2 and LD4 motifs competitively target the same site on the PBD surface. This dissertation also probed the function of paxillin splice variants by comparing their interaction with GIT1 PBD. It seems the paxillin isoforms did not play an important role in determining the affinity to GIT1. We also revealed that paxillin S272 phosphorylation does not influence GIT1 PBD binding in vitro. These results are in agreement with the notion that phosphorylation of paxillin S272 plays an essential role in regulating focal adhesion turnover.This dissertation also computationally derived the complex structures of GIT1 PBD bound with either LD2 peptide or LD4 peptide, based on the experimental binding site information. The LD2 and LD4 peptides bound to GIT1 PBD in a manner similar to the crystal structure of FAT-LD2 complex. The complex structures visualized the reason why both LD2 and LD4 can bind to the same GIT1 binding site. It also addressed the specificity problem in determining paxillin binding to GIT1 versus FAK. Our finding reconciles the controversial observations of earlier studies and provides a clearer picture of focal adhesion regulation. The structural studies of GIT1 PBD presented in this dissertation shed more light on the understanding of GIT functions. The novel findings also allow us to propose a working model regarding FA disassembly

Novel formulations for magnetic-resonance imaging guided theranostics

Recent advances in bioimaging, biochemistry and bioinformatics have facilitated the development of personalized and precision medicine. Theranostics, a combination of imaging modalities and therapeutic agents, have garnered increasing attention in this context, thanks to their potential to monitor and control treatment for individual patients. An attractive strategy to achieve this goal involves the development of therapy guided by magnetic resonance imaging (MRI). MRI, possessing a number of benefits including a high degree of soft tissue contrast, low invasiveness, high depth of penetration and good spatial resolution, could offer advanced imaging-guided therapy enabling precise and time-resolved assessment of disease conditions and therapeutic progression. The goal of this PhD thesis is to develop novel formulations based on polymeric, inorganic or hybrid materials using two pharmaceutical fabrication techniques (electrohydrodynamic atomisation or spray drying), and explore their potential in MRI-guided chemotherapy. Five different types of formulation carrying MRI contrast agents and chemotherapeutic agents were fabricated. Chapter 3 reports the fabrication of pH-responsive formulations via electrodynamic atomization, loaded with superparamagnetic iron oxide nanoparticles (SPIONs) as contrast agents and the model chemotherapeutic carmofur. These platforms are able to protect the cargo from release acidic conditions representative of the stomach, while at neutral pH the relaxivity is tightly correlated to the extent of drug release. Chapter 4 describes a series of dual responsive systems with distinct morphology, comprising of pH-responsive Eudragit shells with SPIONs, and thermo-responsive core loading carmofur. The fibres are found to have better thermo-responsive properties compared to microparticles, and the relaxivity display clear linear relationships with drug release data. Chapter 5 focuses on using spray drying to fabricate nano-in-micro particles based on a synthetic polymer with an upper capital solution temperature. The microparticles encapsulate drug-loaded layered double hydroxide nanosheets, have thermo-sensitive release and relaxivity profile, and in vitro cell studies reveal that the formulations permit synergistic hyperthermia-aided chemotherapy. Chapter 6 details the preparation and characterization of four gadolinium doped layered double hydroxides to develop theranostic platforms carrying chemotherapeutics with high T1-relaxivity. In Chapter 7, polydopamine-coated polycaprolactone/poly(lactic-co-glycolic) acid nanofibers are developed via co-axial electrospinning, which are loaded with dug-loaded LDH nanocomposites in the core. In vitro studies reflect sustained release of chemotherapeutics, and highly effective cytotoxic effects on tumour cells with the polydopamine coated formulations, which was further enhanced at higher levels of glutathione

Novel research methods to examine renewable energy and energy related greenhouse gases: evidence from novel panel methods

In the current time, the most distressing issue is emissions control and environmental recovery. All developed and developing economies are rapidly expanding their industrial sector and increasing energy use while struggling for environmental sustainability. This study aims to analyse whether renewable energy helps BRICS economies reduce energy related emissions. Also, the role of economic growth, research and development, and public–- private partnership investment in energy is investigated during the period from 1990 to 2020. Using various panel data instruments, the results illustrate the slopes heterogeneity, panel crosssection dependence, and the long-run co-integration association between the variables. Using the novel method of moment quantile regression, this study found that economic growth adversely affects environmental quality by triggering energy related emissions. However, renewable energy consumption, research and development, and public–private partnership investment in energy significantly reduce energy related emissions in the region at all quantile (25th, 50th, 75th and 90th). Besides, this study found bidirectional causal nexus between economic growth, renewable energy, research and development, and the energy related greenhouse gas emissions, while unidirectional causality from public–- private partnership investment to energy related emissions. Some relevant policies are suggested that could help tackle the issue of energy related emissions