413 research outputs found

    Investigating the effects of palmitoylation on the dopamine 1 receptor (D1)

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    The dopamine D1 receptor (D1) is a G protein-coupled receptor (GPCR) which regulates various key brain functions like attention, movement, reward, and memory. Understanding D1 signalling may open the horizon for novel treatments for neurological disorders. Upon agonist activation, the heterotrimeric G proteins Gαs activate adenylyl cyclase to increase cAMP/PKA signalling. D1 also engages β-arrestin proteins leading to β-arrestin dependent signalling. The D1 has two palmitoylation sites on cysteines 347&351 in its C-tail domain. However, the distinct roles and implications of palmitoylation on the D1 signalling, trafficking and β-arrestins recruitment are still largely unexplored. A palmitoylation D1 mutant was generated and luminescent based techniques such as BRET and split-Nanoluc complementation assay were employed, to delineate D1 palmitoylation effects on its pharmacology and signalling. The D1 agonists induced 50% less cAMP production in the mutant compared to wildtype (WT) and WT showed a more efficient dissociation of its Gαs. Moreover, the mutant receptor failed to recruit β-arrestin1&2, induced less ERK1/2 activation and internalises in an agonist-independent process while showing an altered intracellular Golgi trafficking. Also, in β-arrestin 1&2 KO HEK 293 cells similar cAMP production levels were reported for D1 WT and palmitoylation mutant. β-arrestin 1&2 KO blocked agonist-induced WT D1 plasma membrane trafficking, indicating that these β-arrestins are driving the differences between WT and the palmitoylation mutant D1. Taken together, our studies indicate that Gαs is the main transducer for D1 cAMP and ERK1/2 signalling and that palmitoylation is essential for its β-arrestin 1&2 interactions and modulating D1 signalling cascades in a drug-dependant process

    Current reviews depicting therapeutic potential of novel drug delivery system in rheumatoid arthritis

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    Modern advancement in science and technology has altered the way we detect, treat and prevent different diseases in all aspects of human life. Rheumatoid Arthritis (RA) is chronic progressive autoimmune disease in which body’s immune system has role to protect the health by attacking foreign bacteria but the virus mistakenly attacking the joints as a result thickened synovium, pannus formation, & destruction of bone, cartilage occurs. Researchers are still researching but are unable to know the exact reason for the disease. Although, it is believed that genes and environmental factors play an important role in the development of RA. In this review the pathophysiology, predictors and factors involved in pathogenesis of RA have been investigated. The conventional drug therapeutic agents and emerging novel drug delivery system (NDDS) like nanoparticles, dendrimers, micelles, microspheres, liposomes and so on are discussed, as these are the tools which show promising effect in overcoming the limitations associated with conventional drug delivery systems. Although several NDDS have been used for various purposes, liposomes have been focused and found to have its potential applications in RA diagnosis and therapy. In addition, the therapeutic effectiveness, and challenges for RA by using these novel drug delivery systems have been reviewed along with its future perspectives

    The Host-Microbiota Axis in Chronic Wound Healing

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    Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and communitycomposition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management

    The study of renal function and toxicity using zebrafish (Danio rerio) larvae as a vertebrate model

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    Zebrafish (Danio rerio) is a powerful model in biomedical and pharmaceutical sciences. The zebrafish model was introduced to toxicological sciences in 1960, followed by its use in biomedical sciences to investigate vertebrate gene functions. As a consequence of many research projects in this field, the study of human genetic diseases became instantly feasible. Consequently, zebrafish have been intensively used in developmental biology and associated disciplines. Due to the simple administration of medicines and the high number of offspring, zebrafish larvae became widely more popular in pharmacological studies in the following years. In the past decade, zebrafish larvae were further established as a vertebrate model in the field of pharmacokinetics and nanomedicines. In this PhD thesis, zebrafish larvae were investigated as an earlystage in vivo vertebrate model to study renal function, toxicity, and were applied in drug-targeting projects using nanomedicines. The first part focused on the characterization of the renal function of three-to four-dayold zebrafish larvae. Non-renal elimination processes were additionally described. Moreover, injection techniques, imaging parameters, and post-image processing scripts were established to serve as a toolbox for follow-up projects. The second part analyzed the impact of gentamicin (a nephrotoxin) on the morphology of the pronephros of zebrafish larvae. Imaging methodologies such as fluorescent-based laser scanning microscopy and X-ray-based microtomography were applied. A profound comparison study of specimens acquired with different laboratory X-ray-based microtomography devices and a radiation facility was done to promote the use of X-ray-based microtomography for broader biomedical applications. In the third part, the toxicity of nephrotoxins on mitochondria in renal epithelial cells of proximal tubules was assessed using the zebrafish larva model. Findings were compared with other teleost models such as isolated renal tubules of killifish (Fundulus heteroclitus). In view of the usefulness and high predictability of the zebrafish model, it was applied to study the pharmacokinetics of novel nanoparticles in the fourth part. Various in vivo pharmacokinetic parameters such as drug release, transfection of mRNA/pDNA plasmids, macrophage clearance, and the characterization of novel drug carriers that were manipulated with ultrasound were assessed in multiple collaborative projects. Altogether, the presented zebrafish model showed to be a reliable in vivo vertebrate model to assess renal function, toxicity, and pharmacokinetics of nanoparticles. The application of the presented model will hopefully encourage others to reduce animal experiments in preliminary studies by fostering the use of zebrafish larvae

    Hansen solubility parameters as a predictive tool for the development of oral polymeric nanoparticle delivery systems

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    Oral drug delivery proves to be the ideal means of drug administration however it is associated with challenges such as drug dissolution and drug permeability, particularly in BCS class IV drugs. These contribute to a reduction in drug bioavailability which accounts for 40% of failures in the developmental stages of oral drug formulations. As a result, drug delivery systems have been designed to alleviate these obstacles and improve oral delivery of various drugs. An example of such drug delivery systems is the use of N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N trimethyl-6-O-glycolchitosan (GCPQ) as nanocarriers. An amphiphilic polymer which has the ability to self-assemble into micelles in solution which are then able to traverse the mucosal layer – a common limitation in oral drug delivery. GCPQ comprises of a range of functional groups which could be chemically modified to achieve required properties. Investigation of various modifications using polymer chemistry allows for a better understanding of the independent effects and relative importance of each individual modification on the process of drug delivery. However, the empirical approach to optimisation of drug delivery systems can be time consuming and resource intensive, potentially also contributing to an increase in time to market. Theoretic predictions have been utilised as a means to evade these challenges. An example of these approaches is the Hansen Solubility Parameters (HSP) predictive tool. HSP was employed using two class IV drugs (curcumin and caspofungin), and their interactions with GCPQ constituents was analysed to guide experimental design. This showed unfavourable predictions despite evidence to support encapsulation capability of GCPQ. Encapsulation experiments carried out with varying degrees of palmitoylation (DP) and quaternisation (DQ) showed data contrary to HSP predictions with EE of curcumin and caspofungin reaching 90% and 96% respectively. A thin film method was used and formulations characterised, with their accelerated stability in aqueous solution studied under two storage conditions for one week. Upon oral administration in vivo, GCPQ-CFG 5mg/kg enhanced bioavailability to a comparable degree as caspofungin acetate, the soluble formulation of caspofungin, with plasma Cmax values of 242±361ng/mL at a Tmax of two hours. Biodistribution analysis showed trace amounts of caspofungin were also found in liver and heart tissues up to 12 hours post dosing at 20 ng/kg and 60ng/kg respectively. An oral CFG formulation has been prepared with the ability to increase oral bioavailability of drug systemically

    Hemodynamic Quantifications By Contrast-Enhanced Ultrasound:From In-Vitro Modelling To Clinical Validation

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    Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function

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    In terms of lipid nanoparticle (LNP) engineering, the relationship between particle composition, delivery efficacy, and the composition of the biocoronas that form around LNPs, is poorly understood. To explore this we analyze naturally efficacious biocorona compositions using an unbiased screening workflow. First, LNPs are complexed with plasma samples, from individual lean or obese male rats, and then functionally evaluated in vitro. Then, a fast, automated, and miniaturized method retrieves the LNPs with intact biocoronas, and multiomics analysis of the LNP-corona complexes reveals the particle corona content arising from each individual plasma sample. We find that the most efficacious LNP-corona complexes were enriched with high-density lipoprotein (HDL) and, compared to the commonly used corona-biomarker Apolipoprotein E, corona HDL content was a superior predictor of in-vivo activity. Using technically challenging and clinically relevant lipid nanoparticles, these methods reveal a previously unreported role for HDL as a source of ApoE and, form a framework for improving LNP therapeutic efficacy by controlling corona composition.</p

    Development of topical ophthalmic formulations for fungal keratitis treatment

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    In the present thesis, different topical-ophthalmic formulations of antifungals and cyclodextrins were developed for the treatment of fungal keratitis. In addition, the safety and permanence on the ocular surface of different cyclodextrins were evaluated in order to know their behavior in the eye and to optimize their use in the development of new ophthalmic formulations
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