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Subject: search.filters.subject.320506 Medical biochemistry - proteins and peptides (incl. medical proteomics)

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Now showing 1 - 6 of 6
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    Investigating the molecular basis of Menke Hennekam syndrome : a structural and functional analysis of the TAZ2 & IDR4 domains of CREB binding protein : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2023) Walsh, Courtney
    Menke Hennekam syndrome (MHS) is a rare genetic disorder characterised by intellectual disability and very specific physical abnormalities. MHS occurs as a result of de novo mutations to the cAMP response element binding protein binding protein (CREB-BP or CBP) gene. CBP is a master gene regulator protein, containing both intrinsic histone acetyltransferase activity and transcriptional coactivator activity. MHS associated mutations are located within exons 30 and 31 of the CBP gene, which encode the TAZ2 and IDR4 domains. The role which the TAZ2 and IDR4 domains play in the gene regulation carried out by CBP are yet to be fully understood. Previous research has established DNA binding activity by both TAZ2 and IDR4 domains, in addition to a lack of HAT specificity in the absence of the TAZ2 domain. It is suggested that the TAZ2 domain and neighbouring residues of the IDR4, may be involved in a regulatory interaction with DNA targets of the HAT domain. This study focused on uncovering more about the molecular basis of MHS by comparing the behaviour of MHS associated mutant CBP fragments of the TAZ2 and IDR4 domains with wild type fragments. Comparisons of structure, stability and DNA binding activity were carried out utilising circular dichroism spectroscopy, electrophoretic shift mobility assays and biolayer interferometry assays. The results produced throughout these experiments displayed that the MHS associated TAZ2-IDR4 mutant fragment contained DNA binding activity similar to the wild type TAZ2-IDR4 CBP fragments, during both EMSA and BLItz DNA binding assays. Structural investigations by circular dichroism spectroscopy of the MHS associated CBP mutants revealed a lack of native folding by the mutant protein fragments, which were prone to aggregation throughout expression and purification.
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    FUNctional characterisation of type 1 ryanodine receptor variants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2021) Hewson, Liam Scott
    Malignant hyperthermia (MH) is a pharmacogenetic disorder that presents as a hypermetabolic response to volatile anaesthetics such as isoflurane, desflurane and sevoflurane, as well as the depolarising muscle relaxant succinylcholine. MH reactions are characterised by a wide variety of clinical symptoms, including but not limited to muscle rigidity, tachycardia, hyperthermia, and hyperkalaemia. If left untreated, an MH reaction can result in cardiac arrest; therefore, those with a family history of MH are encouraged to be tested for susceptibility. The current diagnostic procedure is the in vitro contracture test (IVCT), wherein the contractile response of a patient muscle biopsy after exposure to caffeine and halothane is measured. While considered the “gold-standard” for MH susceptibility testing, the reliance of the test on an excised muscle specimen makes it traumatic for the patient, and a financial burden on the healthcare system. As a result, a cheaper and less traumatic genetic test has been under development to one day replace the IVCT. Genetic testing for MH susceptibility focuses on the allelic heterogeneity of a Ca²⁺ release channel known as the type 1 ryanodine receptor (RYR1). RyR1 functions to regulate Ca²⁺ release for the purposes of muscle contraction. Currently, between 50 and 86% of MH reactions can be attributed to variants in the RYR1 gene. In order for MH susceptibility to be accurately diagnosed using a genetic test, known variants of RyR1 must meet a set of requirements to determine their pathogenicity, including being functionally characterised and shown to be hypersensitive to agonists. Over the course of this project, six RyR1 variants, from families known to be susceptible to MH, were functionally characterised. Of these, one was located in the N-terminal hot spot domain (p.Phe539Leu), three were located in the central hot spot domain (p.Gly2183Glu, p.Cys2237Tyr, p.Arg2458Leu), while two flanked the central domain (p.Arg1707Cys, p.Pro2793Leu). The results obtained indicate that the substitutions p.Cys2237Tyr and p.Pro2793Leu produce hypersensitive channels, and while p.Pro2793Leu remains a variant of unknown significance, p.Cys2237Tyr has met the criteria to be classified as likely pathogenic for MH and should therefore be added to the list of MH diagnostic variants.
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    Nucleosomal arrays : a novel method to detect PD-L1 on the cell surface : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Mohammad, Aseel
    Cancer is a complex group of diseases that can be difficult to therapeutically target as cancer cells develop a myriad of mechanisms to spread and survive. Immune evasion is one such mechanism cancer cells use to bypass the immune system and continue to invade other tissues. Evading the immune system is achieved through over-expression of the immune checkpoint protein programmed death ligand-1 (PD-L1). This protein is used as a predictive bio-marker to determine whether cancer patients are viable for PD-L1 immunotherapy, and over-expression of PD-L1 is determined used immunohistochemistry assays. Given that these assays use different PD-L1 antibodies that recognise different epitopes, it introduces variability to staining patterns and scoring matrices. This study has taken advantage of the endogenous high affinity interaction between PD-L1 and its receptor, programmed death receptor-1 (PD-1), to create a novel technique to detect PD-L1 on the surface of cancer cells. Nucleosomal arrays were formed with PD-1 peptide tagged to histone H2B containing octamers and fluorescently labelled DNA. These nucleosomal assemblies had correct nucleosome formation when using biotin-labelled positioning DNA and were able to be detected by binding Avidin-Alexa 488. These nucleosomal arrays bound specifically to PD-L1 on the surface of cancer cells shown by fluorescent confocal microscopy and demonstrated greater specificity than Wild Type H2B containing nucleosomal arrays. The use of nucleosomal arrays in this manner has an advantage over an antibody as there are a greater number of PD-L1 binding sites, and the fluorescent signal is able to be amplified due to positioning DNA being labelled at multiple sites. Though this study used the PD-L1/PD-1 interaction as a proof of principle, other short specific binding domains could be attached to the N-terminal tail of histones that could detect other extracellular proteins. This novel detection method is efficient and specific, and further optimisation in other fluorescent detection platforms may yield a more consistent and high-throughput method to detect extracellular proteins to determine patient response to immunotherapy treatments.
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    Loss of HP1α alters nuclear integrity to promote cellular invasion : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2019) Solomon, Raoul
    The onset of invasion is a key step towards the development of metastatic cancer. For a cell to invade through interstitial spaces in the tissue requires a reduction in nuclear rigidity as the cell needs to deform to squeeze through small spaces. Heterochromatin Protein 1α (HP1α) is a protein that defines domains of heterochromatin, the highly compact regions of the genome, and is essential for maintaining the appropriate patterns of gene expression and genome stability. Loss or reduction of HP1α has been correlated with an increase in invasive potential in human tumours. Using an established model of Drosophila melanogaster epithelial cell invasion, the causative role HP1α plays in suppressing cellular invasive is confirmed within an epithelial tissue microenvironment. This model also demonstrates that loss of the Drosophila melanogaster HP1 homologue synergistically promotes cellular invasion in conjunction with an activated malignant signalling pathway. Importantly, human HP1α is shown to rescue this highly invasive Drosophila phenotype and demonstrates the relevance of this model to human disease, and its use for exploring protein interactions in a cellular microenvironment. As loss of nuclear integrity has been linked to a reduction in peripheral heterochromatin, the biophysical mechanisms by which HP1α acts as a suppressor of invasive potential were explored in the poorly invasive MCF7 breast cancer cell line with constitutive HP1α knock-down. These cells with reduced HP1α expression had a significant loss of nuclear membrane integrity and stiffness. The underlying nuclear lamina meshwork and associated peripheral heterochromatin was disrupted. This was associated with an increased solubility of lamina proteins, particularly lamin A, as well as the altered localisation of a number of peripheral nuclear proteins. In summary, this work established the important contribution of HP1α to the mechanical integrity of the nucleoskeleton and the role HP1α plays in suppressing malignant signalling pathways that promote cell invasion.
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    Development and applications of filamentous phage-derived particles in immunotherapy and diagnostics : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University (Manawatū), New Zealand
    (Massey University, 2020) Rajič, Marina
    Most vaccines that are currently in clinical use induce antibody-mediated responses. However, for many infectious diseases, T cells are an essential part of naturally acquired protective immune responses. T cell-inducing vaccines, such as the one developed in this research, could additionally be used for treatments in cancer or chronic viral infections. One way to target the immune cells and stimulate their responses is to use filamentous phage particles. Filamentous phages (Ff) are ssDNA viruses that infect Escherichia coli, which have been adapted and used extensively in phage display technology and nanotechnology. In this research, a filamentous phage (Ff)-based vaccine carrier was constructed to allow the tuneable display of non-protein immune adjuvant molecules (BODIPY-α-GalCer) and antigenic peptides (OVA; MHC I + MHC II) on the same particle. For the first time, azide groups were incorporated into the recombinant pVIII phage coat proteins that expressed recombinant peptides. Azide groups were subsequently used to attach fluorescently labelled adjuvant molecules, which were successfully presented to NKT cells in vivo. Additionally, high induction of in vitro proliferation of OVA-specific CD8+ T-cells was achieved. However, the Ff-derived particle use outside the laboratory is hindered because they are genetically modified viruses, which in addition, carry antibiotic resistance genes that can be horizontally transferred to gut bacteria. These limitations were overcome by developing a system for efficient production of non-replicating, controllable-length protein-DNA nanorods, derived from Ff, named BSFnano (~100 × 6 nm). In this research, functionalised BSFnano particles were constructed, and their application in diagnostics was tested in a proof of concept dipstick assay for detection of a soluble analyte (fibronectin). For the first time, an ultrasensitive dipstick assay was achieved with Ff-derived nanorods, detecting the test-analyte at a concentration of as low as 0.04 pg/μL, equivalent to only 100,000 molecules/μL. Overall, while the phage-based vaccine produced in this research elicited CD8+ T-cell responses in vitro, but not in vivo, the Ff-derived nanorods were successfully functionalised and tested in lateral flow immunoassay, with promising implications for use in point of care diagnostics.
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    Functional consequences of RyR₁ variants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2019) Parker, Remai
    Malignant hyperthermia (MH) is an uncommon pharmacogenetic disorder that is asymptomatic until triggered by volatile anaesthetics or depolarising muscle relaxants. Exposure to such a trigger can result in a potentially fatal hypermetabolic crisis in an MH-susceptible individual. With prior diagnosis, MH episodes can be avoided by using alternative anaesthesia. Diagnostic testing requires a morbidly invasive muscle biopsy for those considered at risk based on family history. Linkage of MH-susceptibility to variants in the skeletal muscle calcium release channel ryanodine receptor 1 (RyR₁) has provided an opportunity for DNA testing as an alternative to the muscle biopsy. DNA-based diagnosis is severely limited by the number of diagnostic mutations identified—only 50 mutations have been established as MH-causative from over 300 genetic variants associated with the disorder. Moreover, DNA testing may only diagnose an individual as MH-susceptible; a negative DNA test is insufficient under current guidelines for a negative MH diagnosis. The purpose of this study was to develop molecular tools to investigate the hypothesis that RyR₁ variants associated with MH-susceptibility cause dysregulation of calcium release from intracellular stores. Two experimental approaches were followed with the objective of expanding the capabilities of DNA-based diagnosis for MH. The first technique was the generation of mammalian cell lines stably expressing recombinant RyR₁ variants by use of the Flp-In™ T-REx™ system from Invitrogen, followed by functional analysis. Four of five genetic variants associated with MH or myopathy had altered sensitivities to an RyR₁ agonist and therefore meet the criteria for use as diagnostic variants for MH-susceptibility. The second molecular technique explored was gene editing, with the aim of showing that a single nucleotide change was both necessary and sufficient to cause MH-susceptibility. This was developed by introducing a well-characterised MH-causative variant into the genome of a human skeletal muscle cell line. Preliminary results indicated that gene editing was successful.