Biochemistry and Cell Biology - Doctoral Theses
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Item Investigation of a novel cell penetrating protein for the delivery of biomolecules(University College Cork, 2024) Cantillon, Emer A.; Fleming, John V (Eoin); Sanghamitra, Nusrat; CyGenica Ltd.Cellular delivery of therapeutically valuable macromolecules such as proteins and nucleotides, or genome editing systems such as CRISPR-Cas9, are key to advancing the treatment of many diseases. To make this a therapeutic reality however, the safe delivery of cargo across the cellular membrane remains a key obstacle. Furthermore, the development of molecular tools that target intracellular compartments and tissue specific subtypes is necessary in order to improve the therapeutic efficiency of macromolecules and negate off-target effects. In this context, several viral and non-viral systems have been developed to varying degrees, but there remain problems with immunogenicity, carcinogenesis, toxicity and low in-vivo delivery efficiency (1–3). Here we describe a protein, termed GEENIE, and identify its novel cell penetration capabilities, demonstrating that it can translocate across the plasma membrane of mammalian cells. We show that GEENIE can penetrate cells in a time dependent manner and can proceed in the presence of serum. Additionally, using a combination of biochemical and pharmacological experiments, we demonstrate that the mechanism of GEENIE uptake is not limited by endocytosis and confirm GEENIE uptake in red blood cells that lack endocytic machinery. Having demonstrated that GEENIE can cross the plasma membrane, we then extended its application to the delivery of biomolecules. As for other non-viral delivery systems, we first assessed the ability of GEENIE to deliver protein cargo such as fluorescent proteins. Using recombinant DNA technology, we expressed GFP tagged GEENIE chimeras. This strategy allowed for the quick and affordable purification of chimeric proteins, whereafter their cellular uptake was visualized using confocal microscopy. We were able to successfully demonstrate the ability of GEENIE to deliver GFP protein cargo intracellularly. The delivery of biotherapeutics can often be limited by the off-target effects that cause excessive cytotoxic to healthy, non-diseased cells. The cell specific delivery of therapeutics is therefore an important stipulation for delivery vehicles in order to maximize therapeutic output and reduce off target effects. The development of delivery vehicles that contain a moiety to target and deliver to a specific cell surface receptor is a common strategy to improve cell specific targeting. To this end, we investigated a newly identified peptide that has shown specificity to the HER2 receptor. Incorporating the peptide within GEENIE, allowed for increased cell specificity to HER2 expressing cells (SKBR-3) compared to HER2 negative cells (MDA-MB-468). Having demonstrated that GEENIE can be targeted to cell specific subtypes, can traverse the cellular membrane and deliver protein cargo, we next evaluated a strategy to deliver biomolecules involved in gene therapies. Gene therapies are an important class of therapeutics that have potential in the treatment of a wide variety of diseases, while also providing an opportunity to progress personalized treatment strategies. Two important groups include nucleic acids, such as siRNA, and gene editing toolkits, such as CRISPR. Employing a genetic engineering approach, we produced two separate GEENIE chimeras, R9-GEENIE and Cas9-GEENIE, to achieve delivery of siRNA or Cas9-sgRNA ribonucleotide protein respectively. Despite our optimization attempts we were unable to produce a functional R9-GEENIE to achieve siRNA delivery and protein knockdown. On the other hand, we demonstrated a potential strategy for the delivery of Cas9-RNPs via GEENIE. Future studies to optimize gene knockout may provide extra insight into whether GEENIE can be used for this strategy, and whether it has clinical potential for the delivery of Cas9.Item A family of multi-functional phosphorescent bioprobes based on the PtPFPP scaffolding moiety(University College Cork, 2023) Zanetti, Chiara; Papkovsky, Dmitri B.; Maguire, Anita; Science Foundation IrelandAccurate monitoring of O2 levels is crucial for understanding the growth, differentiation, and function of individual cells and tissues. While phosphorescence-based oxygen sensing offers great potential for such investigations, current probes still show limitations in terms of their bio-distribution and analytical performance. New improved O2 probes are highly needed. In this project we synthesized a family of new hetero-substituted phosphorescent derivatives of Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye, through click-modification of its para-fluorine atoms with 1-β-D-thio-glucose, neutral (mPEG) and carboxy (cPEG) thiol-PEGs, named as PtmPEG31Glc and PtcPEG31Glc respectively, or cysteamine (CA), named PtCA31Glc. Derivatisation with 2-thioelthyl-β-D-glucopyranoside was also performed to evaluate the effects of alternative SH-Glucose structures on cellular uptake, obtaining the corresponding 2Glc derivatives. The new probes comprise one cell-targeting moiety and three polar moieties, forming a hydrophilic shell. We also synthesized the di-glucosilated-di-PEGylated derivative, named as PtmPEG21Glc2 (trans), in which 1-thio-β-D-glucose (1Glc) and thio-methyl-polyethylene-glycol (mPEG) moieties were covalently attached to PtPFPP in trans- position. Tetra-substituted derivatives Pt1Glc4, Pt2Glc4, PtmPEG4 and PtcPEG4 were also synthesised and characterized for benchmarking. The chemical synthesis and purification procedures were optimized for all the new derivatives to achieve high reaction yields and scalability, and new chemical structures were confirmed by HR-MS and NMR. Then, the most promising derivatives were assessed for their spectral properties and cell penetrating ability in 2D mammalian cell cultures using time-resolved fluorescence (TRF) on a standard plate reader, and confocal microscopy in PLIM mode (phosphorescence lifetime imaging). Probes' ability to function as either cell permeable or impermeable was found to depend on polarity, molecular charge, size, and substitution position of the bioconjugate. Notably, the hetero-substituted tri-PEGylated (PtcPEG31Glc, PtmPEG31Glc) and tetra-PEGylated (PtcPEG4, PtmPEG4) derivatives showed reduced cell internalization compared to the 3:1 cysteamine (PtCA31Glc) derivative and 4:1 glycosylated probe (Pt1Glc4). This finding opens possibilities for their extracellular applications. In contrast, PtmPEG21Glc2 (trans), demonstrated efficient cell staining and suitability for intracellular oxygen sensing (IcO2) in murine embryonic fibroblast (MEFs) cells. Moreover, biocompatibility studies indicated no significant cytotoxicity for all probes at any tested concentrations, except for PtCA31Glc which caused reduced cell viability at concentrations above 10 µM. The chemical modifications did not alter absorption and emission spectra of the PtPFPP moiety but reduced its brightness and lifetimes. Some of the PEGylated derivatives showed tendency to aggregate in biological media. PtmPEG4 and Pt1Glc4 were also evaluated in microbial respirometric assays with Escherichia coli (E. coli) cells, however due to the lack of shielding, their performance in complex media was not as good as the macromolecular MitoXpress probe and the nanoparticle based NanO2 probe. Overall, our new phosphorescent bioprobes, particularly the hetero-substituted structures, extend the library of extracellular and intracellular probes for monitoring and imaging O2 in mammalian cell cultures. They are compatible with standard TRF plate readers and PLIM microscopes, making them promising candidates for physiological studies with cells. The established structure-activity relationships (SARs) will also help to develop new probe structures for future studies.Item The gut microbiome of the wild great tit (Parus major): drivers and fitness consequences(University College Cork, 2023) Somers, Shane Edmond; Quinn, John; Ross, R. Paul; Stanton, Catherine; Irish Research Council for Science, Engineering and Technology; European Research Council; Science Foundation IrelandThe gut microbiome plays a vital role in its host’s ecology. Clinical studies have shown gut microbes increase host health and fitness by providing digestive and immune functions, as well as aiding development. Natural variation in the microbiome is widely believed to affect host fitness in the wild but we are lacking experimental studies to test this. The microbiome varies with both host and environmental factors but most studies to date have focussed on individual factors and not adequately addressed the multiple overlapping and hierarchical drivers of microbiome variation working at environmental, host and microbial scales. This thesis investigates the role of the gut microbiota in host fitness, and how this is affected by and varies across contexts. Additionally, we address sources of variation in the gut microbiota at a host and environmental level, accounting for host ecology and drivers at different scales. We find that the host’s weight is correlated with microbiome diversity during development but that the direction of this relationship is context dependent. This shows that the microbiome interacts with the environment to determine host fitness and is important because it helps explain the contradictory findings linking diversity to weight. We also show that the interaction between the host, its microbiome and environment change with developmental stage. Specifically, we found that the microbiome of developed individuals is remarkably resilient to environmental perturbation, while developing individuals are much more sensitive, with important implications for future experiments. We developed a novel method for experimentally perturbing the microbiome that will allow microbiome researchers to begin testing hypotheses linking the microbiome to host ecology and evolution in natural settings. Finally, we show that welfare measures, such as environmental enrichment may interact with the gut microbiota to impact on host health and behaviour. In summary, I show that variation in the microbiome is linked to host ecology and that this variation is linked to host fitness.Item Functional characterisation of LNX1 and LNX2 proteins(University College Cork, 2022) Lenihan, Joan A.; Young, Paul; Irish Research CouncilLigand of Numb protein X1 (LNX1) and LNX2 are E3 ubiquitin ligases that contain a catalytic RING (Really Interesting New Gene) domain and four PDZ (PSD-95, DlgA, ZO-1) domains. LNX1 and LNX2 can interact with Numb – a key regulator of neurogenesis and neuronal differentiation. LNX1 can target Numb for proteasomal degradation, and Lnx mRNAs are prominently expressed in the nervous system, suggesting that LNX proteins play a role in neural development. This hypothesis remains unproven, however, and our understanding of LNX protein function is very limited – largely because LNX proteins are present at very low levels in vivo. Chapter 2 of this thesis addresses this – investigating possible reasons for the low levels of LNX proteins observed in vivo, at both transcriptional and translational levels, and also in terms of protein stability. Luciferase reporter assays show that the 5’ untranslated region of the Lnx1_variant 2 mRNA, that generates the LNX1p70 isoform, strongly suppresses protein production. This effect is mediated in part by the presence of upstream open reading frames (uORFs), but also by a sequence element that decreases both mRNA levels and translational efficiency. By contrast, uORFs do not negatively regulate LNX1p80 or LNX2 expression. Instead, some evidence is presented that protein turnover via proteasomal degradation may influence LNX1p80 levels in cells. To gain functional insights into the LNX family, Chapter 3 details the first physiologically relevant affinity purification/mass spectrometry-based analysis of the LNX interactome. In the context of mammalian cells, this approach identified a large number of novel LNX1-interacting proteins, as well as confirming known interactions with NUMB and ERC2. Many of the novel interactions mapped to the LNX PDZ domains, particularly PDZ2, and many showed specificity for LNX1 over the closely related LNX2. It was shown that LIPRIN-α1, KLHL11, KIF7 and ERC2 are substrates for ubiquitination by LNX1. LNX1 ubiquitination of LIPRIN-α1 is dependent on a PDZ binding motif containing a carboxyl terminal cysteine that binds LNX1 PDZ2. Surprisingly, the neuronally-expressed LNX1p70 isoform, that lacks the RING domain, was found to promote ubiquitination of Liprin-α1 and KLHL11, albeit to a lesser extent than the longer RING-containing LNX1p80 isoform. Of several E3-ligases identified in the LNX1 interactome, interactions of LNX1 with MID2/TRIM1 and TRIM27 were confirmed. On this basis, a model is proposed, whereby LNX1p70 - despite lacking a catalytic RING domain, may function as a scaffold to promote ubiquitination of its ligands through recruitment of other E3-ligases. Proteomic analysis of LNX-interacting proteins in the context of brain tissue identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2, and LIPRIN-s (PPFIA1, PPFIA3), as well as the F-BAR domain proteins FCHSD2 (nervous wreck homolog) and SRGAP2. To examine the role of LNX proteins in vivo, mice lacking both LNX1 and LNX2 expression in the brain were generated. Surprisingly, these mice are viable, fertile and physically healthy (Chapter 4). Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety-related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. The proteomic analysis (Chapter 3) revealed several novel neuronal LNX-interacting protein candidates that might contribute to the anxiolytic phenotype observed. Overall, these findings provide novel functional insights into the LNX protein family and identify promising candidates to mediate LNX functions in the central nervous system.Item An investigation of the development and evaluation of online approaches for improved kinaesthetic learning in science(University College Cork, 2023) Scanlan, Anna M.; McCarthy, Tommie V.; Kennedy, DeclanKinaesthetic learning approaches (KL) offer great potential to enhance learning in the advanced molecular sciences. However, online KL remains under-researched and poorly implemented on affordable, scalable platforms. Furthermore, there appears to be a disconnect between the fields of education psychology and neuroscience when discussing kinaesthetic techniques. This research connects what is known from both disciplines to provide a coherent overview of what constitutes kinaesthetic learning. Here, an online KL assembly model is presented which proved effective for learning advanced molecular science topics as exemplified by three different lessons: the Lac Operon gene regulation system in E. coli, DNA transcription and translation, and Salmonella virulence factors. A mixed-methods study was conducted including three pilot studies, three randomised control trials and two sub-studies. Study participants included over 100 students from a variety of secondary schools (typically aged 16-19 years), over 250 first-year undergraduate science and medicine students, and 18 postgraduate students from both science and non-science disciplines. Topics were chosen for which each cohort would have little to no prior learning. Results show that KL assembly was at least as effective and, in some instances better than, some top learning strategies identified in education psychology namely, computer notetaking (Trafton & Trickett, 2001; Bui et al., 2012; Chi & Wylie, 2014) and retrieval-practice (O’Day & Karpicke, 2021). KL assembly involving both movement and recall was most effective overall for long-term learning retention, and for learning science material that is represented in a complex graphical and text format.