Biochemistry and Cell Biology - Masters by Research Theses

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    Generation of a CRISPR-Cas9 mediated knock-in reporter for the GRIA3 candidate gene for schizophrenia
    (University College Cork, 2022-09-27) Breen, Lisa; McCarthy, Tommie V.
    Glutamatergic neurotransmission impairment is considered a major feature of the neurobiology of Schizophrenia (SZ) and implicates genes in this pathway as potential candidates for the condition. A study on α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor genes found strong evidence of association between the Glutamate Ionotropic Receptor AMPA Type Subunit 3 (GRIA3) gene and SZ. Similarly, a recent report has identified a number of genes, including GRIA3, with ultra-rare disabling variants that promote SZ. The association of a rare disabling GRIA3 variant with SZ indicates that reduced expression of the gene predisposes people to SZ and suggests that increasing the expression GRIA3 could be a potential therapeutic avenue for treatment of the condition. The aim of this thesis was to establish a cell model enabling rapid analysis of GRIA3 expression. Such a model would be of high value and in addition to facilitating expression studies on GRIA3, would enable screening for new drugs that increase GRIA3 expression which could have therapeutic potential. This project aimed to modify the cell line using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR associated protein 9 (Cas9) homology directed repair (HDR) approach so that a donor reporter vector containing the Gaussia secreted luciferase gene and linked green fluorescent protein (GFP) or neomycin resistance gene (Neo) gene would be integrated directly under the control of the endogenous promoter of the GRIA3 gene, in a manner that retains intact expression of the GRIA3 protein. This donor reporter vector was successfully constructed and has significant general use as it facilitates cloning of any pair of homology arms and the insertion of a reporter cassette into any target gene via CRISPR-Cas9 HDR. Flanking GRIA3 homology arms were inserted 5’ and 3’ of the reporter cassette for CRISPR-Cas9 HDR mediated insertion into the GRIA3 locus in human U87 glioblastoma cells. Luciferase activity was monitored post-transfection and was present at low levels suggesting successful HDR events. However, the presence of the donor cassette could not be demonstrated at the GRIA3 locus. It was not possible to distinguish if the luciferase activity resulted from read through of the donor plasmid or if a low number of targeted integration events had occurred. Further work involving isolation of individual clones of the targeted U87 cells and checking for the presence of the donor at the GRIA3 locus will be necessary to resolve this question. Overall, this reporter system should be of high value for targeting other loci and can be improved further by modifications to ensure luciferase is only active when inserted into the targeted locus.
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    Targeting transcription-regulating cyclin dependent kinase 12 for the treatment of breast cancer
    (University College Cork, 2022-11-27) Burns, Martha; Krajewska, Malgorzata
    Cyclin-dependent Kinase 12 (CDK12) with its binding partner Cyclin K regulates transcription through phosphorylation of RNA Polymerase II (RNA Pol II) at serine 2. Previous work showed that inhibition of CDK12 leads to decreased expression of DNA damage response (DDR) genes and sensitizes cancer cells to DNA damage-inducing agents. However, the exact mechanism by which this is achieved remains unclear. This study aimed to investigate CDK12 as a therapeutic target in breast cancer and to identify new drug combinations involving the inhibition of CDK12 and clinically relevant inhibitors of DNA repair. We demonstrated that CDK12 inhibitor SR-4835 is cytotoxic in MCF-7 breast cancer cells at a low nanomolar concentration. The observed toxicity was associated with G2-M cell cycle arrest, increased apoptosis, and DNA damage. We showed that CDK12 inhibition had a minor effect on the phosphorylation of RNA Pol II at serine 2 indicating that global transcription was not affected. Interestingly, we observed that CDK12 inhibition resulted in decreased expression of chromodomain helicase DNA binding protein 2 (CHD2), a potentially new target of CDK12. Next, we tested the combination of CDK12 inhibition with inhibitors of DNA repair including inhibitors of PARP (olaparib) and CHK1 (AZD7762) using a colony formation assay. The preliminary results indicate that combining CDK12 and CHK1 inhibition will likely have greater therapeutic potential than the combination of CDK12 and PARP inhibition. Future studies are required to establish the exact role of CDK12 in transcription and DDR as well as to investigate further the potential of combining CDK12 and CHK1 inhibition in breast cancer.
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    Inhibition of the endosomal recycling pathway to overcome resistance to cancer therapies
    (University College Cork, 2022) Fletcher, Kelsey; Lindsay, Andrew
    Cancer is a major public health threat with incidence and mortality rates continuing to rise every year. Breast and prostate cancer are the most diagnosed cancers in women and men, respectively. Both cancers account for roughly 30% of all cancers diagnosed in each sex. Despite the continuous development of new therapies, drug resistance is a growing problem and is a major cause of cancer treatment failure, accounting for many cancer recurrences and deaths. Therefore, new drugs and treatment regimens are urgently required to overcome this resistance. Recent findings from our own lab and others have found that inhibition of the endosomal recycling pathway may be a promising strategy to downregulate clinically relevant cell surface proteins and to overcome drug resistance. This thesis focuses on two clinically relevant hormone receptors that are strongly linked to the development and progression of breast and prostate cancer linked to disease development and progression, the estrogen receptor alpha and the androgen receptor. The aim of this project was to confirm data obtained from a reverse-phase protein array (RPPA) study performed by our lab that found that the endosomal recycling inhibitor primaquine downregulates ER-α and AR expression. We used Western blot, quantitative RT-PCR, and immunofluorescence microscopy to confirm the RPPA results. We found that another endosomal recycling inhibitor, monensin, also potently downregulates these hormone receptors and that both inhibitors synergise with tamoxifen and enzalutamide, standard-of-care therapies for breast and prostate cancer. Keywords Cancer, prostate, breast, androgen receptor, estrogen receptor, endosomal recycling pathway, primaquine, monensin, drug resistance, tamoxifen, lapatinib, enzalutamide, synergy.
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    The regulation of protein degradation at the endoplasmic reticulum
    (University College Cork, 2021-06-28) O'Nualláin, Fearghal; Fleming, John V (Eoin)
    The majority of membrane and secretory protein synthesis in the cell occurs in the endoplasmic reticulum (ER) where proteins reach their native energy stable folded state and are subsequently transported to their functional location. Aberrations in this process whereby a protein cannot reach its intended location or cannot fold correctly often result in accumulation of that protein in the ER lumen. This is toxic to the cell and ER stress is induced in an effort to remove the misbehaving protein to avoid cellular stress and damage. Chaperones can be recruited to assist protein folding but if this is unsuccessful then the protein is marked for degradation via endoplasmic reticulum associated degradation (ERAD). Ubc6 (UBE2J2 in humans) is an E2 ubiquitin conjugating enzyme which catalyses the attachment of ubiquitin to target proteins which marks them for degradation. However, the specific set of substrates which Ubc6 acts upon and the mechanism for its regulation is yet to be understood. In this study, we construct a protein-protein interaction network (PPIN) of Ubc6 to better understand where this protein fits into its environment and to help elucidate what substrates it mediates degradation for. Here, we demonstrate how Ubc6 has the ability to mediate degradation for both a plasma membrane resident protein E-cadherin and also an ER trapped protein HFE C282 (HFE-M). We also show the increased expression of Ubc6 by MAPK signalling through phorbol-12-myristate 13-acetate (PMA) incubation and constitutive BRaf signalling (V600E). Whether this increased expression results in higher Ubc6 stability and/or increased substrate degradation remains to be seen.
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    An investigation of the differential function of actinin-1 and actinin-4
    (University College Cork, 2020-10-24) Nathamuni Suresh, Apoorva; Young, Paul
    Actinins are involved in actin cross-linking and belong to the spectrin family of proteins. They are highly conserved and exist as dimers within the cell. Of the four isoforms of actinin, Actinin-1 (ACTN1) and Actinin-4 (ACTN4) are the non- muscle isoforms sharing a high sequence identity of nearly 87%. They play a significant role in cellular processes including cell migration, proliferation and adhesion. Apart from this, overexpression of Actinin-4 is also closely associated with the invasive phenotype of many cancers and used as a cancer prognosis marker in many cases. However, the molecular function of Actinin-4 overexpression in cancer is not well understood. Furthermore, the sub-cellular localization of the two non-muscle isoforms also varies considerably as reported in previous studies. The most striking difference was the localization of Actinin-4 to the nucleus, where Actinin-4 reportedly acts to modulate transcription. Actinin-1 has not been detected in the nucleus to date. Though neither Actinin-1 nor Actinin-4 contain a consensus nuclear localization signal sequence (NLS), the hydrophobic spectrin repeats (helical protein motifs acting as interaction sites for structural and signaling proteins) are hypothesized to allow its nucleo-cytoplasmic shuttling. Actinin-4 was found to have a consensus nuclear export sequence (NES) which allows its nuclear export mediated by CRM1. Actinin-1, with high sequence similarity to Actinin-4, also contains such a putative motif in its sequence. These observations raise the question as to why Actinin-4, and not Actinin-1, is localized to the nucleus. A possible explanation for such distinguishing characteristics could be unique interacting partners of Actinin-1 and Actinin-4. Another reason could be a higher affinity of Actinin-1 for its cytoskeletal protein partners, as compared to Actinin-4, which sequesters all available Actinin-1. Also it could also be possible that the putative NES-dependent nuclear export of Actinin-1 is much more efficient than its import, such that there is no Actinin-1 population in the nucleus. This work employs an immunoprecipitation assay to identify differences in the interacting partners of Actinin-1 and Actinin-4 in HEK293T cells to select proteins. The results proved that PDLIM1 can act as a positive control, showing strong interaction with both Actinin-1 and Actinin-4. Otherwise no statistically significant differences were observed between Actinin-1 and Actinin-4, though Actinin-4 shows subtly stronger interaction with AKT1 and RAVER1 as compared to the untransfected control. To study localization, fluorescence and confocal microscopy was employed to assess fluorescently tagged actinins. Though no differences were observed in localization of Actinin-1 and Actinin-4, under the experimental conditions employed, this study establishes an efficient system to study localization of Actinin-1 and Actinin-4 simultaneously, previously unutilized. Understanding the underlying molecular mechanism of the difference between the non-muscle actinin isoforms could help identify potential drug targets for cancer therapy.