3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Comparing changes in gene expression across three myeloid cell lines during monocyte-to-macrophage differentiation(2022) Cosser, Duncan AlexanderMonocytes and macrophages exhibit wide heterogeneity in phenotype and function. Despite this variety, cells from model cell lines are often used in experiments without accounting for these differences. We therefore sought to characterize the gene expression profiles of three model myeloid cell lines as they differentiate from a monocyte-like state to a macrophage-like state in order to identify core pathways across cell lines central to the differentiation process, as well as pathways uniquely involved in differentiation in specific cell lines. Using an RNA-seq analysis pipeline we developed, we examined gene expression in three myeloid cell lines – HL60, U937, and THP-1 – during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation. Using gene expression data from three experiments which induced macrophage differentiation in cells from these cell lines using PMA, we constructed gene expression profiles for the cells before and after PMA treatment. We performed differential gene expression analysis to find the genes that are differentially expressed in differentiation, followed by pathway overrepresentation analysis to identify key pathways involved in this process. We identified 1789 genes which were differentially expressed in all cell lines, as well as 2131 genes marked as differentially expressed only in HL60 cells, 1711 in U937 cells, and 2159 genes uniquely differentially expressed in THP-1 cells. Through subsequent investigation of specific gene clusters, we found that there were conserved pathways involved in the monocyte-to-macrophage transition in all cell lines, particularly those involved in phagocytosis and adherence. All cell lines also downregulated maintenance pathways involved in DNA replication and cell cycle control, a behaviour associated with differentiation. Additionally, we found that there were differences in differentiation between the cell lines: the ribosome and oxidative phosphorylation pathways were strongly down-regulated during the transition in HL60 cells, and multiple different components in the cytokine-cytokine receptor interaction pathway were up- and down-regulated in THP-1 cells. A core gene expression profile is associated with PMA-induced differentiation of myeloid cell lines to a macrophage state. However, differentiation of HL60 and THP-1 cells also induces distinct changes in metabolism and cellular signalling. Our research recommends discretion in cross-comparison between experimental results incorporating gene expression studies of these myeloid cells.Item Oncogene expression in hepatocellular carcinoma and cells(2016) Arbuthnot, Patrick BrianAn investigation has been made into aspects of the expression of oncogenes in normally dividing cells and in hepatoceilular carcinoma (Hee). HOC occurs commonly in Southern Africs, and thf1aetiology ·ofthis tumour lsaseccieted with hepatitis a virus (HBV) infection. c·erbA, c..mva and e-tos but not c~Ha..res mANA were elevatad in tumours and adjacent hepatic tissue from the same petiEJ;htswhen compared to normal liver. Amounts of Fos and MYQ prot~in in the liver tumour specimens were else raised. The"e was some correlation between the patients' serum a..fetoproteirt concentretlons, histological features of tumour differentiatic)t"l, c..mvc and c40s r.ixpression. expression of e-tas and c..myc has been reportec to be elevated after stimulation of cells to alvlde, ,'1$ occurs during liver r19ganeration. This was corroborated by the findin~ that c-mvc, c·fo~· and c-jun mRNA concentratlona "Jere increased it"! cultured 3T6 mouse fibroblasts following treatment with alkaline medium aa a mitogenlo stimulus. The time course of the expression of these oncogenes was similar to that reported after gro\l'l/th factor sttmulation, The H[~V X..gene ma\' be responsible for increased oncogene expression it' YCC as a result of its documented trans activating properties. This vi!'a~ gene is unusual in that it has a codon preferanc";which is similar to that of eukarvotic ceU genes. Also HBV may ha'V& evolved from ti similar ancestral virus to that giving rise to retroviruses. These ideas suggest that the HBV X·gene is a viral oncogene derived from a host homologue. Low stringency Northern brot hybridisation using a X-gene probe denlonstrated a murine transcrlpt in heart and thymus. Attempts to isolate the sequence from mouse heart and thymus eDNA libraries ware unsuccessful despite ext,~n$jve screening with sensitive probes (SP6 palymerfjsa and peR fab(':.lUed X~gen~~fragments). Conserved X~gene \ . I sequences were also used fot the desigr:Jof primers in .~.peR bas£'d method " . II aimed at isolating a mammalian sequence. No sinnificant sequsnce \\ homology was found bet\lveen the HBVI\X..gene and Ol\A ampllfle'd from \1 l! gen(llmic and eDNA I1br'srytemplate sou~\pes.The peR preducts ttppeared to have been artef.,ots of arnplWaation. ~~n'IJreto detect the hQrtll.)logous gene may have resu~ted from poo' complS,JIlentarity between the VIral ant! \\ mammalian secuencec, 1\ \\ Non..~pecific amplification is commonly enct~unter&d when u$1110 PCli'. A qtJick asvmmatrlc re·ampW~catj(ii1 method I,?ssed on eXUOSilin of an " interm.uly' hybrfdising X·gelllapfimar we! davisQ\j to confirm FICRprOdu(,ts. The l"n1ithodwas specific irlthat "ver~ single bas~ mlsmatohe$ betwsen the internal primer and tem1>late re;.,ultad in fatJut~ of dete(;tabla \tUim$f extension.