No description available.

No description available.

No description available.

Alternative splicing functions to generate proteomic diversity and to regulate gene expression in higher eukaryotes. Genome-wide analyses suggest that alternative splicing and transcription typically regulate different gene sets to achieve cell- and tissue-type specificity. However, within individual cell-types, most alternative splicing events occur co-transcriptionally and are impacted by the transcriptional machinery. Despite many focused studies on co-transcriptional regulation of alternative splicing, its mechanisms and functions in regulation of gene expression are still poorly understood. To investigate relationships between transcription and alternative splicing, I performed microarray profiling of alternative splicing and transcript levels during activation of a T cell line. This experiment revealed that different sets of genes and associated functional categories are regulated by alternative splicing and transcription during T cell activation. I next employed inhibitors of RNA polymerase II (Pol II) elongation and microarray profiling to identify genes with coupled changes in splicing and transcript levels when transcription is impeded in activated T cell. Genes that were affected at both levels were significantly enriched in RNA binding and processing functions, and generally displayed increased alternative exon inclusion and decreased transcript levels when transcription elongation was disrupted. Similar effects were observed when transcription was driven by mutant polymerases with reduced elongation activity, and when cells were subjected to stress treatments. Many of the elongation inhibition-sensitive exons from the affected genes introduce premature termination codons into the mRNA, resulting in spliced mRNAs that are substrates of the nonsense-mediated decay pathway and further reduction in mRNA levels. ChIP-Seq experiment demonstrated that Pol II occupancy specifically increased in introns flanking the affected exons. These results provide evidence that a physiological function of transcription elongation-coupled alternative splicing regulation is to regulate the levels of RNA processing factors under conditions that reduce elongation activity, including cell stress. In summary, my thesis research has provided new insights into the integration of transcription and splicing control. While these two regulatory levels can control different gene sets during the activation of T cells, within a given cell type, they are closely coupled to control specific alternative splicing events that appear to coordinate mRNA and RNA processing factors levels.

Alternative splicing functions to generate proteomic diversity and to regulate gene expression in higher eukaryotes. Genome-wide analyses suggest that alternative splicing and transcription typically regulate different gene sets to achieve cell- and tissue-type specificity. However, within individual cell-types, most alternative splicing events occur co-transcriptionally and are impacted by the transcriptional machinery. Despite many focused studies on co-transcriptional regulation of alternative splicing, its mechanisms and functions in regulation of gene expression are still poorly understood. To investigate relationships between transcription and alternative splicing, I performed microarray profiling of alternative splicing and transcript levels during activation of a T cell line. This experiment revealed that different sets of genes and associated functional categories are regulated by alternative splicing and transcription during T cell activation. I next employed inhibitors of RNA polymerase II (Pol II) elongation and microarray profiling to identify genes with coupled changes in splicing and transcript levels when transcription is impeded in activated T cell. Genes that were affected at both levels were significantly enriched in RNA binding and processing functions, and generally displayed increased alternative exon inclusion and decreased transcript levels when transcription elongation was disrupted. Similar effects were observed when transcription was driven by mutant polymerases with reduced elongation activity, and when cells were subjected to stress treatments. Many of the elongation inhibition-sensitive exons from the affected genes introduce premature termination codons into the mRNA, resulting in spliced mRNAs that are substrates of the nonsense-mediated decay pathway and further reduction in mRNA levels. ChIP-Seq experiment demonstrated that Pol II occupancy specifically increased in introns flanking the affected exons. These results provide evidence that a physiological function of transcription elongation-coupled alternative splicing regulation is to regulate the levels of RNA processing factors under conditions that reduce elongation activity, including cell stress. In summary, my thesis research has provided new insights into the integration of transcription and splicing control. While these two regulatory levels can control different gene sets during the activation of T cells, within a given cell type, they are closely coupled to control specific alternative splicing events that appear to coordinate mRNA and RNA processing factors levels.

T helper (Th) cell activation is necessary for the adaptive immune response. Formation of an immunological synapse (IS) between Th cells and antigen-presenting cells is the first step in Th cell activation. In vitro studies indicate that formation of the IS induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca2 influx across the plasma membrane and Ca2 -dependent Th cell activation. Earlier studies have demonstrated that n-3 polyunsaturated fatty acids (PUFA) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFA suppress CD4 T cell mitochondrial translocation during the early stages of IS formation and down-modulate Ca2 dependent Th cell activation. CD4 cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were co-cultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. fat-1 mice exhibited a significantly (P< 0.05) reduced percentage of CD4 T cells with mitochondria which translocated to the IS; fat-1 (30 percent) versus wild type control (82 percent). With respect to an effect on the mitochondrial-to-cytosolic Ca2 ratio, wild type cells showed significant increases at the IS (71 percent) and total cell (60 percent) within 30 min of IS formation. In contrast, fat-1 CD4 T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca2 ratio was observed in wild type cells co-incubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca2 (CRAC) channels, BTP2. Together, these observations provide evidence that n-3 PUFA modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca2 entry.

<p>S. 1-80: sammanfattning, s. 81-159: 7 uppsatser</p> / digitalisering@umu

No description available.

For a long time research has focused on the protein-coding mRNA, but there is a complex world of non-coding RNAs regulating the human body that we yet know very little about. Non-coding RNAs (ncRNAs) are involved in modulation of different cell processes including proliferation, differentiation and apoptosis. In the current study the role of ncRNAs in T cell activation and function was investigated. T cells are important mediators of immune responses, for example upon viral infections. The T helper cells (TH or CD4+ cells) are involved in orchestrating immune processes like aiding the activation of macrophages and enhancement of B cell function. The TH1 cell subtype is generally pro-inflammatory and IFNγ-secreting. There are regulatory T (Treg) cells that are involved in downregulation of TH1 cells, to decrease or terminate the immune response. It has been shown that upon repeated stimulation TH1 cells can switch into a Treg-like IL10-secreting anti-inflammatory phenotype. In the IL10-secreting Treg-like cells the microRNA 150 (miR-150) was found upregulated compared to IFNγ-secreting TH1 cells. Thus, miR-150 was believed to be a candidate in key regulation of the switch between the two phenotypes. Predicted target genes of miR-150 were identified using mRNA arrays investigating down-regulated genes in the IL10-secreting Treg-like subpopulation. In this thesis predicted targets of miR-150 were investigated using luciferase assays. Unfortunately no targets were identified. Upon isolation of IFNγ-secreting TH1 cells and Treg-like IL10-secreting cells, it was found that the ncRNA 886 (nc886) was upregulated in these activated cells, compared to resting TH cells. This indicates that nc886 has an important role in T cell activation. Nc886 has been shown to inhibit PKR activation in other cell types. The effect of nc886 on protein kinase R (PKR) was therefore investigated. PKR shuts down translation upon activation in response to viral double-stranded RNA or cellular stress. We showed that in an activated T cell phenotype nc886 is affecting PKR upon activation by dsRNA from HIV or synthetic origin. The PKR activation pattern is reversed in a resting T cell phenotype.

Humans are exposed to high levels of amorphous silica on a daily basis, via the diet and the use of cosmetic and pharmaceutical products. Amorphous silica particles (10-200 nm) have also been developed for use in biomedical applications, including as binding agents in tissue repair, drug and gene therapy delivery agents, coatings for medical contrast agents and as vaccine adjuvants. Numerous studies have already been conducted to evaluate the cellular toxicity of these silica particles but still little is known about their effects both in vitro and in vivo, especially of nanosilica particles under 10 nm in diameter. The aim of this thesis was to investigate the cellular and in vivo activity of < 10 nm diameter nanosilica particles with different properties (e.g., size and dissolution rate in dilute conditions) as it may infer upon safety after exposure via the diet and intravenous administration (biomedical applications). First, the cytotoxicity of sub-10 nm nanosilica particles, fully characterized by size, dissolution rate, zeta-potential and by NMR spectroscopy, on immune cell function was assessed using transformed and cancerous cell lines and primary cells. The particles were toxic to the immune cells in a dose dependent manner and impaired certain cellular functions. Primary cells were most susceptible to nanosilica induced death and, of the primary cells, phagocytes were most susceptible to its cytotoxicity. Further investigations were conducted to assess the effect of nanosilica on T cells, as there was evidence suggesting that nanosilica particles were directly interacting with these cells. Nanosilica particles 3.6 nm in diameter were found to have a significant effect on T cell function. The particles induced numerous markers of T cell activation, including CD25 and CD69 on CD4 T cells, CD8 T cells, gamma-delta T cells and NK/NKT cells, CD95 on CD4 and CD8 T cells, CD40L, FoxP3, LAP, GARP on CD4 T cells, and IFN-gamma production, but it did not induce T cell proliferation. The particles were found to activate T cells regardless of their antigenic specificity. Further investigations showed that nanosilica interacts with the T cell receptor complex, the first documented case of a non MHC-coated nanoparticle directly interacting with this receptor complex. The nanoparticulate induced signalling through Zap70, LAT, and, eventually, through NFAT but not through MAPK. Similar signalling in the literature has been shown to induce a hyporesponsive T cell state (anergy) or activation induced cell death. The induction of the CD25 and CD69 T cell activation markers was limited to nanosilica particles below 10 nm in size, while similarly sized iron hydroxide nanoparticles (3-5 nm) only induced low levels of CD69 expression on T helper cells. Finally, it was shown that nanosilica is capable of inducing T cell activation in whole blood, though the T cell responses were greatly attenuated. Although identification of activation pathway in vivo remains elusive, the nanosilica particles were shown to have therapeutic value, decreasing murine subcutaneous tumour growth rate and significantly reducing the formation of lung metastases. Whether these in vivo responses are related to T cell activation identified in vitro remains unclear.