<p>The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown.</p><p>Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In <em>paper I, </em>genetic alterations in <em>Notch1 </em>and the Notch1 regulating gene <em>CDC4 </em>were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating <em>Notch1</em> mutations in 39% of the lymphomas, suggesting that <em>Notch1 </em>is<em> </em>an important target gene for mutations in chemically induced lymphomas.<em></em></p><p>While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes <em>per se</em> are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In <em>paper II</em>, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including <em>Notch2</em>, were transcriptionally downregulated. We also confirmed binding of Lef1 to <em>Notch2</em> as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the <em>Notch2</em> promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target <em>cyclin D1</em>. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines.</p><p>In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases.</p> / Studies on molecular genetic alterations in colorectal cancer

Gene therapy has emerged as a field to modulate cell functions by introducing genes of interest to target cells. An emerging focus in this field is to employ non-viral vectors to deliver immunosuppressive cytokines to dendritic cells (DCs) to attenuate damaging immune responses. DCs serve as potential targets for suppression of T cell responses. In this work, we investigated the ability of polycation-modified polymeric particles complexed with interleukin-10 (IL-10) gene to modulate DCs. The delivery systems (designated as PSO10H6 and PLGAO10H6) were formed by coating cationic peptide O10H6 (O: ornithine; H: histidine) on the polystyrene (PS) and poly (lactic-co-glycolic acid) (PLGA) particulates. A mouse IL-10 encoding plasmid (pIL-10) was loaded on the surface of PSO10H6 and PLGAO10H6 via ionic interactions. Physical characterization of these particles revealed stable colloidal dispersions (diameters: 297.2&#177;14nm in PLGAO10H6-pIL-10 and 126.0&#177;8nm in PSO10H6-pIL-10). DNA molecules carried by PSO10H6 and PLGAO10H6 were protected from serum digestion. Results from in vitro gene transfection studies showed two-fold enhancement of IL-10 expression in bone marrow-derived DCs transfected with PSO10H6-pIL-10 and PLGAO10H6-pIL-10 compared to untransfected DCs. Their suppressive functions were evaluated in an in vitro mixed lymphocyte model. Results indicated that PSO10H6-pIL-10 and PLGAO10H6-pIL-10 modified DCs elicited weakest proliferation of allogeneic bulk T cells as well as CD4 and CD8 T cells among all the delivery modes. Using cell-embedded Matrigel as a surrogate graft, we showed that IL-10 gene-modified DCs suppressed host cell infiltration in vivo. These data suggested PSO10H6-pIL-10 and PLGAO10H6-pIL-10 deliver an overriding suppressive signal to T cells. Further studies revealed T cells stimulated by the IL-10 gene-modified DCs exhibited characteristics of regulatory T (Treg) cells, as evident by up-regulation of a Treg cell marker forkhead-type transcription factor 3 (Foxp3). This result was concomitant with an increase in of transforming growth factor beta (TGF-beta) production. &lt;br&gt;Taken together, this work demonstrated that PSO10H6 and PLGAO10H6 are effective in delivering pIL-10 to modulate DCs to suppress T cell responses. Collectively, the results raise the prospects of using PSO10H6 and PLGAO10H6 as vectors to deliver immunosuppressive genes to modulate T cell responses in vivo. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmaceutics / PhD / Dissertation

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 122-135).

Multiple sclerosis (MS) is an immune-mediated prevalent chronic demyelinating and neurodegenerative disease of the central nervous system that begins with an abrupt onset during early adulthood. MS is idiopathic, but many factors are thought to influence the pathogenesis of the disease, which include genetic, gender and environmental factors. To date, there is much evidence that suggest that both the onset and progression of MS is facilitated by both viral infections and stress. Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus that upon inoculation into susceptible strains of mice (i.e. SJL and CBA) causes a persistent infection which, in turn, results in an early acute encephalomyelitis followed by a late chronic immune-mediated demyelinating and neurodegenerative disease that pathologically resembles MS. In contrast, resistant mice (i.e C57BL/6 and BALB/c) are able to clear the virus from the CNS, and consequently do not develop chronic demyelination. Previous studies indicated that stress during early infection of susceptible mice can increase CNS viral titers and alter dissemination of TMEV, decrease early cytokine and chemokine expression in the spleen and CNS, and result in an exacerbated late demyelinating disease. The studies herein, focused on the hypothesis that chronic stress during early infection with TMEV infection would lead to drastic immunosuppression of both innate and adaptive arms of immunity, and that this immunosuppression may overcome the genetically controlled resistance of C57BL/6 mice to Theiler’s virus-induced demyelination. In these series of studies, we were able to show that stress, regardless of mouse strain susceptibility, decreases NK cell activity, and increased viral titers at day 1 p.i. Furthermore, after seven days of stress, susceptible mice demonstrated decreased virus specific T-cell effector function in both the CNS and spleens as indicated by a globalized reduction in type 1 and type 2 cytokines, as well as transcription factors. Importantly, these decreased responses were, in part, attributable to the actions of glucocorticoids. However, stress during early infection of C57BL/6 mice did not alter resistance to demyelination. These results begin to shed light on how stress, infection, and genetics can influence the onset of human MS.

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.</p>

Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

T lymphocytes comprise one arm of the adaptive immune system and are critical for immunity to neoplasia and infection. A full understanding of their development has important implications for the treatment of autoimmunity, immunodeficiency, and leukemias arising from T cell developmental intermediates. The Notch signaling pathway is already known to be absolutely required for T cell commitment and development, but its collaboration with other factors is poorly understood. Unfortunately, deficiency in many of the genes critical to hematopoiesis, including Notch, causes early embryonic lethality by disrupting multiple developmental processes. This complicates the study of such genes by in vivo models or ex vivo hematopoietic progenitors. To circumvent these difficulties, this thesis describes the use of in vitro-differentiated embryonic stem cell-derived T progenitors to examine the roles of two GATA family members during early T cell development. GATA-2, while not required for T cell development, is shown to act downstream of Notch signals to inhibit myelopoiesis. These findings both characterize a novel role for GATA-2, and demonstrate that T progenitor maturation and exclusion of non-T cell fates are distinct and separable events. GATA-3, in contrast to GATA-2, is absolutely required for T lymphopoiesis. However, the current literature does not distinguish between a requirement for GATA-3 in homing to the thymic environment, committing to the T cell fate, or surviving such a commitment event. This thesis demonstrates that GATA-3 is dispensable for commitment itself, but required to permit survival and proliferation after commitment. Taken together, the results presented in this thesis employ a novel model system to characterize the interactions of two important collaborators with Notch signals during T cell development, and further dissect the stages through which early T cell development is enacted.

The serine protease factor Xa (FXa) plays an integral role in the coagulation cascade and has recently been implicated in a variety of proinflammatory roles, establishing it as a link between coagulation and inflammatory processes. In this thesis, I elaborate on previous literature by characterizing further the response of primary human T lymphocytes to FXa. Building on previous literature that describes the effect of FXa on whole T cell populations, I describe here the effect of FXa on both antigen-independent and antigen-dependent proliferation and costimulation of primary CD4+ and CD8+ T cells, thereby establishing an immunological role for FXa. Further, I show that FXa elicits an immediate and direct effect on T cells demonstrated by the rapid upregulation of the signalling cascade kinases, ERK1 and ERK2. Lastly, I demonstrate that the protease activated receptor 2 (PAR2) is involved in the mediation of this direct FXa effect.

The serine protease factor Xa (FXa) plays an integral role in the coagulation cascade and has recently been implicated in a variety of proinflammatory roles, establishing it as a link between coagulation and inflammatory processes. In this thesis, I elaborate on previous literature by characterizing further the response of primary human T lymphocytes to FXa. Building on previous literature that describes the effect of FXa on whole T cell populations, I describe here the effect of FXa on both antigen-independent and antigen-dependent proliferation and costimulation of primary CD4+ and CD8+ T cells, thereby establishing an immunological role for FXa. Further, I show that FXa elicits an immediate and direct effect on T cells demonstrated by the rapid upregulation of the signalling cascade kinases, ERK1 and ERK2. Lastly, I demonstrate that the protease activated receptor 2 (PAR2) is involved in the mediation of this direct FXa effect.

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.