This work explored features of canine mast cell tumours (MCT) to improve prognosis and to discover potential therapeutic targets. Subcutaneous MCT - a subset of these tumours arising in the subcutis - are usually grouped with cutaneous MCT, but there is evidence that they may be clinically different. The first objective was to develop a grading scheme for subcutaneous MCT. Over 300 canine subcutaneous MCT were evaluated retrospectively and parameters were correlated with clinical outcomes, making this the largest retrospective survival study of these tumours to date. The results of the study showed that the majority of subcutaneous MCT had excellent outcomes and key prognostic markers were identified (mitotic index, surgical margins and degree of infiltration). A subset of the subcutaneous MCT from the retrospective study was further evaluated to assess the cellular localization of KIT - a receptor tyrosine kinase (RTK) which is dysregulated and constitutively activated in some cutaneous MCT - as well as Ki67, a proliferation marker. In addition, evaluation of mutations of c-KIT, the gene for KIT, was determined for each MCT. Cytoplasmic KIT localization and high Ki67 values were predictive of decreased survival time and time to local reoccurrence, but no c-KIT mutations were detected. The majority of canine MCT do not appear to depend solely upon KIT for tumour progression and few other RTK targets have been studied in canine MCT. Based on evidence that vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR) - may play a role in the progression of canine MCT; the expression and distribution of these RTK were evaluated. The results showed that canine MCT have unique expression profiles and activity of KIT, VEGFR2 and PDGFR. Two novel mast cell tumour cell lines were generated and used to assess signalling of KIT and VEGFR2 in vitro. Stimulatory and inhibitory responses were assessed and found to be different in both cell lines. Both had autophosphorylated VEGFR2 and an autocrine VEGF/VEGFR2 signalling pathway existed for both cell lines. These findings are unique and the first that identify autocrine VEGF signalling as a possible survival mechanism for canine MCT. / Pet Trust Foundation, Ontario Veterinary College

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression. / Thesis (Master, Biochemistry) -- Queen's University, 2011-09-14 11:49:32.871

Durability tests are important to ensure the safety and reliability of a ground vehicle and involve frequently driving a vehicle through a series of events that simulate different road conditions or obstacles encountered during actual driving. Since durability tests are costly in-terms of time and money, accelerated durability lab tests can be used to spot failures before actual road tests. Signals of different events of the actual durability road tests generate three continuous time series data, that can be used to conduct accelerated durability lab tests. The actual analysis of these time series is very challenging because they are (i) of high frequency (ii) very noisy and (iii) inconsistent. The purpose of this study was to identify the patterns of signals from the noisy and inconsistent time series data collected from the field tests. The Box-Jenkins methodology was used to identify models corresponding to different events. Due to complex structures of the real data, ARMA modelling was considered after testing stationarity of the given time series. While the time series data in vertical direction was used to identify the first three events, the time series in vertical, longitudinal and lateral directions were used to identify other four events.

Diffuse large B-cell lymphoma (DLBCL), which is the most common type of lymphoma, is characterised by its aggressiveness and poor outcome without adequate treatment and also for its biological and clinical heterogeneity. It is therefore highly desirable to gain a more profound understanding of the underlying biology of the disease, as well as predictive factors for the guidance of treatment. The studies presented here attempt to gain an overall grasp on DLBCL, from the epidemiological level down to the genomic level. The tumour microenvironment consists of both tumour cells and normal infiltrating cells in a delicate interplay. By assessing the number of infiltrating mast cells (MCs) in the microenvironment, a correlation between low numbers of MCs and poorer prognosis of DLBCL was found. However, malignant cells are not only affected by environmental conditions but also by intrinsic factors, such as small non-coding microRNAs. A low expression level of microRNA-129 was found to correlate with poor survival of DLBCL and the finding remained significant even for rituximab-treated patients. An even smaller intracellular genomic unit is one single nucleotide. The single nucleotide polymorphism 309 (SNP309) is a T to G change in the promotor region of MDM2, a regulatory protein in the p53 pathway, which results in increased transcription of MDM2 and thus decreased levels of p53. It was found that homozygous T allele patients had longer overall survival, as well as disease-specific survival and disease-free survival. However, treatment with rituximab eliminated the predictive value of the SNP309 polymorphism. In the last project presented in this thesis we used epidemiological methods to analyse all DLBCL cases diagnosed 2000-2013 in Sweden. Here it was possible to categorically show that higher age is an adverse prognostic factor, and most importantly, this starts from a young age. In conclusion, within this thesis I have applied different laboratory and analysis techniques to examine DLBCL biology in relation to the clinic. I have identified potential new prognostic markers, contributed to an enhanced understanding of DLBCL biology and described epidemiological data from one of the largest DLBCL cohorts ever presented. All of these aspects provide important information for a deeper understanding of the disease DLBCL.

Durability tests are important to ensure the safety and reliability of a ground vehicle and involve frequently driving a vehicle through a series of events that simulate different road conditions or obstacles encountered during actual driving. Since durability tests are costly in-terms of time and money, accelerated durability lab tests can be used to spot failures before actual road tests. Signals of different events of the actual durability road tests generate three continuous time series data, that can be used to conduct accelerated durability lab tests. The actual analysis of these time series is very challenging because they are (i) of high frequency (ii) very noisy and (iii) inconsistent. The purpose of this study was to identify the patterns of signals from the noisy and inconsistent time series data collected from the field tests. The Box-Jenkins methodology was used to identify models corresponding to different events. Due to complex structures of the real data, ARMA modelling was considered after testing stationarity of the given time series. While the time series data in vertical direction was used to identify the first three events, the time series in vertical, longitudinal and lateral directions were used to identify other four events.

Despite recent advances in oncology and extensive research efforts, gliomas remain essentially incurable. Glioblastoma multiforme (GBM, WHO grade IV) is the most common glioma and may arise de novo or progress from a lower-grade lesion. GBM is characterized by invasive growth, aberrant angiogenesis and necrosis. The heterogeneity of GBM is further complicated by the contribution of the inflammation that is facilitated by immune cells that reside in and infiltrate this immuno-privileged organ. One of the cells types present in the tumor microenvironment are mast cells (MC) that accumulate in the tumor in a grade-dependent manner. GBM cells secrete a plethora of cytokines acting as chemoattractants in MC recruitment and to a lesser degree induce MC proliferation in situ. Expression of one of the cytokines secreted by GBM cells - macrophage migration inhibitory factor (MIF) - correlates with MC accumulation in vivo. GBM cells invade the surrounding parenchyma making complete resection impossible. Here, migration was studied with the focus on RAP1 and its negative regulator RAP1GAP. Activation of RAP1 signaling by lentiviral silencing of RAP1GAP lead to decrease in cell migration and a shift in expression of SOX2 and GFAP, presumably enhancing stem cell phenotype. MicroRNAs are small non-coding RNAs known to regulate the mRNA network. miR-21 is highly overexpressed in the majority of cancers including GBM. Its expression is strictly regulated during embryonic development of the brain. SOX2 is co-regulated with miR-21 demarcating a cell population with neural/glial progenitor/stem cell properties. In an experimental mouse model, expression of miR-21 can be sustained by forced expression of PDGF-BB leading to gliomagenesis. GBM cells seem to be addicted to oncogenic properties of miR-21 as its knockdown leads to extensive apoptosis. This observation combined with the fact that miR-21 is absent in the normal adult mammalian brain suggest miR-21 to be an excellent therapeutic target. Effects of conventional therapy (surgery combined with radiochemotherapy) on prolonging patient survival have reached a plateau. New effective personalized therapeutic modalities need to be designed and implemented. Targeting the tumor microenvironment as well as cell intrinsic properties like invasive potential, stemness and onco-miR addiction studied in this thesis will hopefully lead to efficient disruption of GBM’s aberrant ecosystem.

Mast cells (MCs) play a central role in inflammation by releasing mediators following activation. S100A8 and S100A9 are abundantly expressed in inflammatory sites such as asthmatic lung, sunburnt skin and atherosclerosis where MCs are involved in pathogenesis; roles of S100A8 in MC function are undetermined. The aims of this thesis were to determine effects of S100A8 on MC activation, particularly provoked by IgE and UVB. Initially, effects of UVB on MC activation were investigated as detailed functions were unclear. Cord blood-derived human mast cells (CBMCs) were treated in vitro with varying doses of UVB and production of multiple cytokines and viability investigated. UVB exposure selectively increased levels of IL-8 (CXCL8), and to a less extent IL-1β, but not eight other cytokines tested. New protein synthesis partially contributed and IL-8 production was p38 MAPK-dependent. UVB dose-dependently induced MC apoptosis indicating a potential regulatory mechanism of MC function. The ability of recombinant S100A8, S100A9 or S100A8/9 heterodimer to modulate IgE/antigen (DNP/anti-DNP)-mediated activation of a murine MC line, and of bone marrow-derived (mBM) MC activation was determined. The S100s did not directly induce degranulation or induce IL-6. S100A8 significantly inhibited DNP/anti-DNP-provoked degranulation, and IL-6 and TNF mRNA and protein induction. S100A8 did not alter FcεRIα expression. S100A9 was less effective; and the S100A8/9 complex was also suppressive. S100A8 only weakly suppressed non-specific MC degranulation. Mutation of Cys41 in S100A8 negated its suppressive activity. Because S100A8 scavenges oxidants via this reactive Cys residue, we propose that this may mediate its ability to downmodulate IgE-dependent MC responses. Similar to the thiol scavenger N-acetyl-L-cysteine, S100A8 but not the Ala41 mutant, attenuated DNP/anti-DNP-provoked LAT phosphorylation. However, the disulfide-bonded S100A8 dimer and S100A8 containing a sulfinamide bond between Cys41 and Lys34/35 also reduced MC activation, indicating an additional pathway(s). S100A8 did not suppress antigen/IgE-induced responses of CBMC possibly because these may not truly reflect fullymature human tissue MCs. S100A8 did not alter UVB-induced IL-8 release by CBMCs, or affect apoptosis. Murine S100A8 may have anti-inflammatory properties by regulating MC activation in an activator-specific manner, at least partially by scavenging ROS to suppress intracellular signalling.

Mast cells are known to play an important role in allergic events and in other inflammatory reactions through varied intracellular signaling transduction proteins. RasGRP4 is a mast cell-restricted guanine nucleotide exchange factor (GEF) and diacylglycerol (DAG)/phorbol ester receptor. Interleukin (IL) -13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Rα, IL-13Rα1 and IL-13Rα2. IL-13Rα2 has been reported to be a decoy receptor for IL-13. My experiments indicate that the mast cell specific RasGRP4 protein regulates the level of IL 13Rα2 and controls IL-13/ IL 13Rα1-mediated intracellar signaling events in mast cells. Phosphorylation of STAT6 plays an important role in airway hyperresponsiveness and asthma. The development of therapeutics that can regulate RasGRP4 could be used to modulate the IL-13-induced phosphorylation of STAT-6 that may be used as therapy in patients with asthma. SLE is a complex, heterogeneous systemic autoimmune disease characterized by the presence of high levels of autoantibodies. Dysregulation of RasGRP1, a Ras active gene, in mice resulted in a SLE-like disorder. Yasuda and coworkers demonstrated that a defective isoform of RasGRP1 (Δ11) was present in a subset of patients with SLE. My experiments indicate that RasGRP1 upregulates the expression of IL2RG in T cells. In contrast the Δ11 RasGRP1 isoform expressed in a subset of SLE patients leads to defective expression of IL2RG. The IL2RG chain is a common chain which forms part of a number of different receptors eg. IL-2, 4, 7, 9, 15, 21. IL-2 as well as IL-21, which shares sequence homology with IL-2, has been reported to be involved in the generation of regulatory T cells (Tregs). In SLE patients, CD4 + CD25+ Tregs, which play an essential role in controlling immunologic tolerance to self-antigens and preventing autoimmunity, are significantly decreased when compared with healthy controls. The accumulative evidence suggests that the defective isoform of RasGRP1 (Δ11) downregulates expression of IL2RG in SLE patients?? T cells and this could effect the generation of CD4 + CD25+ Tregs. This may be another immunological mechanism in loss of tolerance observed in patient with SLE.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 2003.