Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

Atherosclerosis is an inflammatory disease of the cardiovascular system. Discoidin domain receptor 1 is a receptor tyrosine kinase that binds collagens. Previous work in our lab has shown that deleting DDR1 in a mouse model results in attenuation of atherosclerosis, with fewer macrophages in the plaque. The aim of this study was to determine what changes in macrophage behaviour due to the lack of DDR1 was attenuating plaque development. In order to carry out experiments, primary mouse peritoneal macrophages were used. DDR1-deficient macrophages adhered significantly less to type IV collagen and fibronectin compared to DDR1-expressing cells. In addition, when plated on type IV collagen and fibronectin, DDR1-deficient macrophages produced decreased levels of MCP-1 protein, a cytokine known to be important in plaque development, particularly in leukocyte recruitment to plaque. These results suggest that DDR1 is an important mediator in macrophage adhesion to matrix and macrophage cytokine production

Short linear motifs (SLM) play critical roles in cell signaling and are associated with important biochemical events such as phosphorylation, glycosylation, and other post translational modifications. The primary aim of this thesis is to develop a new computational method (“ConDens”) to predict kinase substrates by assessing the evolution of phosphorylation SLM’s in a novel manner. This method could predict yeast Cdc28 kinase substrates that were not confidently detected by several other prediction methods published in literature and was demonstrated to be generalizable to other kinases. Genome-wide predictions experiments with this method also revealed potentially interesting novel substrates of Mec1, Prk1, PKA, and CKII.

Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

The chemical synapse is an asymmetric structure consisting of presynaptic and postsynaptic terminals in direct apposition to each other. Synapses function to mediate the transmission of signals between neurons and their targets. The formation of synapses is a tightly regulated process requiring the interaction of many genes and molecular pathways. I am interested in identifying genes and signaling pathways that are required for proper synapse formation. Using the GABAergic neuromuscular junctions of C. elegans as a model system, I have identified fsn-1 (F-box synaptic protein), a gene required for the control of synaptic growth. fsn-1 mutants exhibit a synaptic defect characterized by both synaptic over differentiation and under differentiation. FSN-1 is an F-box protein with a SPRY (SPla and RYanodine receptor) domain that functions cell-autonomously in neurons to regulate synaptic growth. I have shown that it functions in an E3 ubiquitin ligase-like complex with the RING-H2 finger protein RPM-1 (Regulator of presynaptic morphology), SKR-1 and Cullin. The composition of this complex is similar to SCF (Skp1, Cullin, F-box) E3 ubiquitin ligases. We hypothesize that this complex controls synapse formation by down regulating synapse promoting factors through an ubiquitin mediated process. We have identified two receptor tyrosine kinases that genetically interact with fsn-1, the Anaplastic Lymphoma Kinase homolog SCD-2 (Suppressor of Constitutive Dauer) and the C. elegans insulin receptor DAF-2 (abnormal DAuer Formation). Loss of function mutations in scd-2 or daf-2 partially suppress the synaptic differentiation defects of fsn-1 mutants, suggesting that they participate in signaling pathways whose activities are normally inhibited by FSN-1 during synapse formation. Unlike FSN-1 that functions in GABAergic neurons, I found that SCD-2 and DAF-2 have cell non-autonomous functions at GABAergic neuromuscular junctions. SCD-2 is required in the nervous system in the RID interneuron where it could modulate synapse formation through ligands present on the motoneuron cell surface. The DAF-2/insulin pathway functions in postsynaptic muscle cells to regulate FSN-1 dependent presynaptic growth likely through a retrograde or feedback mechanism. I propose a model where FSN-1 regulates synapse formation by attenuating signals that converge upon the presynaptic terminal to stimulate or inhibit synaptic growth.

Objective: Study the clinical, genetic and serological factors associated with new onset ileal inflammation in ulcerative colitis (UC) patients who have undergone ileal pouch-anal anastomosis (IPAA). Methods: Retrospective assessment of UC patients registered in the MSH surgical database, questionnaire, chart review and serological and genetic analysis of blood sample. Results: There was a positive association between gASCA antibodies, anti-L, smoking status and axial arthritis/ankylosing spondylits with a Crohn’s Disease (CD)-like phenotype post-op in UC/IPAA patients. Associations between alleles of selected genes including ECM1, PTPN2, and CD14 and post-operative phenotype were identified. Conclusions: gASCA is typically associated with CD rather than UC which raises the possibility that gASCA positivity may delineate a subset of UC patients at risk for developing a CD-like phenotype after IPAA. The identification of genotype/phenotype associations in UC/IPAA will help to find those at risk for poor post-operative outcome. Prospective studies are needed to confirm these findings.

Objective: Study the clinical, genetic and serological factors associated with new onset ileal inflammation in ulcerative colitis (UC) patients who have undergone ileal pouch-anal anastomosis (IPAA). Methods: Retrospective assessment of UC patients registered in the MSH surgical database, questionnaire, chart review and serological and genetic analysis of blood sample. Results: There was a positive association between gASCA antibodies, anti-L, smoking status and axial arthritis/ankylosing spondylits with a Crohn’s Disease (CD)-like phenotype post-op in UC/IPAA patients. Associations between alleles of selected genes including ECM1, PTPN2, and CD14 and post-operative phenotype were identified. Conclusions: gASCA is typically associated with CD rather than UC which raises the possibility that gASCA positivity may delineate a subset of UC patients at risk for developing a CD-like phenotype after IPAA. The identification of genotype/phenotype associations in UC/IPAA will help to find those at risk for poor post-operative outcome. Prospective studies are needed to confirm these findings.

The overall aim of my thesis is to gain insight into the cellular and molecular basis of the hierarchical organization of the human blood system, and how these normal development processes are subverted into leukemogenesis. To date, the major cellular classes that comprise human blood remain ill defined as rigorous clonal analysis required to define the self-renewal and lineage potential of single cells has not yet been performed. Here, identification CD49f as a novel marker of human HSC led to the ability to transplant single human HSC in NOD-scid IL2Rgc-/- mice. Loss of CD49f and Thy1 uniquely demarcated multi-potent progenitors (MPP) from HSC. The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision during lineage restriction from HSC. The validity of this model in the mouse has been questioned; however, little is known about the lineage potential of human progenitors. By clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow, human multi-lymphoid progenitors (MLP) were identified as a distinct population of Thy1-/loCD45RA+ cells in the CD34+CD38- stem cell compartment that can give rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells. This indicates that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation. While non-genetic mechanisms govern cell-fate commitment and lineage specification, hematopoietic malignancies are often initiated by aberrant gene rearrangements that can subvert normal cellular processes. Full transformation requires the accumulation of multiple genetic lesions. Most tumours exhibit dramatic genetic heterogeneity downstream of the initiating oncogenic event and are composed of pockets of genetically distinct clonal subpopulations. However little is known of how diversity evolves or the impact diversity has on functional properties. Here, using xenografting and DNA copy number alteration (CNA) profiling of human BCR-ABL1 lymphoblastic leukaemia, it was demonstrated that genetic diversity occurs in functionally defined leukaemia-initiating cells (L-IC) and that many diagnostic patient samples contain multiple genetically distinct L-IC subclones. Reconstructing the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multi-clonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, while in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A/B, and a trend to poor patient outcome. Our findings link clonal diversity with L-IC function and underscore the importance of developing therapies that eradicate all intratumoural subclones.

In a previous study, three determinants of fitness were identified as mutant alleles (each designated "e") that arose in yeast populations propagated in divergent environments. In a low-glucose environment, MDS3e and MKT1e interacted positively to confer a fitness advantage. PMA1e from a high-salt environment interacted negatively with MKT1e in low glucose, indicating a mechanism of reproductive isolation. In this thesis, I demonstrated that the negative interaction between PMA1e and MKT1e is mediated by alteration in intracellular pH and likely by a delay of the cell division cycle, while the positive interaction between MDS3e and MKT1e is mediated by changes in gene expression affecting glucose transporter genes. I also confirmed the evolutionary significance of the positive interaction by showing that an MDS3e genetic background is required for the recapitulation of the MKT1e mutation. Collectively, these results illustrate how epistasis can play a central role in both adaptation and speciation.

Studies of symbiotic bacteria have demonstrated that they provide multiple benefits to their hosts. These studies, however, have overlooked the importance of interactions with other bacteria and environmental factors that affect bacterial assemblages. To understand what shapes bacterial assemblages, I manipulated the diet of ants from the genus Cephalotes and disturbed their gut microbiome. I found that a deficit of nitrogen reduces bacterial densities. Furthermore, the data suggest that bacterial abundance may influence ant survival. I followed this experiment up by manipulating a putative protein source in the field. Our lab assigned Allomerus octoarticulatus ant colonies to treatments in which potential prey were present or absent. I collected data on foraging behaviour, colony performance, and composition of the bacterial community. The absence of prey increased ant recruitment to protein-rich baits; these ants were also less fit than ants that had insect prey but their bacterial assemblages were not affected.