Because stem rust of wheat is of great economic importance, much attention has been given to its control, especially by means of resistant varieties. However, there is very little information upon the nature of this resistance. At the same time, there are several reasons for believing that the differences in resistance between varieties are due to differences in their chemical composition. So far, attempts to find a correlation between the resistance of plants and the presence of certain compounds in them have failed. This failure may possibly be explained by the dependence of resistance on the presence of compounds which may be difficult to detect owing to either their low concentration or their instability.

There are a number of reasons for the choice of the epidermis of the mouse as the site for experimental tumour production in this study. Experimental epidermal tumours have been studied so intensively that a vast amount of useful information about them is available. The past work has characterized these tumours so well as to give a firm foundation to any expansion of previous achievements. There are furthermore some specific reasons for the use of this tissue of the mouse in experimental carcinogenesis.

Cancer is a disease recognized in antiquity. Its name, “Kapkivos”, a crab, was derived from the ancient description of breast cancer. Until the Nineteenth Century, it was entirely within the therapeutic province of the physician and surgeon. According to Bett (1957), Bernard Peyrilhe (1735-1804) was the first to conduct a systemic experimental investigation of the causation of cancer, in which he attempted to identify its toxin, and to trace the spread of the disease in order to devise better methods of treatment. Although more than two hundred years have passed since Peyrilhe's attempt, research into the cause of cancer is still one of the most intricate scientific activities of modern times.

The problem of carcinogenesis is an extremely wide and complex one and is unlikely to be solved by one man using one experimental model. On the other hand, it seems preferable to choose one model and explore its possibilities thoroughly before discarding it in the hope that there may be a common denominator to all carcinogenic processes. This thesis will attempt to show that by the use of a well-established experimental model, but by adding newer techniques, new and valuable information can be obtained. The two-stage production of papillomata in the skin of mice was chosen for several reasons. Training in this subject under experienced guidance is available in a favourable university environment. The basic experimental technique has been well established, and much information on it is available.

Bone formation during fracture repair inevitably initiates within, or around, extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone healing by promoting inflammatory and mesenchymal progenitor egress into the zone of injury. However, a failure of efficient fibrin clearance from wound fields can also be detrimental to normal tissue repair. The precise contribution of fibrin to fracture repair, be it supportive or detrimental, is unknown. Thus, in this dissertation I tested the hypothesis that fibrin is indispensable for the initiation of fracture repair but failure to remove fibrin impedes late stages of repair. I monitored fracture healing in mice with either a complete genetically fibrinogen deficiency or plasminogen deficiency and are thus unable to clear fibrin at the fracture site. Unexpectedly, fibrin was not required for long bone fracture repair. However, a failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluding bone union and resulted in robust heterotopic ossification. Notably, pharmacological fibrinogen depletion in plasminogen-deficient animals restored normal fracture healing, rescued impaired fracture vascularization, and significantly limited heterotypic ossification. Fibrin is therefore not essential for fracture repair but inefficient fibrinolysis impairs fracture healing by interfering with endochondral angiogenesis and ossification.

Invariant natural killer T (iNKT) cells are unique group of lymphocytes that secrete copious amounts of immunoregulatory cytokines upon activation. This allows iNKT cells to modulate a wide range of mouse models that model human disorders such as multiple sclerosis, metastatic cancer, atherosclerosis, etc. The activation of iNKT cells relies on the presentation of glycolipids in the context of major histocompatibility molecule CD1d on antigen presenting cells (APCs). The mechanisms and cellular factors involved in the preparation of glycolipids for iNKT cell activation remain to be defined, yet expression of molecules involved in lipoprotein metabolism have been shown to play a role in iNKT cell activation. In the current study, we investigated the expression of low density lipoprotein receptor-related protein (LRP) and determined that macrophages (MFs) are the main cell type expressing LRP. Using a mouse model with a genetic deletion of LRP in MFs, we demonstrated that in vivo activation of iNKT cells with prototypical iNKT ligand, a-galactosyl-ceramide (aGC), leads to decreased serum levels of interleukin-4 (IL-4) and decreased production of IL-4 by iNKT cells. Using a fluorescently labeled glycolipid we show that LRP deletion increases the rate of glycolipid flux and through generation of bone marrow chimaeras determined that the decrease of the IL-4 response is iNKT cell extrinsic. Additionally, our studies revealed that in WT mice, challenge with aGC leads to a decrease in LRP levels that is iNKT cell and interferon-gamma dependent. These data demonstrate that LRP expression in MFs is critical for the activation of iNKT cells and that iNKT cell derived interferon-gamma can modulate LRP expression in WT MFs.

Cystic fibrosis (CF) is a debilitating, multi-organ, autosomal recessive disease caused by mutations leading to absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells. Chronic pulmonary disease, characterized by excessive inflammation and recurrent infections, is the primary source of morbidity and mortality in individuals with CF. Altered polyunsaturated fatty acid (PUFA) biosynthesis, resulting in increased metabolism of linoleic acid (LA) to arachidonic acid (AA), plays a key role in CF pathophysiology. Increased AA biosynthesis contributes to excessive inflammation in the lungs of individuals with CF. The mechanism by which absence of functional CFTR leads to aberrant PUFA biosynthesis has been elusive. This dissertation is the first delineation of a pathway linking PUFA metabolism to CFTR. Through studies in human bronchial epithelial cells, this dissertation demonstrates that absence of functional CFTR leads to increased activation of AMP-activated protein kinase (AMPK) by calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Elevated AMPK activity leads to increased expression and activity of Δ6-desaturase (Δ6D) and Δ5-desaturase (Δ5D). Increased activity of Δ6D and Δ5D results in increased metabolism of LA to AA.

The oncogenes Mdm2 and Mdmx are often overexpressed in many human cancers. In addition to being negative regulators of the tumor suppressor p53, there is evidence these proteins have important p53-independent functions that are not clearly understood. Recently, a novel function of Mdm2 was identified where overexpression of Mdm2 inhibited double-strand DNA break repair through interacting with an important DNA repair protein, Nbs1, and this function of Mdm2 was independent of p53. Little was known about p53-independent functions of Mdmx and the contribution these functions may have on tumorigenesis. Here, I identify a novel interaction between Mdmx and Nbs1. Furthermore, I characterize a novel function of Mdmx that inhibits DNA damage response signaling and double-strand DNA break repair independent of both p53 and Mdm2. Specifically, Mdmx overexpression results in a decrease in the phosphorylation of the histone variant H2AX in addition to serine/glutamine or theronine/gluatamine motifs on substrates of the DNA-damage induced kinase ATM. Additionally, Mdmx overexpression promotes genome instability and confers transformation potential. Because increased levels of Mdm2 and Mdmx inhibit DNA break repair and promote genome instability, targeting this family of proteins pharmacologically has the potential to increase the efficacy of genotoxic drugs. Here, I demonstrate that the small molecule, Nutlin-3, causes an increase in Mdm2 protein levels, which results in an inhibition of double-strand DNA break repair and DNA damage response signaling independent of p53. In ovarian cancer cells with inactivated p53, Nutlin-3 combined with genotoxic agents, such as cisplatin, has a cooperative effect resulting in increased apoptosis. My results provide insight into generating novel treatment options for cancers with inactivated p53. Overall, my findings illustrate a conserved function of Mdm2 and Mdmx in the inhibition in double-strand DNA break repair. This function can be beneficial for developing novel therapeutic options for cancers with inactivated p53.

Normal tissue repair involves a series of highly coordinated events that include inflammation, granulation tissue formation, revascularization, and tissue remodeling. The transcriptional co-factor, ankyrin repeat domain protein 1 (Ankrd1), is rapidly and highly up regulated by wounding and tissue injury in mouse skin. Ankrd1 is also strongly elevated in human wounds. Overexpression of Ankrd1 in wounds by adenoviral gene transfer enhances wound healing. Ankrd1 has dual roles: a transcriptional co-regulator of several genes and a structural component of the sarcomere, where it forms a multi-component complex with the giant elastic protein, titin. Deletion of Ankrd1 results in a wound healing phenotype characterized by impaired wound closure and reduced granulation tissue thickness. In vitro studies confirmed the importance of Ankrd1 for proper cell-matrix interaction. We identified two Ankrd1-target genes, Collagenase-3 (MMP-13) and Stromelysin-2 (MMP-10). Both, MMP-13 and MMP-10 are important players in matrix turnover during physiological and pathological events. In summary, Ankrd1 regulates genes involve in remodeling of the extracellular matrix and is essential for proper interaction with the extracellular matrix in vitro.

Factor XI (FXI) is the zymogen of a protease (FXIa) that contributes to blood coagulation by activating factor IX (FIX). This thesis presents several novel observations regarding the structure and function of FXI that will enhance our understanding of this molecule. First, a new mechanism is described for FIX activation by FXIa based on a rigorous kinetic analysis. FXIa cleaves FIX initially after Arg145 to form FIXá, which is released, and then quickly rebinds to FXIa, allowing cleavage after Arg180 to form the active protease FIXaâ. Catalytic efficiency for cleavage after Arg180 is significantly greater than after Arg145, limiting FIXá accumulation. Using site-directed mutagenesis and protein modeling, the structural basis for the interaction of FIX and FXIa was identified. The ù-loop of FIX appears to bind to an area on FXIa comprised of residues from the N- and C-termini of the A3 domain in the heavy chain. These residues are buried in zymogen FXI, and are exposed upon activation to permit FIX binding. FXI is a homodimeric zymogen that is converted to a protease with one (1/2-FXIa) or two (FXIa) active subunits by factor XIIa (FXIIa) or thrombin. The in vitro and in vivo studies presented in this thesis indicate that FXIIa and thrombin activate FXI by different mechanisms. The dimeric structure of FXI is essential for normal activation by FXIIa, but is not required when thrombin is the activating protease. Activation of FXI is accelerated by polyanions such as polyphosphate released from platelet dense granule. Indeed, FXI can undergo autoactivation in the presence of polyphosphate. We determined that polyphosphate enhances FXI activation through a template-type mechanism requiring FXI and an activating protease to binding in proximity to each other on the polyphosphate. Two anion binding sites on the A3 and catalytic domains of FXI are required for optimal enhancement. Work with mice indicates that the anion binding sites contribute to FXI-dependent thrombotic processes in vivo. The work in this thesis provides a foundation for future studies to understand the role of FXI in normal and pathologic coagulation, and for developing therapeutic agents to treat or prevent thrombotic disorders.