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Interleukin-11 (IL-11) is a cytokine that promotes intestinal epithelial resistance to injury, however the mechanisms remain incompletely understood. Evidence from the Ropeleski Lab supports IL-11 dependent activation of the transcription factor Nuclear Factor кB (NFкB), without the degradation of the inhibitor кB (IкB), which deviates from the classical mechanism involving proteolytic processing of IкB. Also, IL-11 mediates the modulation of genes associated with healing, such as cyclooxygenase-2 (COX-2). It was hypothesized that IL-11 activates NFкB in intestinal epithelial cells by direct modulation of NFкB which, in a physiological setting, stimulates restitution during the healing response in the gut. Both cultured rat IEC-18 and human HIEC epithelial crypt cells were used as models to investigate whether the effect of IL-11 was species-specific. Activated NFкB is targeted to the nucleus therefore immunoblotting of nuclear extracts for expression of NFкB protein subunits including p65, activated p65 (phospho-p65Ser536), p50, and RelB, as well as by immunofluorescent detection of p65 were used. Inhibition of IL-11 signaling was carried out using various pharmacological inhibitors in order to determine their effect on p65 phosphorylation. Mechanically wounded cells were used as a model of gut injury and restitution where immunoblotting was used to examine IL-11 dependent effects on phospho-p65Ser536 and COX-2 expression. The binding of p65 to the кB binding site on DNA was detected with an ELISA-based system. IL-11 treatment was associated with the nuclear accumulation of phospho-p65ser536 in epithelial cell lines. Inhibition of PI3K/Akt signaling with LY294002 and AktiVIII suggested a partial reduction in phospho-p65Ser536 while inhibition of MEK1,2 signaling with U0126 indicated almost a complete abrogation of phospho-p65Ser536 accumulation in the nucleus. Inhibition of inhibitor of кB kinase-β (IKKβ) with SC-514 also revealed a strong attenuation of IL-11 induced phospho-p65Ser536. Inhibition of p90RSK1 with SL0101 was inconsistent but suggested a partial blockage of phospho-p65Ser536 whereas inhibition of Src kinase with PP2, did not affect phospho-p65Ser536 in IL-11 treated IEC-18 cells. There was no increased binding of p65 to the кB binding motif on DNA after IL-11 treatment. In mechanically wounded cells treated with IL-11, nuclear phospho-p65Ser536 was unaffected; however there was an evident potentiation of wound-induced COX-2 expression compared to untreated cells. In conclusion, IL-11 activates NFкB signaling in a non-classical manner through the phosphorylation of the p65 subunit. The predominant pathway appears to involve IKK and MEK signaling. Also, IL-11 modulates COX-2 expression in response to wounding in intestinal epithelial cells. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-07-31 10:12:18.794

Previously, we have demonstrated that a DNA helicase-like protein, termed RTEL (regulator of telomere length) is essential for the maintenance of genomic stability. RTEL deficiency induced telomere loss and genomic instability, leading to embryonic lethality. However, the role of RTEL in these biological pathways is largely unknown. To uncover RTEL’s function(s), we applied several approaches to identify the proteins that could interact with RTEL. Proliferating Cell Nuclear Antigen (PCNA), the key regulator of the replication fork, was found to be a strong candidate. In this study, we have demonstrated the interaction between RTEL and PCNA. Further characterization of the interaction between RTEL and PCNA revealed that the interaction is important for maintaining genomic stability. Due to the essential role of PCNA in nucleic acid metabolism as a component of the replication and repair machinery, its interaction with RTEL could be the key to the role of RTEL in the maintenance of genomic stability and mouse development. Along with a bioinformatics approach, we have employed several biochemical approaches to identify the interaction of PCNA with RTEL. Using co-immunoprecipitation, we have demonstrated that RTEL can specifically interact with PCNA. A PCR-based mutagenesis method was used to mutate the PCNA-interacting motif (PIP) in RTEL. Further we have demonstrated that several key amino acids in the PIP motif are responsible for mediating RTEL/PCNA interaction by using co-immunoprecipitation and immunofluorescence studies. Using a gene-targeting approach, we have specifically knocked-in a mutant RTEL with a mutation in PIP motif into mouse genome. Thus we have developed a transgenic mouse model to study the significance of the interaction between RTEL/PCNA in vivo. This study not only validated the interaction of RTEL with PCNA, via the PIP box, but also generated the RTEL PIP mutant alleles for further functional analysis by transgenic approaches. We have employed biochemical and cytogenetic studies to characterize the phenotypes in RtelI1169A/I1169A mouse. This is the first direct genetic approach to address whether PCNA is an important downstream mediator of RTEL’s function in the regulation of genomic integrity

<p> Spermatogenesis is governed by vitamin A, as shown by vitamin A deficient (VAD) testes, which lack advanced germ cells. Vitamin A signaling is mediated by retinoid receptors. There are two families of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with alpha, beta and gamma subtypes. Retinoic acid receptor alpha (RARA), plays a significant role in the testis such that <i>Rara</i>-null males are infertile because of severe germ cell loss. </p><p> Striking similarities of the testicular phenotypes are detected between <i> Rara</i>-null and VAD mice: severely degenerated testes, lack of germ cells, sloughing of mature spermatids, and infertility. To discern the molecular function of RARA in germ cells, <i>Rara</i> was conditionally deleted using stimulated by retinoic acid 8 (STRA8)-iCRE. With RARA function disabled in germ cells, morphological abnormalities detected in the testes included lack of germ cell organization, lack of lumen, sloughing cells, and vacuolization. Not surprisingly, germ-cell specific <i>Rara</i> conditional knockout mice (cKO) had a dramatic reduction in epididymal sperm number. Further analysis of cKO testes demonstrated decreased spermatogonial proliferation and differentiation, while meiotic defects such as reduced synapsis, synaptonemal fragmentation, and unrepaired double strand breaks were increased. Furthermore, functional spermatogonial transplantation assays pointed to the possibility that RARA regulates spermatogonial stem cell colonization and proliferation, as shown by the reduction of donor-derived spermatogenesis from the cKO donor germ cells. The lack of RARA in the testes clearly shows quantifiable deficiencies during spermatogonial proliferation, differentiation, and meiosis. </p><p> Microarray gene expression studies of mRNAs from the enriched germ cells from wild type and cKO mice provided molecular evidence that RARA regulates spermatogonial differentiation at postnatal day 4 (P4) and meiosis at P8. Cell differentiation, cell adhesion, cell migration, and other pathways related to the early steps of spermatogonial differentiation were found to be functional categories significant in germ cells from P4. These were very distinct from synapsis, synaptonemal complex formation, and crossover formation related to meiosis, which were functional categories significant in germ cells from P8. In conjunction with phenotypic abnormalities, we provide gene expression evidence that RARA mediates retinoic acid function during spermatogonial proliferation, differentiation, and meiosis.</p>

<p>Soft Tissue Sarcomas are a rare group of mesenchymal tumors with over 50 recognized subtypes. These tumors are a diverse group of malignancies that primarily arise from the connective tissue, fat and muscle. In the United States, there are estimated to be approximately 11,000 new diagnoses a year with an annual mortality rate approaching 40%. Unfortunately, with such a diversity of subtypes of soft tissue sarcoma, and the relative scarcity of patient samples, there is a need for animal models that faithfully recapitulate the biology of these tumors. Such animal models would be useful for dissecting the underlying biology of soft tissue sarcomas and to evaluate novel therapies. One such model is the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue sarcoma. These tumors are generated in a spatial and temporally restricted fashion and closely mimic the natural history of human soft tissue sarcomas, including a predilection to develop lung metastases. Here I will characterize this model of soft tissue sarcoma by: 1) performing cross species genomic comparisons to show that the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue sarcoma most closely resembles Undifferentiated Pleomorphic Sarcoma , 2) utilizing this mouse model to identify cathepsin proteases as molecular markers of soft tissue sarcoma. I will then use cathepsin activated imaging probes for intraoperative molecular imaging to identify microscopic residual cancer in real time. Finally, 3) I identify a novel mechanism through which MAPK signaling regulates miRNA biogenesis and the development of distant metastases in the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue sarcoma.</p> / Dissertation

<p>Soft tissue sarcomas are rare malignancies that derive from connective tissue. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, while undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the cell(s) of origin of these sarcomas in the myogenic lineage, I used the tamoxifen-inducible CreERT2-loxP system in vitro and in vivo. Pax7-CreERT2 and MyoD-CreERT2 mice were utilized to transform Pax7+ and MyoD+ myogenic progenitors by expressing oncogenic K-rasG12D and deleting p53 in vivo. After injection of systemic tamoxifen into Pax7-CreERT2 and MyoD-CreERT2 mice, primary myogenic sarcomas including mouse rhabdomyosarcoma (mRMS) and mouse UPS (mUPS) developed within 2 to 6 months at various anatomical sites. Using unsupervised gene expression analysis, mRMS from Pax7+ myogenic progenitors clustered separately from the mUPS generated from the Pax7+ myogenic progenitors, as well as the mUPS generated by MyoD+ myogenic progenitors. These results suggest that Pax7+ and MyoD+ myogenic progenitor cells are tumor-initiating cells mUPS and that Pax7+MyoD- progenitors are tumor initiating cells for mRMS. These results demonstrate that mRMS and mUPS lie along a continuum. Furthermore, by comparing these tumors to their cell of origin, we find that Hedgehog signaling is dysregulated by increased expression of activated Gli3 in the sarcomas. Knockdown of Gli3 in cell lines derived from mouse and human sarcomas blocks tumor cell proliferation. I have established two novel mouse models of sarcoma with rapid onset and high penetrance, which may be useful for identifying novel therapies in sarcoma.</p> / Dissertation

<p>Quinolone treatment induces stabilized cleavage complexes (SCCs), consisting of a covalent gyrase-DNA complex, and processing of these complexes is thought to cause double-strand breaks and chromosome fragmentation. SCCs are required but not sufficient for cytotoxicity; the mechanism that converts SCCs to double-strand breaks is not clearly understood. Evidence of chromosome fragmentation due to quinolones comes from indirect measures such as sedimentation analysis of nucleoids and measurements of lysis viscosity. This work outlines a method that combines agarose plugs, conditional lysis and field inversion gel electrophoresis to allow direct visualization of chromosomal fragmentation resulting from quinolone treatment. We are able to distinguish between latent breaks within the stabilized cleavage complex and irreversible breaks that result from downstream processing.</p><p>When seeking to understand the genetic requirements for quinolone-induced SOS response, we found that a dnaQ mutant has a specific defect in SOS induction following nalidixic acid. The product of dnaQ is the &epsilon; subunit of DNA polymerase III, which provides 3' &rarr; 5' exonuclease activity. In addition to the nalidixic acid-specific SOS defect, &delta;dnaQ has multiple phenotypes: slow growth, high mutation frequency, and constitutive SOS. We propose that &epsilon; has a role in the quinolone response beyond the normal proofreading function of the subunit in the polymerase III core. Using a unique transposon mutagenesis system, we created a library of dnaQ mutants with 15 base pair insertions that were scored phenotypically. We identified mutants that separated the various phenotypes, arguing strongly that &epsilon; has multiple functions. The isolation of a stable dnaQ mutant with SOS phenotypes allows the study of this function without confounding results from spurious mutations throughout the chromosome. We also isolated a novel class of SOS "hyper-inducible" mutants. Additionally, my findings with weak and strong &beta;-clamp binding mutants provides the first in vivo characterization of these &epsilon; mutants and gives insight into the SOS response following nalidixic acid treatment.</p> / Dissertation

<p> The research presented in this dissertation deals with the phenomena of bacterial efflux pump inhibition by natural products and plant extracts. Bacterial efflux pumps are active transport proteins, primarily deriving their energy source from the proton motive force, which functions to export toxic compounds outside the cell. This is a natural defense mechanism that bacteria utilize to protect themselves from toxic environments. The over-production of efflux pumps is one mechanism by which bacteria can evolve resistance to clinical antibiotics, as well as other antimicrobials. Thus, the study of efflux pump inhibitors is important because it holds the potential to reverse some forms of antibiotic resistance. In light of this importance, a series of studies were designed to improve the ability to study this phenomenon, to investigate the distribution of efflux pump inhibition in land plants, and to improve our ability to identify an important class of efflux pump inhibitors, the flavonoids. </p><p> The first aim of this research was to develop an improved method for experimentally quantifying efflux pump inhibitory activity of small molecules. Preexisting methods made this difficult due to several limitations including: the collection of indirect results, time consuming materials handling techniques, and/or matrix interference problems pertaining to the quenching of fluorescent signal. An improved method relying on mass spectrometry measurements was developed that addressed the aforementioned limitations. The importance of this improved method lies in its ability to produce data sets useful in calculating IC₅₀ values for a wider range of samples than was previously possible. </p><p> The second aim was to evaluate the presence of efflux pump inhibitor production across the land plant lineage. This is important to botanical science and the understanding of plant-microbe interactions and plant evolutionary biology. The most ancient lineages of land plants have not been previously evaluated for efflux inhibitory activity. Additionally, land plants play an important role in many traditional medicinal systems and in modern complementary and alternative medicine. Thus, understanding the distribution of efflux pump inhibitor production in this group increases our understanding of these common forms of medical treatment. In order to gain these data, a set of 14 plant species spanning the major lineages within the land plant group (bryophytes, pterophytes and lycophytes, gymnosperms, and angiosperms) were extracted and assayed to determine efflux pump inhibitory activity of the extracts. Positive results (indicating the presence of an efflux pump inhibitor) were observed for many (but not all) of the plant species tested. The observation of activity in extracts prepared from the most ancient plants tested (bryophytes--the liverworts and mosses) lends credence to the hypothesis that the production of efflux pump inhibitors is of great antiquity in land plants. </p><p> The last component of this work was the evaluation of methods for the analysis of flavonoids via mass spectrometry. This is of importance to this study due to the commonality of flavonoids in the literature pertaining to efflux pump inhibitors, and the consistency activity of the flavonoids evaluated in Chapter II. The goal of this work was to compare two methods for the tentative identification of signals in complex data produced via high-resolution mass spectrometry that could be labeled as "possible flavonoids." The methods evaluated were firstly the use of mass spectrometry fragmentation spectra to identify key diagnostic fragments of the flavonoid ring structure and secondly the use of mass defect filtering directly applied to high resolution data to select a short list of signals for further processing. The former method was not fruitful due to a combination of the frequent poor fragmentation and the dependency on standards for all samples. The latter proved more useful, successfully producing a list of potential flavonoids to be carried forward to other methods such as database searching and molecular formula calculation. This method was also successfully applied to a complex extract of <i>Hydrastis canadensis </i>, identifying three flavonoids known from previous work to be present. Further, the mass defect method is an intrinsic property of molecules, and therefore does not change with experimental conditions. For all of these reasons mass defect was selected as the more useful of the two methods evaluated for the identification of "possible flavonoid" signals in crude extracts.</p>

<p> Remodeling of the mouse mammary gland is a highly coordinated process that occurs after the removal of suckling pups from the mother. Involution, or shrinking of the mammary gland, after removal of the pups has been linked to apoptotic events within the mouse mammary tissue during forced weaning. Several transcription factors are hypothesized to be involved in this process. A transcription factor known as GATA-3, which was first identified in the thymus, is also important for maintenance of various tissue types within the mouse mammary gland; its loss leads to epithelial cell detachment and eventual death. Another transcription factor known as fetal zinc liver finger protein 1, or Fliz1, has been found to regulate GATA-3 in T-cells. This interaction had not been elucidated during involution in mouse mammary tissue. I hypothesized that Fliz1 is expressed at heightened levels during mouse mammary gland involution following forced weaning of pups, and that this expression correlates with a decrease in GATA-3 levels, with increased expression of the pro-apoptotic protein BAD. Using qRT-PCR, immunoblotting and immunohistochemistry I have shown that Fliz1 is indeed expressed in involuting mouse mammary gland tissue as well as several other tissue types. However, levels of Fliz1 remain fairly constant during involution. The findings also show that Cathepsin L, a known apoptotic marker for mammary gland involution, is substantially up-regulated during the process of mammary gland involution in the mouse. The study also revealed that GATA-3 levels as hypothesized decrease substantially during the process of mouse mammary gland involution, indicating that GATA-3 is required for maintenance of the mouse mammary gland.</p>

<p> In the presence of arachidonic acid (AA), <i>Saccharomyces cerevisiae </i> produces prostaglandin E₂ (PGE₂). <i> S. cerevisiae</i> and its metabolites may be consumed in products manufactured using the yeast (e.g. beer). Neutrophils are immune cells present in the gastrointestinal (GI) tract during inflammation. As a lipid-signaling molecule, PGE₂ can potentially modify neutrophil functions and exacerbate pre-existing inflammation. As neutrophil migration is a hallmark of inflammation, we investigated the impact of PGE₂ on neutrophil chemotaxis. Chemotaxis assays were performed on neutrophils isolated from human whole blood using the chemotactic agents f-Met-Leu-Phe (fMLP) or interleukin-8 (IL-8). Neutrophil chemotaxis was concentration dependent as it was enhanced 3.5-fold at low concentrations of PGE₂ (0.1 nM-10 nM) and reduced 3.0-fold at higher concentrations of PGE₂ (100 nM).</p><p> The biochemical pathway utilized by <i>S. cerevisiae</i> to produce PGE₂ is unknown. Identifying enzymes that metabolize AA may direct approaches to reduce the impact that yeast PGE₂ may have on neutrophils. <i>S. cerevisiae</i> does not have genes homologous to those involved in mammalian AA metabolism. We employed RNAseq transcriptome sequencing to study the lipid biosynthetic pathway in <i>S. cerevisiae </i> and observed 1248 genes upregulated in yeast that were cultured in the presence of AA relative to yeast that were cultured without AA. Notably, genes that mediate beta-oxidation of fatty acids (<i>Pot1, Pox1, Faa1 and Faa2</i>) were upregulated up to 2.3-fold.</p><p> The results demonstrate that low concentrations of PGE₂ enhance neutrophil chemotaxis that is mediated by fMLP or IL-8, suggesting that PGE₂ may aid in recruiting neutrophils from regions that are distant to a site of inflammation. Once a higher concentration of PGE₂ is encountered by neutrophils, neutrophils may halt their migration and engage effector functions such as phagocytosis and superoxide production. Increased expression of genes involved with fatty acid metabolism points to enzymes that may utilize AA to produce PGE₂ in <i>S. cerevisiae</i>. Experiments testing PGE₂ levels in knock-out strains of yeast will identify genes involved in PGE₂ production. Results of this study have implications to reduce potential off-target effects caused by yeast PGE₂ in consumables.</p>