<p>Epigenetic mechanisms, including DNA methylation, play an important role in regulation of stem cell fate and tumorigenesis. The Ten-Eleven-Translocation 2 (TET2) is a core enzyme for DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydromethylcytosine (5hmC). It has been shown that TET2 is the main regulator of hematopoietic stem cell homeostasis and loss of TET2 is highly associated with hematopoietic malignancies. Our previous work has also shown that loss of TET2 expression is linked to promotion of an epithelial-mesenchymal-transition phenotype and expansion of a breast cancer stem cell-like population with skewed asymmetric cell division in vitro; however, the in vivo role that TET2 plays in regulation of mammary stem cell (MaSC) fate and development of mammary pathology has yet to be determined. Here, using our newly established mammary-specific Tet2-knockout mouse model, the data reveals for the first time that TET2 plays a pivotal role in mammary gland development via directing MaSC to luminal lineage commitment in vivo. Furthermore, we find that TET2 coordinates with FOXP1 to target and demethylate FOXA1, GATA3, and ESR1, key transcription factors that orchestrate mammary luminal lineage specification and endocrine response and are often silenced by DNA methylation in aggressive human breast cancers. Finally, loss of TET2 expression leads to promotion of mammary tumor development with defective luminal cell differentiation and tamoxifen resistance in a PyMT;Tet2 deletion breast cancer mouse model. As a result, this study provides a previously unidentified role for TET2 in governing luminal lineage specification and endocrine response that underlies resistance to anti-estrogen treatments.</p>

No description available.

The CD-ROM disc containing supplementary material is kept in the cardboard box in the Systems Office. / The heterogeneity of breast cancer (disease stage and phenotype) makes it challenging to differentiate between each subtype; luminal A, luminal B, HER2, basal-like and claudin-low, on the basis of a single gene or protein. Therefore, a collection of markers is required that can serve as a signature for diagnosing different types of breast cancer. New developments in proteomics have provided the opportunity to look at phenotype-specific breast cancer cell lines and stage-specific liquid biopsies (nipple aspirate fluid [NAF], plasma samples) to identify disease and phenotype specific signature. An 8-plex iTRAQ quantification strategy was employed to compare proteomic profiles of a range of breast cancer and ‘normal-like’ cell lines with primary breast epithelial cells. From this, 2467 proteins were identified on Orbitrap Fusion and Ultraflex II, of which 1430 were common. Matched pairs of NAF samples from four patients with different stages of breast cancer, were analysed by SCX-LC-MS and a total of 1990 unique gene products were identified. More than double the number of proteins previously published data, were detected in NAF, including 300 not detected in plasma. The NAF from the diseased patients have 138 potential phenotype biomarkers that were significantly changed compared to the healthy volunteer (7 for luminal A, 9 for luminal B, 11 for HER2, 14 for basal-like and 52 for claudin-low type). The average coefficient of variation for triplicate analyses by multiple reaction monitoring mass spectrometry (MRM-MS), was 9% in cell lines, 17 % in tissue biopsies, 22% in serum samples and 24% in NAF samples. Overall, the results provide a strong paradigm to develop a clinical assay based on proteomic changes in NAF samples for the early detection of breast cancer supplementary to established mammography programmes. / The supplementary material submitted with the thesis is not available online.

Retinoids are a class of compounds derived from the metabolism of vitamin A and β-carotene. Retinoid signaling has vital functions in both vertebrate and invertebrate embryogenesis such as the formation of body axes and the control of organogenesis. Genetic evidence suggests a role of retinoids in cell fate decision, maturation and homeostasis of the prostate epithelium. Knockout (KO) of the retinoic acid receptor gamma (RARG) gene in mice leads to growth deficits and male sterility due to squamous metaplasia and keratinization of the seminal vesicles and prostate. Noteworthy, synergistic antitumor effects of retinoids and vitamin D have been described in prostate cancer cell lines, although a mechanistic link between retinoic acid (RA) signaling and prostate epithelium differentiation and tumorigenesis has not yet been elucidated. Here, taking advantage of mouse prostate organoids (mPrOs), we report an essential role for RA in the differentiation and integrity of the periurethral and proximal luminal compartments of the prostate epithelium. Mechanistically, RA, through the activation of RARγ, promotes the expression of Foxa1, a pioneer transcription factor that cooperates with androgen receptor (AR) in directing progenitor cells towards the luminal lineage. Reduced RA signaling in organoids leads to downregulation of key structural and polarity proteins along with a loss of luminal identity, a phenotype that is fully rescued by constitutive expression of exogenous Foxa1. Overall, our study demonstrates the importance of RA signaling in prostate epithelium differentiation and homeostasis. In addition to the tumorigenic role of Foxa1 mutations recently described in several human cancers, alteration in RA pathway due to altered uptake/absorption/metabolism of vitamin A and β-carotene, or depending on specific molecular dysfunctions (e.g., epigenetic RARB silencing), could represent a critical rheostat for prostate tumorigenesis.

No description available.

<p>Adhesion of the blastocyst to the uterine wall is a highly sensitive phenomenon referred to as implantation. Novel-males are capable of disrupting the success of this process (the Bruce effect). A leading hypothesis invokes the transfer of estradiol from the male to the female via urine. This estradiol has direct effect on the uterus which may include morphology and molecular dynamics. Estradiol has been related to closure of the uterus around the blastocyst during implantation, which may assist in bringing the blastocyst close to the uterine wall for strong adhesion. E-cadherin, a cellular adhesion molecule, is found on both blastocyst and uterine surfaces and has been suggested to be involved in their interaction during implantation. Estradiol has been observed to reduce e-cadherin expression in hormonally sensitive tissues like the mammary glands, ovaries and uteri. Here, male-induced disruption of implantation was examined across days 2-8 of gestation. Luminal area was quantified in isolated and male-exposed females as a measure of extent of luminal closure. This area was larger in male-exposed animals. E-cadherin was found to have reduced expression on luminal epithelial cells. I suggest that the reduction in e-cadherin may lead to weaker attachment of the blastocyst to the uterine wall as well as reduced adhesion between opposing uterine walls leading to the “opening” of the uterus observed in male exposed animals. Together, these data may in part explain the blastocyst implantation failure observed in male-exposed animals during the Bruce effect.</p> / Master of Science (MSc)

<p>The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important.</p><p>In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect.</p><p>The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP >> 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. </p><p>In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells. </p>

The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important. In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect. The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP &gt;&gt; 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells.

The lumen-to-cell transport, cellular accumulation, and toxicity of ionic cadmium (109Cd2+) and cadmium-cysteine conjugate (Cys-S-109Cd-S-Cys) were studied in isolated perfused S2 segments of the proximal tubule of the rabbit kidney. All perfusion solutions were HEPES buffered and contained 3H-L-glucose which functioned as a volume and leak marker along with 250 nM FD & C Green dye as a vital dye. When ionic cadmium, 0.73µM Cd2+, or 0.73µM cadmium-cysteine conjugate (Cys-S-109Cd-S-Cys) containing solution was perfused through the lumen of the tubule there was no visual evidence of toxicity such as blebbing of the luminal membrane, cellular vital dye uptake, and cellular swelling. Ionic Cd2+ transport was temperature dependent (87% reduction at 22°C and 100% at 11°C) and inhibited by FeCl2 (42% reduction at 10µM) and ZnCl2 (48% reduction at 20µM), and high Ca2+ concentrations (27% reduction at 1.95mM and 69% at 2.6mM). The ionic Cd2+ transport was not affected by verapamil and diltiazem. The cadmium conjugate (Cys-S-Cd-S-Cys) transport was also temperature dependent (76% reduction at 22°C and 100% at 11°C) and inhibited by the amino acids L-cystine and L-arginine (55% and 50% respectively), stimulated by L-methionine (56%), but not affected by L-aspartate, L-glutamate and Gly-Sar. 2, 3-Dimercaptopropane-1-Sulfonate (DMPS) co-perfused with Cd2+ decreased absorption of 20µM Cd2+ (39% reduction at 30 µM and 94.6% reduction at 200 µM), while DMPS added to the bathing solution has no effect on the luminal transport of Cd2+. DMPS co-perfused with 20 µM Cys-S-Cd-S-Cys substantially reduced Cd2+ transport (62% reduction at 30 µM). We conclude that cadmium can be transported at the luminal membrane of the S2 segment of the proximal tubule by multiple mechanisms, depending on the form which it is presented to membrane. Ionic cadmium appears to be transported by iron (DCT1), zinc (ZTL1) transporters and some kind of calcium-selective channel while cadmium conjugate of L-cysteine appears to be transported by L-cystine transporters (system b0+). Dipeptide transporter is not involved in the transport of cadmium. DMPS appears to be a chelator for cadmium.

Obesity is pandemic. Pharmacological treatment development depends on modeling the regulation of feeding, particularly by free fatty acids (FFA). Most models have been employed in the rat in vivo, and show FFA-stimulated intestinal satiety signals are dependent on the fat’s acyl chain-length, involve cholecystokinin (CCK) secretion, and are mediated by vagal afferents. I hypothesized that an in vitro mouse model could be employed, with sensitivity to measure afferent responses to nutrient stimuli. Male C57BL/6N mice were killed, the intestine harvested en bloc, and a jejunal section dissected with neurovascular mesenteric arcade emanating centrally. The tissue was placed in a Krebs-superfused chamber, the lumen cannulated with the outlet open to drain, and Krebs or other mediators were continuously perfused intraluminally. The dissected afferent nerve was placed in a suction electrode for extracellular recording. Afferent responses to distension and the perfusion of mediators (e.g. CCK or FFA) were tested. Preparations from normal mice (no surgery), or from mice following chronic subdiaphragmatic vagotomy or sham operation, were used to assess vagal afferent contributions. Luminally-perfused CCK (100 nM) increased afferent firing. This response was abolished with the CCK-1 receptor antagonist lorglumide (10 µM). The short-chain fatty acid (SCFA) sodium butyrate (30 mM) potentiated firing. The long-chain fatty acid (LCFA) sodium oleate (1-300 mM) activated concentration-dependent firing (EC50=25.35 mM) that was significantly greater at 30 mM than that evoked by butyrate. Lorglumide (30 µM) abolished the oleate (30 mM) response. The L-type Ca2+ channel (LTCC) inhibitor nicardipine (3 µM), intraluminally, potentiated the oleate response, while bath application abolished it. Vagotomy attenuated the oleate response. Vagotomy abolished the intraluminal CCK (100 nM) response, and attenuated the response to bath-superfused CCK. These findings support FFA chain-length-dependent mesenteric afferent activation and CCK involvement in oleate-induced firing, and suggest LTCC mediation of excitatory and inhibitory oleate response transduction pathways. The murine oleate response was shown to be mostly vagally-mediated, with some spinal contribution, and both vagal and spinal contributions to CCK responses were suggested. These data provide a basis for further investigation in vitro of cellular and molecular mechanisms of afferent satiety signals, and ultimately of obesity pathogenesis. / Thesis (Master, Physiology) -- Queen's University, 2010-06-29 15:56:08.387