Connexins are a family of gene products sharing a similar topology that mediate transmembrane and intercellular diffusion of ions and small molecules through hemichannels (HCs) and gap junction channels (GJCs), respectively, and participate in intracellular signaling through protein-protein interaction. Connexin 37 (Cx37) and Cx40 are co-expressed by endothelial cells, suggesting a unique role for each connexin in regulating cellular function. Through gene knockout studies in mice, Cx37 was found to regulate vascular cell proliferation while Cx40 regulates conduction of upstream vasomotor signals and leukocyte infiltration. The unique functions of Cx37 and Cx40 result in contrasting effects of ischemic injury in mice lacking expression of either Cx37 or Cx40 genes. Cx37 knockout mice (Cx37-/-) have significantly improved recovery due to an increase in both vasculogenesis and angiogenic processes. In the current studies, the mechanism by which Cx37 mediates cellular proliferation is explored by examining the role of functional GJCs and HCs. Channel function is necessary, but HC function is not sufficient for Cx37-mediated suppression of proliferation. These results suggest that Cx37 requires a competent GJC that supports a specific conformation of the carboxyl terminus that is able to interact with necessary growth regulatory protein(s). Conversely, Cx40-/- mice have reduced angiogenic and arteriogenic processes and impaired post-ischemic recovery. In the current study, we explored the inflammatory response following the femoral artery ligation (FAL) surgery, in wildtype (WT) and Cx40-/- mice. Cx40-/- mice had an excessive infiltration of activated neutrophils to the ischemic gastrocnemius muscle and a prolonged presence of activated macrophages. We depleted mice of circulating neutrophils during the induction of ischemia to evaluate if the excessive infiltration of neutrophils impaired recovery and found no improvement in post-ischemic recovery in either WT or Cx40-/- mice. These data indicate that, although Cx40-/- mice have a pro-inflammatory state that results in an excessive and prolonged presence of leukocytes post-ischemia, reduction of acute leukocyte infiltration does not improve post-ischemic recovery. Additional information is necessary to determine the mechanisms by which Cx37 and Cx40, individually or in concert with each other, regulate endothelial cell function and arterial vascular response to ischemic injury.

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<p><font size="3"> <p>The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.<font size="3">The aim of this study was to investigate </font><i><font size="3" face="Times New Roman,Times New Roman"><font size="3" face="Times New Roman,Times New Roman">in vitro </font></font><font size="3">effects of pure and street MA &quot / tik&quot / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. <font size="3">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. <font size="3">Results of this study showed that:&nbsp / <font size="3">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05). <font size="3">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. <font size="3">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA. </font></font> <p>&nbsp / </p> </font>&nbsp / </font></font></font> <p>&nbsp / </p> </i></p> </font></p> <p>&nbsp / </p>

<p><font size="3"> <p>The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.<font size="3">The aim of this study was to investigate </font><i><font size="3" face="Times New Roman,Times New Roman"><font size="3" face="Times New Roman,Times New Roman">in vitro </font></font><font size="3">effects of pure and street MA &quot / tik&quot / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. <font size="3">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. <font size="3">Results of this study showed that:&nbsp / <font size="3">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05). <font size="3">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. <font size="3">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA. </font></font> <p>&nbsp / </p> </font>&nbsp / </font></font></font> <p>&nbsp / </p> </i></p> </font></p> <p>&nbsp / </p>

Kaunas sea contains more than 500 mln. m3 of Nemunas water. The Kaunas' sea Regional park was established at 1992. The purpose - to retain ecosystem, landscape, and inheritance of culture. Water molluscs - invertebrate organisms. They are very important in such ecosystem. Outspread of water molluscs is interconnected with climatic and edafical factors and is not influenced by such factors as competition.

<p><span style="font-size: 11.5pt">The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB. </span></p> <div><span style="font-size: 11.5pt">The aim of this study was to investigate <i>in vitro </i>effects of pure and street MA &ldquo / tik&rdquo / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. </span></div> <div><span style="font-size: 11.5pt">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. </span></div> <div><span style="font-size: 11.5pt">Results of this study showed that: </span></div> <div><span style="font-size: 11.5pt">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05)&nbsp / </span><span style="font-size: 11.5pt">&nbsp / </span></div> <div><span style="color: windowtext / font-size: 11.5pt">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). </span></div> <div style="margin: 0cm 0cm 25pt"><span style="color: windowtext / font-size: 11.5pt">3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) </span><span style="color: windowtext / font-size: 11.5pt">4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. </span><span style="color: windowtext / font-size: 11.5pt">5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase.&nbsp / </span></div> <div style="margin: 0cm 0cm 25pt"><span style="line-height: 115% / font-size: 11.5pt">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA.</span></div>

Cardiac progenitor cells (CPCs) are abundant in the embryonic heart and have hallmark features which include a rapid rate of cell division and the ability to differentiate into mature heart muscle cells (cardiomyocytes). Based on these features, CPCs are considered an attractive candidate cell type for transplantation therapies which aim to replenish the diseased heart muscle tissue (myocardium) with new muscle forming cells. A better understanding of how pharmacological drugs and endogenous hormones/signalling molecules modulate the balance between proliferation and differentiation of CPCs could be used to develop more effective cell based therapies for myocardial repair. Furthermore, this information could provide valuable new insight into molecular mechanisms regulating normal cardiogenesis during the embryonic period. The specific aims of the present study were to characterize the effects of the Ca2+ channel blocking drug nifedipine and the endogenous hormone/paracrine factor atrial natriuretic peptide (ANP) on CPC proliferation and differentiation. Results showed that primary cultured CPCs, isolated from the ventricles of embryonic day (E) 11.5 mouse embryos, underwent a reduction in cell cycle activity following exposure to nifedipine. Furthermore, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs) was associated with smaller graft sizes compared to control animals that did not receive the drug. Results from the present study also demonstrated that ANP receptor mediated signalling systems are biologically active in E11.5 ventricular cells and have an antiproliferative effect on cultured E11.5 CPCs. Moreover, preliminary data provided evidence that genetic ablation of the ANP high affinity receptor (NPRA) may be associated with impaired development of the ventricular cardiac conduction system. Collectively, work from this thesis provides evidence that interactions between transplanted cells and pharmacological drugs could have a significant impact on the effectiveness of cell based therapies and that ANP signalling systems may play a critical role in cardiac ontogeny by regulating the balance between CPC proliferation and differentiation.

As intestinal epithelial cells mature, they continuously transition from proliferation to differentiation to apoptosis under the influence of cell signalling pathways including c-Jun N-terminal kinase (JNK). Glutathione S-transferases (GSTs) are cytoprotective detoxification enzymes, some of which, including GSTA1, also sequester and inhibit JNK through complex formation. Thus, GSTA1 may be a key sensor of cellular state and regulator of responses to cell stress stimuli. The focus of this research study was to investigate the functional importance of GSTA1 in two contexts: 1) modulating complex integrity with JNK and activation of JNK by oxidative stress, 2) controlling cellular transitioning between proliferation, differentiation and apoptosis. In the first study, the impact of GSTA1 levels on dissociation of GSTA1-JNK complexes and JNK activation in response to cellular stress was investigated in the human colonic adenocarinoma Caco-2 cells. The pro-oxidant menadione caused GSTA1-JNK complex dissociation in preconfluent Caco-2 cells, whereas postconfluent cells were relatively resistant to this effect. Preconfluent cells were more sensitive than postconfluent cells to menadione-induced cytotoxicity. Additionally, menadione-induced JNK activation was transient since removal of the stimulus resulted in re-association of GSTA1 with JNK and significantly reduced cytotoxicity. Over-expression and knockdown of GSTA1 affected the degree of GSTA1-JNK complex association without altering the JNK activation. However, enhanced GSH levels by N-acetyl cysteine blocked menadione-induced complex dissociation and JNK activation in Caco-2 cells. The results suggest that the mechanism of menadione-mediated JNK activation involves the production of reactive oxygen species, likely superoxide anion, and that the level of intracellular GSH plays an important role in preventing menadione-induced GSTA1-JNK complex dissociation and subsequent JNK activation. The functional importance of GSTA1 in controlling cellular proliferation, differentiation and apoptosis was investigated. Sodium butyrate (NaB) is a short-chain fatty acid, physiologically present in the human large intestine and modulates transitioning of cell states in colon cancer cell lines. GSTA1 levels increased in association with differentiation markers in postconfluent Caco-2 cells. Forced expression of GSTA1 significantly reduced cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increased cells in S-phase and associated cell proliferation. NaB (1 mM) reduced Caco-2 cell proliferation, increased differentiation and up-regulated GSTA1 activity. In contrast, higher dose of NaB (10 mM) caused toxicity in preconfluent cells via apoptosis through caspase-3 activation in association with reduced GSTA1 activity. GSTA1 down-regulation by siRNA did not alter NaB-induced differentiation or the sensitivity of Caco-2 cells to NaB-induced apoptosis. Furthermore, NaB (10 mM) caused GSTA1-JNK complex dissociation but did not affect JNK activation. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.

The Hippo-LATS signaling pathway originally identified in Drosophila is conserved in mammalian systems and serves essential roles in mediating size control as well as tumorigenesis. In humans, the core kinase cassette consisting of adaptor proteins WW45 and MOB1, and Ser/Thr kinases MST1/2 and LATS1/2 signal by phosphorylating and inactivating transcriptional co-activators YAP and TAZ, causing cell growth arrest. As the central kinases within the Hippo-LATS pathway, examining the cellular and molecular phenotypes of LATS1 and LATS2 (LATS) will provide insight into the role of this pathway in tumorigenesis. By simultaneously knocking down both LATS1 and LATS2, genes that were differentially expressed were identified through a whole human genome microarray screen. The multitude of genes identified including CYR61, MYLK, CDKN1A, SLIT2, and TP53INP1 not only provide further evidence for the role of LATS in cell proliferation and apoptosis, but also implicate LATS in novel functions such as cell motility. Loss of LATS1 and/or LATS2 enhances cell migration whereas overexpression of LATS1 dramatically inhibits cell migration in multiple cell lines. The ability of LATS to regulate cell migration occurs through two potential mechanisms. Firstly, LATS functions through its kinase substrates YAP and/or TAZ, or alternatively, LATS1 directly binds actin and inhibits actin polyermization. Thus, through loss of functions studies, we identified a novel role for LATS in regulating cell migration as well as novel mechanisms of LATS function. As an important signaling molecule within the cell, LATS and the Hippo-LATS pathway are tightly regulated. Using clues from the Drosophila pathway, we examined how the previously uncharacterized gene, hEx, functions within this pathway. Importantly, this thesis characterizes hEx as a putative tumor suppressor showing that it can inhibit cell proliferation, sensitize cancer cells to Taxol treatment as well as inhibit tumor growth in nude mice. However, unlike Drosophila expanded, hEx functions independently of the Hippo-LATS pathway, suggesting that the mammalian signaling pathway is more complicated. The research findings from this thesis enhance our knowledge of the Hippo-LATS pathway in tumorigenesis by elucidating new functions and mechanisms of LATS functions as well as by exploring how upstream components function in relation to this pathway. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-09-23 10:06:54.687

No description available.