Luminescent techniques for studying free radical production and cell viability in relation to cardiovascular disease and HRT

Epidemiological studies have suggested that hormone replacement therapy (HRT) offers protection from atherosclerosis, a precursor of cardiovascular disease (CVD), in postmenopausal women. There is good evidence that oxidation of low-density lipoprotein (LDL) by leucocyte-derived reactive oxygen species plays a key role in development of an atherosclerotic plaque. Therefore we have investigated whether the possible protection against CVD by HRT could be due to immunomodulation, specifically of free radical production. The study involves 2 approaches: I) analysing the production of free radicals by leucocytes from women on HRT, 2) investigating the effect of I7p-oestradiol and progesterone on cultured myeloid cells (HL60 and U937). Free radical production by leucocytes was determined using a recently developed bioluminescent assay. In the assay, Pholasin® emits light in the presence of free radicals produced by the NADPH oxidase system of leucocytes stimulated with PMA or fMLP. Cell viability was also investigated using a bioluminescent assay (Cell Titer-Glo®) in which cytosolic ATP levels were measured by the production of luminescence in the presence of Luciferin/Luciferase reagent. Studies of leucocytes from HRT patients showed considerable variation in free radical production, which appeared to be dependent on HRT regime. Studies on the cultured cells showed that there was no cell proliferation at low hormone concentrations, while high concentrations caused cytotoxicity. The effect of hormones on free radical production in this in vitro model system is currently being investigated. The results show that the effects of the hormones on cells of the immune system are very dose dependent, and that both beneficial and adverse effects may occur. In conclusion, luminescent techniques offer a valuable and sensitive approach to studying inflammatory and oxidative processes both in vivo and in vitro.

Viability Kernels of Higher Order

AMS subject classification: Primary 34A60, Secondary 49K24.

Time-Dependent Differential Inclusions and Viability

AMS subject classification: Primary 34A60, Secondary 49J52.

Viability, Optimality and Stability of Dynamical Systems and Estimation of Convergence of Numerical Schemes

AMS subject classification: 49N35,49N55,65Lxx.

Nicotine exposure reduces cell viability but induces anti-inflammatory effects in human cystic fibrosis bronchial epithelial cells

Background: Mouse models of cystic fibrosis (CF) fail to truly represent the respiratory pathology. We have consequently developed human airways cell culture models to address this. The impact of cigarette smoke within the CF population is well documented, with exposure being known to worsen lung function. As nicotine is often perceived to be a less harmful component of tobacco smoke, this research aimed to identify its effects upon viability and inflammatory responses of CF (IB3-1) and CF phenotype corrected (C38) bronchial epithelial cells. Methods: IB3-1 and C38 cell lines were exposed to increasing concentrations of nicotine (0.55-75μM) for 24 hours. Cell viability was assessed via Cell Titre Blue and the inflammatory response with IL-6 and IL-8 ELISA. Results: CF cells were more sensitive; nicotine significantly (P<0.05) reduced cell viability at all concentrations tested, but failed to have a marked effect on C38 viability. Whilst nicotine induced anti-inflammatory effects in CF cells with a significant reduction in IL-6 and IL-8 release, it had no effect on chemokine release by C38 cells. Conclusion: CF cells may be more vulnerable to inhaled toxicants than non-CF cells. As mice lack a number of human nicotinic receptor subunits and fail to mimic the characteristic pathology of CF, these data emphasise the importance of employing relevant human cell lines to study a human-specific disease.

Viability Theory and Economic Modeling

A brief introduction into the theory of differential inclusions, viability theory and selections of set valued mappings is presented. As an application the implicit scheme of the Leontief dynamic input-output model is considered.

Population viability analyses of Chamaecrista keyensis (Leguminosae: Caesalpinioideae), a narrowly endemic herb of the lower Florida Keys: Effects of seasonal timing of fires and the urban -wildland interface

This research first evaluated the effects of urban wildland interface on reproductive biology of the Big Pine Partridge Pea, Chamaecrista keyensis, an understory herb that is endemic to Big Pine Key, Florida. I found that C. keyensis was self-compatible, but depended on bees for seed set. Furthermore, individuals of C. keyensis in urban habitats suffered higher seed predation and therefore set fewer seeds than forest interior plants. ^ I then focused on the effects of fire at different times of the year, summer (wet) and winter (dry), on the population dynamics and population viability of C. keyensis. I found that C. keyensis population recovered faster after winter burns and early summer burns (May–June) than after late summer burns (July–September) due to better survival and seedling recruitment following former fires. Fire intensity had positive effects on reproduction of C. keyensis. In contrast, no significant fire intensity effects were found on survival, growth, and seedling recruitment. This indicated that better survival and seedling recruitment following winter and early summer burns (compared with late summer burns) were due to the reproductive phenology of the plant in relation to fires rather than differences in fire intensity. Deterministic population modeling showed that time since fire significantly affected the finite population growth rates (λ). Particularly, recently burned plots had the largest λ. In addition, effects of timing of fires on λ were most pronounced the year of burn, but not the subsequent years. The elasticity analyses suggested that maximizing survival is an effective way to minimize the reduction in finite population growth rate the year of burn. Early summer fires or dry-season fires may achieve this objective. Finally, stochastic simulations indicated that the C. keyensis population had lower extinction risk and population decline probability if burned in the winter than in the late summer. A fire frequency of approximately 7 years would create the lowest extinction probability for C. keyensis. A fire management regime including a wide range of burning seasons may be essential for the continued existence of C. keyensis and other endemic species of pine rockland on Big Pine Key. ^

Análise estrutural e avaliação do efeito condroitim sulfato extraído de tilápia (Oreochromis niloticus) em modelo de peritonite aguda

Chondroitin sulfate (CS) is a naturally glycosaminoglycan found in the extracellular matrix of connective tissues and it may be extracted and purified those tissues. CS is involved in various biological functions, which may be related to the having structural variability, despite the simplicity of the linear chain structure from this molecule. Researches in biotechnology and pharmaceutical field with wastes from aquaculture has been developed in Brazil. In recent decades, tilapia (Oreochromis niloticus), native fish from Africa, has been one of the most cultivated species in various regions of the world, including Brazil. The tilapia farming is a cost-effective activity, however, it generates large amount of wastes that are discarded by producers. It is understood that waste from tilapia can be used in research as a source of molecules with important biotechnological applications, which also helps in reducing environmental impacts and promote the development of an ecofriendly activity. Thus, nile tilapia viscera were subjected to proteolysis, then the glycosaminoglycans were complexed with ion exchange resin (Lewatit), it was fractionated with increasing volumes of acetone and purified by ion exchange chromatography DEAE-Sephacel. Further, the fraction was analyzed by agarose gel electrophoresis and nuclear magnetic resonance (NMR). The electrophoretic profile of the compound together the analysis of 1H NMR spectra and the HSQC correlation allow to affirm that the compound corresponds to a molecule like chondroitin sulfate. MTT assay was used to assess cell viability in the presence of CS tilapia isolated and showed that the compound is not cytotoxic to normal cells such as cells from the mouse embryo fibroblast (3T3). Then, this compound was tested for the ability to reduce the influx of leukocytes in model of acute peritonitis (in vivo) induced by sodium thioglycolate. In this context, it was done total and differential leukocytes counting in the blood and peritoneal fluid collected respectively from vena cava and the peritoneal cavity of the animals subjected to the experiment. The chondroitin sulfate for the first time isolated from tilapia (CST ) was able to reduce the migration of leukocytes to the peritoneal cavity of inflamed mice until 80.4 per cent at a dose 10µg/kg. The results also show that there was a significant reduction (p<0.001) of the population of polymorphonuclear leukocytes from peritoneal cavity in the three tested doses (0.1µg/kg; 1µg/kg and 10µg/kg) when it was compared to the positive control (just thioglycolate). Therefore, since the CST structure and mechanism of action has been completely elucidated, this compound may have potential for therapeutic use in inflammatory diseases

Caracterização estrutural e atividades farmacológicas do alginato obtido da alga Dictyopteris delicatula (J.V. Lamouroux., 1809) e seu derivado sulfatado

Marine algae are rich sources of various structural compounds which recently has been increasingly studied as a new source of bioactive substances. The alginate, as come as fucans, are considered the main acidic polysaccharides found in brown seaweed. This molecule consists a linear natural polysaccharide, non-sulfated, and presents monosaccharides: acid β-D-mannuronic (M) and α-L-guluronic acid (G); in a vast amount compositions and threads. Alginate has been widely applied in food and pharmaceutical industries because of its ability to retain water, forming films and gels as well as thickening, stabilizing and form emulsions. In this work we aimed to extract, structurally characterize, compare and analyze the possible pharmacological activities of native alginate molecule obtained from brown seaweed Dyctiopteris delicatula (DYN), and its chemically sulfated derivative (DYS). The alginate structure and composition molecule can be proven through chemical dosing, that showed low protein contamination and high sugar level, existence and separation of M and G blocks in the descending paper chromatography, infrared spectroscopy and nuclear magnetic resonance. Molecule sulfation was proven with sulphate dosage, resulting in 28.56% sulphate in molecule; electrophoresis, verify metachromasia with toluidine blue; and infrared spectroscopy, that showed a characteristic band at 1221cm-1 corresponding a sulfate group vibration. For the pharmacological activities the tests was: antioxidant activity, changes in cell function (MTT test) and anticoagulant test. In the antioxidant activity we observed that DYN showed better results in the kidnapping of hydroxyl radicals and ferric chelation compared to DYS, this had the best result in the total antioxidant capacity. Both showed similar activity in reducing power and the kidnapping radicals DPPH. In MTT test DYN and DYS had not proliferative and cytotoxic activity in fibroblast cells (3T3) and showed antiproliferative and cytotoxic activity in cancer cell lines HeLa and B16 melanoma. In anticoagulant assay DYN showed good activity in the intrinsic pathway of blood coagulation, and a small activity in the extrinsic pathway, in the other hand DYS showed only a very small activity in the extrinsic pathway, but cannot come to be regarded as an anticoagulant agent. From these results it can be concluded that the alginate was extracted and sulfated, revealing a potential compound to be used in the pharmaceutical industry as an anticoagulant agent, antioxidant and antitumor and the sulfation has not been conclusively important to performance in the tested pharmacological activities

Neuroprotection and regeneration of adult lesioned retinal ganglion cells by the modulation of fibroblast growth factor-2 and receptor tyrosine phosphatase-sigma

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me2SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me2SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me2SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me2SO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.

Neuroprotection and regeneration of adult lesioned retinal ganglion cells by the modulation of fibroblast growth factor-2 and receptor tyrosine phosphatase-sigma

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Climate change and the long-term viability of the World’s busiest heavy haul ice road

Climate models project that the northern high latitudes will warm at a rate in excess of the global mean. This will pose severe problems for Arctic and sub-Arctic infrastructure dependent on maintaining low temperatures for structural integrity. This is the case for the economically important Tibbitt to Contwoyto Winter Road (TCWR)—the world’s busiest heavy haul ice road, spanning 400 km across mostly frozen lakes within the Northwest Territories of Canada. In this study, future climate scenarios are developed for the region using statistical downscaling methods. In addition, changes in lake ice thickness are projected based on historical relationships between measured ice thickness and air temperatures. These projections are used to infer the theoretical operational dates of the TCWR based on weight limits for trucks on the ice. Results across three climate models driven by four RCPs reveal a considerable warming trend over the coming decades. Projected changes in ice thickness reveal a trend towards thinner lake ice and a reduced time window when lake ice is at sufficient thickness to support trucks on the ice road, driven by increasing future temperatures. Given the uncertainties inherent in climate modelling and the resultant projections, caution should be exercised in interpreting the magnitude of these scenarios. More certain is the direction of change, with a clear trend towards winter warming that will reduce the operation time window of the TCWR. This illustrates the need for planners and policymakers to consider future changes in climate when planning annual haulage along the TCWR.

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.