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Application Of Coastal And Marine Ecological Classification Standard (Cmecs) To Remotely Operated Vehicle (Rov) Video Data For Enhanced Geospatial Analysis Of Deep Sea EnvironmentsRuby, Caitlin A 06 May 2017 (has links)
The Coastal and Marine Ecological Classification Standard (CMECS) provides a comprehensive framework of common terminology for organizing physical, chemical, biological, and geological information about marine ecosystems. Federally endorsed as a dynamic content standard, all federally funded data must be compliant by 2018; however, applying CMECS to deep sea datasets and underwater video have not been extensively examined. The presented research demonstrates the extent to which CMECS can be applied to deep sea benthic habitats, assesses the feasibility of applying CMECS to remotely operated vehicle (ROV) video data in near-real-time, and establishes best practices for mapping environmental aspects and observed deep sea habitats as viewed by the ROV’s forwardacing camera. All data were collected during 2014 in the Northern Gulf of Mexico by the National Oceanic and Atmospheric Administration’s (NOAA) ROV Deep Discoverer and ship Okeanos Explorer.
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Exposure of cardiac microvascular endothelial cells to harmful stimuli : a study of the cellular responses and mechanismsGenis, Amanda 04 1900 (has links)
Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Exposure to harmful stimuli can render vascular endothelial cells dysfunctional, characterised by
reduced nitric oxide (NO) bioavailibility. Endothelial dysfunction (ED) is a reversible precursor of
ischaemic heart disease (IHD), and understanding the mechanisms underlying the development of
ED could lead to clinical strategies in preventing/treating IHD. Very little is known about the
responses of cardiac microvascular endothelial cells (CMECs) to pro-ED stimuli, as most studies are
conducted on macrovascular endothelial cells.
The current dissertation set out to comprehensively investigate the responses of cultured primary
adult rat CMECs to known harmful stimuli, viz. hypoxia and tumor necrosis factor-alpha (TNF-α; proinflammatory
cytokine). We were interested to investigate whether this distinct endothelial cell type
would develop classical features of ED, and if so, what the underlying mechanisms were. First we
aimed to establish a baseline characterization of the CMECs under control conditions. Next, we
developed a model of hypoxia-induced cell injury and measured apoptosis/necrosis, intracellular NO
and reactive oxygen species (ROS), expression and activation of signalling proteins involved with NObiosynthesis,
hypoxia and apoptosis, and differential regulation of proteins. Finally, we characterised
CMEC responses to treatment with TNF-α. We assessed apoptosis/necrosis, intracellular NO and ROS
levels, NO-biosynthesis pathway proteins and large-scale differential protein regulation. The above
measurements were performed by morphological assessment (light and fluorescence microscopy),
FACS analysis, western blotting and large-scale proteomic analyses.
Data showed that CMECs shared many baseline features with other endothelial cell types, including
morphological appearance, LDL-uptake, NO-production, and expression of eNOS protein. In a novel
observation, proteomic analysis revealed the expression of 1387 proteins. Another novel finding was
the high abundance of structural mitochondrial proteins, suggesting that CMECs require
mitochondria for non-respiration purposes as well. High expression of vesicle, glycolytic and RAS
signalling proteins were other features of the baseline CMECs. CMECs exposed to hypoxia responded
by increased apoptosis/necrosis and expression of the hypoxia-marker, HIF-1α. Interestingly, hypoxic
CMECs showed increased eNOS-NO biosynthesis, associated with increased mitochondrial ROS and
reduced anti-oxidant systems, suggestive of oxidative stress. In accordance with the literature,
several glycolytic proteins were up-regulated. A novel finding was the up-regulation of proteins
involved with protein synthesis, not usually described in hypoxic cell studies. The CMECs responded
to TNF-α-treatment by exhibiting hallmarks of ED, namely attenuated biosynthesis of PKB/Akt-eNOSderived
NO and the development of outspoken response to oxidative stress as indicated by the up-regulation of several anti-oxidant systems. The data showed that TNF-α treatment elicited classical
TNF-Receptor 1-mediated signalling characterized by the dual activation of pro-apoptotic pathways
(BID and caspase-3) as well as the protective, pro-inflammatory IKB-alpha–NF-KB pathway.
In conclusion, this is the first study of its kind to describe a comprehensive characterisation of CMECs
under baseline and injury-inducing conditions. On the whole, although it appeared as if the CMECs
shared many responses and mechanisms with more frequently researched endothelial cell types, the
data also supplied several novel additions to the literature, particularly with the application of
proteomics. We believe that this dissertation has provided more insights into endothelial
heterogeneity in the vascular system and into the mechanisms adopted by CMECs when exposed to
stimuli typically associated with cardiovascular risk. / AFRIKAANSE OPSOMMING: Blootstelling aan skadelike stimuli kan tot disfunksionaliteit van vaskulêre endoteelselle lei wat deur
verlaagde biobeskikbaarheid van stikstofoksied (NO) gekenmerk word. Endoteeldisfunksie (ED) is ‘n
omkeerbare voorganger van isgemiese hartsiekte (IHD), en ‘n beter begrip van die onderliggende
meganismes van ED kan lei tot die ontwikkeling van kliniese strategieë vir die
voorkoming/behandeling van IHD. Baie min is bekend oor die respons wat in kardiale
mikrovaskulêre endoteelselle (CMECs) uitgelok word na blootstelling aan pro-ED stimuli, omdat
meeste studies op makrovaskulêre endoteelselle uitgevoer word.
Die huidige proefskrif het daarna gemik om die respons van primêre kulture van volwasse rot CMECs
op bekende skadelike stimuli, nl. hipoksie en tumor nekrose faktor-alfa (TNF-α; pro-inflammatoriese
sitokien) in diepte te ondersoek. Ons was veral geïnteresseerd om vas te stel of hierdie spesifieke
endoteelseltipe die klassieke kenmerke van ED sou ontwikkel, en indien wel, wat die onderliggende
meganismes sou wees. Eerstens het ons beoog om ‘n basislyn karaterisering van CMECs onder
kontrole toestande daar te stel. Vervolgens het ons ‘n model van hipoksie-geïnduseerde selskade
gevestig en apoptose/nekrose, intrasellulêre NO en reaktiewe suurstofspesies (ROS), sowel as die
uitdrukking en aktivering van proteine betrokke by NO-biosintese, hipoksie en apoptose en
differensiële regulering van proteine gemeet. Laastens het ons die respons van CMECs op
behandeling met TNF-α gekarakteriseer. Ons het apoptose/nekrose, intrasellulêre NO en ROS
vlakke, NO-biosintese-seintransduksieproteïene en grootskaalse differensiele regulering van proteïene gemeet. Bg. metings is uitgevoer deur gebruik te maak van morfologiese evaluasie (lig -en
fluoressensiemikroskopie), vloeisitometriese analises, western blot analises en proteomiese analises.
Data het getoon dat die basislyn eienskappe van CMECs grootliks met dié van ander endoteelseltipes
ooreenstem, insluitende morfologiese voorkoms, LDL-opname, NO-produksie en die uitdrukking van
eNOS proteïen. In ‘n nuwe waarneming, het die proteomiese data die uitdrukking van 1387
proteïene aangetoon. ‘n Ander nuwe bevinding was die voorkoms van ‘n groot aantal strukturele
mitokondriale proteïene, wat daarop dui dat die CMECs mitokondria ook vir nie-respiratoriese
doeleindes gebruik. ‘n Hoë uitdrukking van vesikulêre, glikolitiese en RAS-seintransduksie proteïene
was ook kenmerkend van die basislyn CMECs. CMECS wat aan hipoksie blootgestel is, het reageer
met ‘n verhoging in apoptose / nekrose en verhoogde uitdrukking van die hipoksie merker, HIF-1α.
‘n Interressante bevinding was dat eNOS-NO biosintese sterk toegeneem het in die hipoksiese
CMECs wat met verhoogde mitokondriale ROS en verlaagde anti-oksidant sisteme (aanduidend van
oksidatiewe stres) gepaardgegaan het. In ooreenstemming met die literatuur, is verskeie glikolitiese
proteïene opgereguleer. ‘n Nuwe waarneming was die opregulering van proteïene wat betrokke is
by proteïensintese, iets wat nie normaalweg in hipoksie-studies beskryf word nie. Die CMECs het op
TNF-α behandeling gerespondeer deur tekens van ED te toon, naamlik ‘n afname in die NO
afkomstig van PKB/Akt-eNOS biosintese en die ontwikkeling van uitgesproke reaksie op oksidatiewe
stres soos aangedui deur die opregulering van verskeie anti-oksidant sisteme. Die data het ook
aangedui dat TNF-α behandeling tot klassieke TNF-reseptor 1 bemiddelde seintransduksie gelei het,
wat gekenmerk was deur die tweeledige aktivering van pro-apoptotiese seintransduksiepaaie (BID
en kaspase-3) sowel as die beskermende, pro-inflammatoriese IKB-alpha-NF-KB seintransduksiepad.
Ten slotte: hierdie is die eerste studie van sy soort wat die kenmerke en response van CMECs onder
basislyn en pro-besering omstandighede in diepte beskryf. Alhoewel dit oor die algemeen wil
voorkom asof die CMECs baie in gemeen het met ander, beter nagevorste endoteelseltipes, het die
data egter ook verskeie nuwe bevindinge tot die bestaande literatuur gevoeg, spesifiek die data
afkomstig van die proteomiese analises. Ons glo dat hierdie proefskrif meer insig verleen t.o.v. die
heterogeniteit van vaskulêre endoteelselle asook t.o.v. die megansimes wat deur CMECs aangewend
word wanneer hulle aan skadelike stimuli (geassosieer met kardiovaskulêre risiko) blootgestel word.
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