Master of Science / Department of Clinical Sciences / Rose M. McMurphy / Hypoventilation (PaCO2 > 45 mmHg) and large P(A-a)O2 gradients due to V/Q mismatch and shunt, are common during isoflurane anesthesia in horses. A fraction of inspired oxygen < 50% has been shown to improve ventilation and decrease intra-operative atelectasis in humans and some animals. The study compared the effects of two different fractions of inspired oxygen, 50% versus > 95%, on ventilation, respiratory pattern, and P(A-a)O2 gradient in isoflurane anesthetized horses. Eight mature horses were sedated with IV xylazine (1.0 mg/kg) and anesthetized with diazepam (0.05 mg/kg) and ketamine (2.2 mg/kg) twice. Anesthesia was maintained with isoflurane (ET1.5 vol%) in either 50 or > 95% oxygen for 90 minutes. Both treatments were randomly assigned to each horse with a one week interval in between treatments. Horses were positioned in dorsal recumbency, connected to a preloaded circle breathing system and allowed to spontaneously ventilate. Measurements included inspiratory and expiratory peak flow and time, tidal volume, respiratory frequency, ETCO2, CO2, O2, PaO2, PaCO2, pH, SaO2, heart rate, and arterial blood pressure. Calculated values included PAO2, P(A-a)O2, P(A-a)O2 rate of change, and physiologic dead space. FiO2 of 50% resulted in a lower PaO2, SaO2, PAO2, and P(A-a)O2. No significant change in PaCO2, ventilatory pattern, or any remaining measured variables was observed (p<0.05). The use of 50% oxygen and nitrogen as the carrier gas did not significantly change the ventilatory characteristics or improve oxygenation in isoflurane anesthetized horses. Repeatable respiratory rhythms characteristics were observed for horses while inspiring 50% and > 95% oxygen. A high A-a oxygen gradient with an equal rate of change overtime was still observed during both treatments.

Through complex metabolic interactions aquatic microbial life is essential as a driver of ecosystem functions and hence a prerequisite for sustaining plant and animal life in the sea and on Earth. Despite its ecological importance, infor­mation on the complexity of microbial functions and how these are related to environmental conditions is limited. Due to climate change and eutrophication, marine areas facing oxygen depletion are increasing and predicted to continue to do so in the future. Vertically steep oxygen gradients are particularly pronoun­ced in the Baltic Sea. In this thesis, therefore, the ecosystem functions of micro­bial communities were investigated, using metagenomics, to understand how they were distributed along the steep oxygen gradient at the Landsort Deep, the deepest point of the Baltic Sea. Furthermore, microbial communities from the Lands­ort Deep transect were compared to microbial communities of other marine environments to establish whether the environment at this site resulted in a characteristic community. To reveal what microbial community functions and taxa were active in the anoxic sediment a metatranscriptomic approach was used. Results showed a marked effect of the coupled environmental parameters dissolved oxygen, salinity and temperature on distribution of taxa and par­ti­cularly community functions. Microbial communities showed functional capa­cities consistent with a copiotrophic life-style dependent on organic ma­terial sinking through the water column. The eutrophic condition with high organic load was further reflected in the metatranscriptome of the anoxic sedi­ment com­munity, which indicated active carbon mineralisation through ana­erobic hetero­trophic-autotrophic community synergism. New putative linkages between nitro­gen and- sulphur metabolisms were identified at anoxic depths. Further­more, viable Cyanobacteria in the anoxic sediment was evident from the tran­script analyses as another reflection of marine snow. High abundance and expres­­sion of integron integrases were identified as a charac­teristic feature of the Lands­ort Deep communities, and may provide these communities with a mech­an­ism for short-term-adaptation to environmental change. In summary, this thesis clearly documents what impact eutrophication and oxygen depletion have on microbial community functions. Furthermore, it specifically advances the mechanistic insight into microbial processes in anoxic deep-water sediment at both genomic and transcriptional level. Given the predicted progress of oxygen depletion in marine and brackish environments, this work advances information necessary to estimate effects on marine and in particular brackish ecosystem functions where anoxic conditions prevail. / Mikroorganismer är essentiella för fungerande ekosystemfunktioner i akvatiska miljöer och därmed en förutsättning för övrigt växt- och djurliv på vår planet. Trots deras ekologiska nyckelroll är kunskapen om mikroorganismernas funk­tion och komplexitet samt hur dessa är relaterade till miljön begränsad. På grund av eutrofiering och klimatförändringar har marina områden som lider av syrebrist ökat och en ytterligare utbredning av marina och bräckta områden med syrebrist är predicerad i framtiden. Stora områden av Östersjön kännetecknas av vertikala syregradienter med syresatt ytvatten och anoxiskt bottenvatten. I denna avhandling undersöktes därför med metagenomik hur mikrobiella ekosystems funktioner var utbredda längs den vertikala syregradienten i Östersjöns djupaste del, Landsortsdjupet. Dessutom jämfördes de mikrobiella samhällena från Lands­­­ortsdjupet med mikrobiella samhällen från andra marina miljöer för att utröna om den karakteristiska miljön i Landsortsdjupet återspeglade de mikro­biella samhällen som lever där. För att undersöka vilka mikroorganismer samt vilka mikrobiella ekosystemfunktioner som var aktiva i det anoxiska sedimentet i Lands­ortsdjupet användes metatranskriptomik. Resultaten visade en stark kor­re­lation mellan miljöparametrarna syrehalt, salinitet och temperatur och för­del­ningen av mikrobiell taxa och i synnerhet mikrobiell funktion längs Lands­orts­djupets transekt. De mikrobiella samhällena uppvisade en funktionell kapa­citet förenlig med en livsstrategi beroende av organiskt material som sjunker genom vattenkolonnen som en konsekvens av eutrofiering. Eutrofa förhållanden med hög halt av organiskt material var även återspeglad i metatranskriptomet från det anoxiska sedimentet, som indikerade aktiv mineralisering av organiskt kol genom anaerob heterotrof-autotrof synergism. Nya möjliga kopplingar mellan kväve- och svavelmetabolism identifierades i det anoxiska vattnet. Vidare visade resultat från metatranskriptom-analys att livsdugliga cyanobakterier var abun­danta i det mörka och anoxiska sedimentet, vilket även detta kan vara en konse­kvens av sjunkande organiskt material. Hög abundans och hög transkribering av integrongener kunde identifieras som ett karakteristiskt kännetecken hos de mikro­biella samhällena i Landsortsdjupet vilket skulle kunna förse dem med en me­kanism för anpassning till miljöförändringar. Sammanfattningsvis dokumen­terar denna avhandling tydligt vilken påverkan eutrofiering och syrebrist har på mikrobiella funktioner. Dessutom för den specifikt kunskapen om mikrobiella processer i anoxiska djupvattensediment framåt på både genom- och transkrip­tions­nivå. Mot bakgrund av en predicerad ökning av syrebristen i marina mil­jöer, bidrar denna avhandling med information som är viktig för att kunna förutse vilka effekter anoxiska förhållanden kan komma att få på ekosystemfunktioner i marina miljöer och i brackvattenmiljöer i synnerhet.

Precise control of stem cell fate is a fundamental issue in the use of human embryonic stem (hES) cells in the context of cell therapy We examined three ways in which the microenvironment can be controlled to alter hES cell behavior, providing insight into the best conditions for maintenance of pluripotency and neural differentiation in developmental and therapeutic studies. We first examined the effects of polydimethylsiloxane (PDMS) growth surfaces on hES cell survival and maintenance of pluripotency. Lightly cured, untreated PDMS was shown to be a poor growth surface for hES cells. Some of the adverse effects caused by PDMS could be mitigated with increased curing or UV treatment of the surface, but neither modification provided a growth surface that supported pluripotent hES cells as well as polystyrene. This work provides a basis for further optimizing PDMS for hES cell culture, moving towards the use of microdevices in establishing precise control over stem cell fate. The second study explored the use of an easily constructed diffusion-based device to grow hES cells in culture on a defined, physiologic oxygen (O₂) gradient. We observed greater hES cell survival and higher levels of pluripotency markers in the lower O₂ regions of the gradient. The greatest benefit was observed at O₂ levels below 5%, narrowing the potential optimal range of O₂ for the maintenance of pluripotent hES cells. Finally, we developed a small molecule-mediated adherent and feeder-free neural differentiation protocol that reduced the cost and time scale for in vitro differentiation of neural precursors and functional neurons from human pluripotent cells. hES cell-derived neural precursors transplanted into a murine model of focal ischemic stroke survived, improved neurogenesis, and differentiated into neurons. Transplant also led to a more consistent and measurable sensory recovery after stroke as compared to untransplanted controls. This protocol represents a potentially translatable method for the generation of CNS progenitors from human pluripotent stem cells.