Environmental threats to fisheries are a rising concern for fishery managers. Gulf of Mexico hypoxia is particularly concerning. In order to properly manage Gulf fisheries, the sustainability of species stock and its natural capital values should be analyzed. As an empirical effort, I use blue crab in Louisiana waters as a case study to build a bioeconomic model of blue crab under hypoxia. I apply the natural capital valuation approach to estimate the blue crab value function under hypoxia in order to assess blue crab sustainability. I find that blue crab management in Louisiana under hypoxia is not sustainable, being less so under increasing hypoxia severity. In order to manage blue crab fisheries more stably under hypoxia, regulatory agencies along the Mississippi River should continue current efforts to reduce hypoxia, and take steps to sustain more blue crab stocks by management regimes reflecting the severity of hypoxia.

The vasculature is a highly intricate system of "highways" that shuttles blood from the heart to every tissue and organ in the human body. These vessels are responsible for carrying oxygen, trafficking hormones, delivering nutrients, and removing waste products from the body. The formation of a functioning vascular system depends on the close coordination of many cell types and, on the capillary level, specifically endothelial cells and pericytes as well as the surrounding protein microenvironment, known as the extracellular matrix (ECM). Impaired coordination amongst the cellular and protein constituents results in the improper functioning of the vascular network and can eventually contribute to the failure of organ systems. This dissertation research focuses on how improper ECM deposition affects vascular assembly. We utilized several approaches to affect ECM composition, specifically: 1) hypoxia exposure and 2) reducing ECM pharmacologically and utilizing lentiviral-mediated silencing of Type IV Collagen (Col-IV, gene Col4a1) expression. In these experimental settings, we observed downstream changes in the coordination between endothelial cells and pericytes while forming vascular networks. In short, this dissertation work suggests that excess ECM deposition, and particularly that of Col-IV, has unique deleterious effects on the developing vasculature as compared to reduced ECM deposition. The findings from this work suggest mechanisms underlying how the vasculature may be destabilized in hypoxia-associated pathologies, such as preeclampsia. / Doctor of Philosophy / Every tissue and organ in the human body receives blood from the heart via the extremely complex network of "highways" known as the vasculature. These vessels oversee moving nutrients, oxygen, hormones, and waste materials out of the body. At the capillary level, endothelial cells and pericytes, as well as the surrounding protein milieu known as the extracellular matrix (ECM), are required for the development of a functional vascular system. If the vascular network fails to develop and operate properly because of poor protein and cellular coordination, it can eventually lead to the failure of organ systems. The study for this dissertation focuses on how vascular development is impacted by insufficient ECM deposition. We used several strategies to modify the composition of the ECM, including 1) hypoxia exposure, 2) pharmaceutical ECM reduction, and 3) lentiviral-mediated delivery of shRNA to silence Type IV Collagen (Col-IV, gene Col4a1) production. We noticed alterations in the coordination between endothelial cells and pericytes as vascular networks were being formed in these experimental environments. In summary, this dissertation work contends that, in contrast to reduced ECM deposition, excess ECM deposition, and specifically that of Col-IV, has distinct detrimental consequences on the developing vasculature. The results of this study offer methods by which diseases associated with hypoxia, such preeclampsia, may cause the vasculature to become unstable.

The use of human adipose-derived mesenchymal stem cells (ASCs) for cell-based therapeutic approaches, in terms of repair and regeneration of various tissues and organs, offers an alternative therapeutic tool in the field of regenerative medicine. The ability of ASCs to differentiate along mesenchymal lineages is not the only property that makes these cells particularly attractive for therapeutic purposes. Their promising functions in promoting angiogenesis, reducing inflammation as well as in functional tissue restoration are largely related to the trophic effects of a broad panel of secreted cytokines and growth factors. However, in cell-based approaches, the cell-loaded construct often is exposed to an ischemic microenvironment characterized by severe oxidative and nutritional stress after transplantation due to the initial lack of vascular connection, resulting in reduced cell viability and altered cell behaviour. Therefore, the effective use of ASCs in regenerative medicine first requires a comprehensive characterization of the cells in terms of their viability, differentiation capacity and especially their secretory capabilities under ischemia-mimicking conditions in order to better understand their beneficial role. Accordingly, in the first part of this work, ASCs were investigated under different ischemic conditions, in which cells were exposed to both glucose and oxygen deprivation, with respect to viability and secretory function. Using mRNA gene expression analysis, significantly higher expression of selected angiogenic, anti-apoptotic and immunomodulatory factors (IL-6, VEGF, STC-1) could be demonstrated under harsh ischemic conditions. These results were reflected at the protein expression level by a significantly increased secretion of these factors. For stanniocalcin-1 (STC-1), a factor not yet described in ASCs, a particularly high expression with significant secreted amounts of the protein could be demonstrated under harsh ischemic conditions. Thus, the first part of this work, in addition to the characterization of the viability, provided first insights into the secretory response of ASCs under ischemic conditions. The response of ASCs to glucose deficiency in combination with severe hypoxia has been little explored to date. Thus, the focus of the second part of this work was on a more detailed investigation of the secretory response of ASCs under glucose and oxygen deprivation. For a more comprehensive analysis of the secretion profile, a cytokine antibody array was performed, which allowed the detection of a broad panel of secreted angiogenic factors (IL-8, ANG), matrix-regulating proteins (TIMP-1, TIMP-2), chemokines (MCP-1/CCL2, IP-10/CXCL 10) and other factors under ischemic conditions. To verify these results, selected factors were examined using ELISA. The analysis revealed that the secretion of individual factors (e.g., STC-1, VEGF) was significantly upregulated by the combination of glucose and oxygen deprivation compared to oxygen deprivation alone. In order to investigate the impact of the secretome of ischemic ASCs on cell types involved in tissue regeneration, the effect of conditioned medium of ischemia-challenged ASCs on both endothelial cells and fibroblasts was investigated in subsequent experiments. Significantly increased viability and tube formation of endothelial cells as well as activated migration of fibroblasts by the secreted factors of ischemic ASCs could be demonstrated. A direct correlation of these effects to STC-1, which was significantly upregulated under ischemic conditions and has been described as a regulator of key cellular functions, could not be verified. The particular secretory capacity of ASCs provides a valuable tool for cell-based therapies, such as cell-assisted lipotransfer (CAL), where by enriching fat grafts with isolated ASCs, a significantly improved survival rate of the transplanted construct is achieved with less resorption of the fat tissue as well as a reduction in adverse implications, such as fibrosis and cyst formation. In order to better understand the function of ASCs in CAL, an autologous transwell-based lipograft-ASC co-culture was established in the last part of this work, in which first investigations showed a markedly increased secretion of VEGF compared to lipografts without added ASCs. As the stability rate of the fat tissue and thus the success of CAL is presumably also dependent on the preparation of the tissue before transplantation, the conventional preparation method of fat tissue for vocal fold augmentation in laryngoplasty was additionally evaluated in vitro in a pilot experiment. By analyzing the viability and tissue structure of the clinically prepared injection material, a large number of dead cells and a clearly damaged tissue structure with necrotic areas could be demonstrated. In comparison, the preparation method of the fat tissue established in this work as small tissue fragments was able to provide a clearly intact, vital, and vascularized tissue structure. This type of adipose tissue preparation represents a promising alternative for clinical vocal fold augmentation. In conclusion, the results of this work contribute to a comprehensive characterization of ASCs under ischemic conditions, such as those prevalent at the transplantation site or in tissue regeneration. The results obtained, especially on the secretory capacity of ASCs, provide new insights into how ASCs mediate regenerative effects in an ischemic milieu and why their use for therapeutic purposes is highly attractive and promising. / Der Einsatz von humanen mesenchymalen Stammzellen aus dem Fettgewebe (ASCs) für zell-basierte Therapieansätze zur Reparatur und Regeneration von verschiedenen Geweben und Organen bietet eine alternative therapeutische Lösung im Bereich der regenerativen Medizin. Die Fähigkeit der ASCs zur Differenzierung in verschiedene mesenchymale Zelltypen ist jedoch nicht die einzige Eigenschaft, die diese Zellen für therapeutische Zwecke besonders attraktiv macht. ASCs sezernieren vielmehr ein breites Spektrum an Zytokinen und Wachstumsfaktoren, die z.B. durch Förderung der Angiogenese oder der Reduktion von Entzündungsprozessen eine wichtige Rolle bei regenerativen Therapien spielen können. Allerdings ist in zellbasierten Ansätzen, das zellbeladene Konstrukt nach der Transplantation – durch den anfänglich fehlenden Gefäßanschluss und die damit einhergehende mangelnde Versorgung des implantierten Gewebes – starkem oxidativem und ernährungsbedingtem Stress, einem ischämischen Milieu, ausgesetzt, was zu einer reduzierten Zellviabilität und einem veränderten Zellverhalten führt. Der effektive Einsatz der ASCs in der regenerativen Medizin erfordert demnach zunächst eine umfassende Charakterisierung der Zellen in Bezug auf deren Lebensfähigkeit, Differenzierungsfähigkeit und insbesondere die sekretorischen Fähigkeiten unter simulierten ischämischen Bedingungen, um ihren therapeutischen Effekt besser verstehen und optimieren zu können. Dazu wurden im ersten Teil dieser Arbeit die ASCs unter verschiedenen ischämischen Bedingungen, bei denen die Zellen sowohl einem Glukose- als auch Sauerstoffmangel ausgesetzt waren, hinsichtlich der Viabilität und der sekretorischen Funktion in vitro untersucht. Durch mRNA Genexpressionsanalysen konnte für ausgewählte angiogene, anti-apoptotische und immunmodulatorische Faktoren (IL-6, VEGF, STC-1) eine signifikant höhere Expression unter stark ischämischen Bedingungen gezeigt werden. Diese Ergebnisse spiegelten sich gleichermaßen auf Proteinebene durch eine signifikant erhöhte Sekretion der Faktoren wider. Für Stanniocalcin-1 (STC-1), einen Faktor, dessen Rolle bislang im Zusammenhang mit ASCs noch nicht beschrieben ist, konnte eine besonders hohe Expression mit signifikanten sezernierten Mengen des Proteins bei hoher ischämischer Belastung der Zellen gezeigt werden. Somit konnten im ersten Abschnitt der Arbeit neben einer ersten Charakterisierung der ASCs auch erste Erkenntnisse über das sekretorische Verhalten der Zellen in einem ischämischen Milieu gewonnen werden. Die Reaktion von ASCs auf Glukosemangel in Kombination mit Hypoxie ist bislang wenig untersucht. Somit lag der Fokus im zweiten Teil dieser Arbeit auf der detaillierteren Untersuchung des Sekretionsverhaltens von ASCs unter Glucose- und Sauerstoffdeprivation. Für eine umfassende Analyse des Sekretionsprofils wurde ein Zytokin-Antikörper-Array durchgeführt, mit welchem die Sekretion eines breiten Panels von angiogenen Faktoren (IL-8, ANG), matrixregulierenden Proteinen (TIMP-1, TIMP-2), Chemokinen (MCP-1/CCL2, IP-10/CXCL 10) sowie weiterer Faktoren unter ischämischen Bedingungen nachgewiesen werden konnte. Zur Verifizierung dieser Ergebnisse wurden ausgewählte Faktoren mittels ELISA untersucht. Durch diese Analyse konnte gezeigt werden, dass die Sekretion einzelner Faktoren (z.B. STC-1, VEGF) durch die Kombination von Glukose- und Sauerstoffentzug deutlich hochreguliert wird, z.B. gegenüber nur dem Entzug von Sauerstoff. Um die Wirkung des Sekretoms von ischämischen ASCs auf Zelltypen, die in der Regeneration von Geweben eine Rolle spielen, zu untersuchen, wurde in nachfolgenden Experimenten die Wirkung von konditioniertem Medium ischämischer ASCs sowohl auf Endothelzellen als auch auf Fibroblasten untersucht. Dabei konnte sowohl eine deutlich gesteigerte Röhrenbildung („tube formation“) von Endothelzellen als auch eine aktivierte Migration von Fibroblasten durch die sezernierten Faktoren der ischämischen ASCs nachgewiesen werden. Ein direkter Zusammenhang dieser Effekte mit dem unter ischämischen Bedingungen signifikant hochregulierten Faktor STC-1, welcher als Regulator zellulärer Schlüsselfunktionen beschrieben wird, konnte hingegen nicht nachgewiesen werden. Die besondere Sekretionsfähigkeit von ASCs stellt ein wertvolles Werkzeug für zellbasierte Therapien dar, wie z.B. den zellassistierten Lipotransfer (CAL), bei dem durch die Anreicherung von Fetttransplantaten mit isolierten ASCs eine deutliche Verbesserung der Überlebensrate des transplantierten Konstrukts mit einer geringeren Resorption des Fettgewebes sowie einer Verringerung von unerwünschten Folgen, wie Fibrosen und Zystenbildung, erzielt wird. Um die Funktion der ASCs im CAL besser charakterisieren zu können, wurde im letzten Teil dieser Arbeit eine autologe Transwell-basierte Lipograft-ASC-Kokultur etabliert, in welcher durch erste Untersuchungen eine signifikant erhöhte Sekretion von VEGF im Vergleich zu den Lipografts ohne Zusatz von isolierten ASCs gezeigt werden konnte. Da die Stabilitätsrate des Fettgewebes und damit der Erfolg des CAL mutmaßlich auch von der Aufbereitung des Gewebes vor der Transplantation abhängig ist, wurde in einem Pilot-Experiment die konventionelle Präparationsmethode von Fettgewebe für die Stimmlippenaugmentation in der Laryngoplastik in vitro evaluiert. Durch Analysen zur Viabilität und Gewebestruktur konnte bei dem klinisch aufbereiteten Injektionsmaterial eine große Anzahl abgestorbener Zellen sowie eine deutlich geschädigte Gewebestruktur mit nekrotischen Arealen nachgewiesen werden. Im Vergleich dazu konnte mit der in dieser Arbeit etablierten Präparationsmethode des Fettgewebes als kleine Gewebsfragmente eine deutlich intakte, vitale und vaskularisierte Gewebestruktur erhalten werden. Damit bietet diese Art der Aufbereitung von Fettgewebe eine vielversprechende Alternative für die klinische Stimmlippenaugmentation.  Zusammengefasst tragen die Ergebnisse dieser Arbeit zu einer umfassenden Charakterisierung von ASCs unter ischämischen Bedingungen bei, wie sie beispielsweise am Transplantationsort oder in der Geweberegeneration vorliegen können. Die gewonnenen Ergebnisse, insbesondere zu den sekretorischen Fähigkeiten der ASCs, liefern neue Erkenntnisse darüber, wie ASCs regenerative Effekte in einem ischämischen Milieu vermitteln und weshalb deren Verwendung für therapeutische Zwecke besonders attraktiv und vielversprechend ist.

No description available.

The objective of this thesis is to examine the relationship between CAIX (a biomarker for insufficient oxygen in tumor microenvironment) and CD8+ T cells (the immune cells for killing cancer cells) for ovarian cancer. We approach the problem from two perspectives. The first approach is to set up count models such as Poisson, negative binomial, and zero-inflated Poisson models to examine the cell counts between CAIX and CD8+ T cells in the tumor microenvironment. The second approach is to apply the cross-K function, which is a second-order property of the point pattern process. We find that the tissue microarray (TMA), which is a technique to assemble hundreds of tissue samples on one TMA block, has a fixed effect on the CD8+ T cell counts. There are two TMA blocks A2 and B1. The relationship between CAIX and CD8+ T cells highly depends on TMAs. On TMA B1 stroma, a negative relationship between CAIX and CD8+ T cell counts is observed in the negative binomial models. When taking the spatial domain into account and comparing the estimated cross-K function of CAIX and CD8+ T cells to the simulated envelopes generated by a homogeneous Poisson process, we find that CAIX and CD8+ T cells are regulated and repel each other on TMA B1. Tissue category also plays an influential role in analyzing the relationship. The estimated cross-K function of CAIX and CD8 + T cells is more dispersed on tumors than on stroma. / Graduate

No description available.

Organisms are often exposed to multiple stressors in their natural habitat. While the effects of individual stressors alone are generally well-studied, their combined interactions are often unknown. One such unknown interaction is that between copper, an essential micronutrient that is toxic in high levels, and hypoxia, a commonly experienced environmental stressor. This project sought to examine the effects that copper has on the hypoxic response as well as the combined effects of copper and hypoxia exposure using freshwater-acclimated adult killifish (Fundulus heteroclitus), a model euryhaline teleost. It was found that copper blunts the acute ventilatory response to hypoxia, ammonia, and combined hypoxia and ammonia exposure. Gill morphometry was also examined and it was found that while copper alone has no effect, when combined with hypoxia exposure it eliminates the increase in filament length and cross-sectional area seen when fish were exposed to hypoxia alone. Furthermore, when neuroepithelial cell size was examined, copper decreased cell projection area with or without the combined presence of hypoxia. Taken as a whole, this thesis indicates that copper exposure can significantly and negatively impact the ability of aquatic organisms to mount appropriate physiological responses to hypoxia. / Thesis / Master of Science (MSc)

The bristlenose pleco (Ancistrus cirrhosus) is a species of armored catfish in the Loricariidae family that breathes air facultatively when the aquatic environment becomes hypoxic. The bristlenose pleco uses its highly vascularized stomach as an air breathing organ. The two main goals of this developmental study were to determine the size of onset of air breathing and to determine the frequency of air breathing behavior in bristlenose plecos from juveniles to adults. Developing juveniles reach functional maturity within four to six months of hatching and grow to an adult size of eight to ten cm in length. To examine the developmental timing for the onset of air breathing, we tested different sized juveniles beginning at one cm up until 8 cm in length. The developmental timing for the onset of air breathing was measured by exposing each fish to a slowly decreasing aquatic oxygen content from 100% air saturation down to 8% air saturation. Fish were first able to breathe air at just over 2 cm and 1 gram in mass. There was a weak negative correlation between fish length and % air saturation at which air breathing began. When exposed to 15% air saturation, frequency of air breathing was negatively correlated with fish length. Armored catfish are becoming an invasive species in the southern US, outcompeting local fauna potentially because of this adaptation. This research provides important insight into the development of the air breathing adaptation that may allow these fish to outcompete the others.

High altitude is characterized by chronically low ambient temperatures and oxygen. To survive, highland native deer mice (Peromyscus maniculatus) are capable of high rates of prolonged thermogenesis due to elevated aerobic capacity (V̇O2max) in hypoxia. Deer mice primarily use fats to fuel their high metabolic rates for heat production. Carnitine palmitoyl-transferase 1 (CPT-1) is a rate-limiting step in mitochondrial fat oxidation, and a reduction in CPT-I sensitivity for its substrate L-carnitine is associated with a reduction in muscle fat use during high intensity exercise in mammals. Sensitivity of mitochondrial metabolism to ADP also changes with exercise. It is currently unknown whether similar mechanisms underpin regulation of fuel use during shivering, but I predicted that sensitivities to ADP and L-carnitine would be greater in highlanders than lowlanders and increase with acclimation. To address this question, I examined mitochondrial sensitivity to substrates involved in the fat oxidation pathway in low- and high- altitude deer mice born and raised in common laboratory conditions. Mice were also acclimated to high altitude condition of cold hypoxia to examine if the plasticity of these traits were affected by altitude ancestry. Consistent with previous findings, both high and lowland mice increased their cold-induced V̇O2max following cold hypoxia acclimation and rely primarily on lipids to fuel thermogenesis. High- and low-altitude deer mice responded differently to chronic cold hypoxia with highlanders showing a ~7-fold greater ADP sensitivity than lowlanders following acclimation. In contrast to the expected outcome, highlander deer mice tended to have a reduced sensitivity to L-carnitine compared to lowlanders that approached statistical significance. Neither sensitivity to palmitoylcarnitine sensitivity nor mitochondrial expression of FAT/CD36, thought to aid in mitochondrial fat delivery, showed differences between population or changes with acclimation, indicating that limitations to lipid oxidation during shivering likely occur at, or upstream of, CPT-I in the deer mouse. / Thesis / Master of Science (MSc) / Some animals can survive extremely harsh climates, such as high altitude. High altitude is characterized by unremitting cold and thin air, and these challenges can constrain aerobic activities in mammals. The North American deer mouse can thrive at high altitude by actively generating large amounts of body heat in a process known as thermogenesis. The deer mouse relies primarily on fats as fuel to support thermogenesis, but the cellular mechanisms that regulate the use of lipids to power thermogenesis remain unclear. To address this question, I induced shivering in deer mice from both high- and low-altitude populations that I exposed to simulated high- or low-altitude conditions. I then examined the effects of these treatments on the ability of shivering muscle to consume oxygen and fuel for thermogenesis. My thesis contributes to the current understanding of how mammals manage their energy supply to survive in a challenging environment.

No description available.