The research in this thesis investigated the role of the hypoxia-inducible factor (HIF) family of transcription factors in metabolism and cardiopulmonary physiology. Specifically, the effects of HIF on ventilatory control, carotid body morphology, and cardiac metabolism and function were studied using a murine model of a genetic disorder of oxygen sensing known as Chuvash polycythaemia. HIF coordinates oxygen-regulated gene expression throughout all organ systems, thereby orchestrating cellular, tissue and systemic responses to hypoxia. HIF is primarily regulated by oxygen-dependent prolyl hydroxylase-domain enzymes (PHDs) that initiate its degradation via the von Hippel-Lindau protein (VHL). In Chuvash polycythaemia, a homozygous VHL mutation in humans causes generalised stabilisation of HIF in euoxia, resulting in profound changes in cardiopulmonary physiology, exercise and metabolism. The Chuvash mouse model provides an opportunity to further characterise the role of HIF in different organ systems. Chapter 2 of this thesis introduces the murine model, demonstrating an increase in haemoglobin and haematocrit in the Chuvash mice as well as a marked reduction in body weight. Chapter 3 describes the ventilatory and carotid body study. Chuvash mice were shown to have elevated baseline ventilation in euoxia and marked ventilatory sensitivity to hypoxia. These findings were accompanied by changes within the carotid body, including hyperplasia, hypertrophy and altered ultrastructure of the oxygen-sensing type I cells. Chapter 4 of this thesis describes the study into cardiac metabolism, energetics and function. Chuvash hearts were found to have increased glycolytic flux and lactate production (the latter both in and ex vivo), with altered myocardial energetics. Despite this, left ventricular function remained normal, although in vivo cine MRI revealed clear evidence of pulmonary hypertension and right ventricular hypertrophy. Overall, this thesis provides evidence that the PHD-VHL-HIF axis plays a major role in calibrating the hypoxic response in the principal organ systems responsible for oxygen uptake, delivery and utilisation.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction: Among the cancers that afflict females world-wide, neoplastic disease of breast tissue is the most frequently diagnosed form and the leading cause of cancer-related death. Conventional treatment entails the use of doxorubicin, an anticancer agent belonging to the anthracycline family of chemotherapeutic drugs. Cancer cells are becoming increasingly resistant to doxorubicin therapy. The existence of hypoxic zones, which is a common feature of solid tumours, has been shown to promote the selection of therapy resistant clones in proliferating cancer cells. By modifying cellular homeostasis, neoplastic cells are capable of tolerating the hypoxic insult and thriving within the hostile microenvironment of the tumour. This adaptation is known as ‘the hypoxic response’ and is mediated through the action of the transcriptional regulator, HIF-1. Its expression in cancer tissue has been associated with a dismal prognosis as it promotes the degree of malignancy to an advanced stage. Hypothesis & Aims: We hypothesized that the targeting of HIF-1α would circumvent the ‘protective’ hypoxic response conferred upon breast cancer and improve the cytotoxicity of doxorubicin treatment. In this study, the first aim was to identify the hypoxic conditions at which the MCF-7 breast cancer cell line manifests a doxorubicin-resistant phenotype. This was followed by examination of the molecular pathways contributing to the hypoxic resistance by elucidating the potential relationship with the hypoxic regulator HIF-1α. Once the involvement of HIF-1α was established, the next aim was to evaluate whether the attenuation of HIF-1α would terminate the resistant phenotype and sensitize the neoplastic MCF-7 cells to doxorubicin treatment. Finally, the reproducibility of the in vitro experiment and efficacy of treatments within an animal model was evaluated. 2-Methoxyestradiol is a naturally occurring metabolite originating from 17β-estradiol. It has recently been exploited as an anticancer agent due to its anti-proliferative and anti-angiogenic properties. Among its various mechanisms of action, this compound has been shown to inhibit the expression of HIF-1α. It is for this reason that this study employed 2-methoxyestradiol in the adjuvant therapeutic treatment, along with doxorubicin. Methods: The in vitro experimental model employed the use of the breast adenocarcinoma estrogen receptor (ER-positive cell line, MCF-7. These neoplastic cells were propagated under standard culture conditions until reaching ~70-80% confluency, after which treatment commenced. The treatment regime comprised a 12 hour exposure to the doxorubicin (1 μM) chemotherapeutic agent, either alone or in combination with HIF-1α inhibitors, 2-methoxyestradiol (10 μM) or siRNA duplex (400 nM), with parallel incubations under normoxic (21%) and hypoxic (~0.1%) conditions. To serve as a positive control for HIF-1α expression, cells were treated with CoCl2 (100 μM). Molecular techniques employed included the Caspase-Glo® 3/7 Assay, western blotting, and the bioreductive MTT Assay. Mitochondrial integrity was assessed by live cell imaging/fluorescent microscopy. Cellular viability was monitored at all times. The experiment was then translated into a pre-clinical in vivo model where C57BL/6 mice bearing E0771 xenografts (4 week growth) were allocated into the following treatment groups: (1) control (2) doxorubicin (5 mg.kg-1), (3) 2-methoxyestradiol (45 mg.kg-1), and (4) the combination of the two previously mentioned groups. Body weight and the rate of tumour growth were monitored throughout the experiment. Results: Treatment with CoCl2 effectively stabilized HIF-1α under normoxic conditions. 2-Methoxyestradiol was capable of attenuating HIF-1α expression under both normoxia and hypoxia as compared with siRNA transfection, which was only effective under normoxia. HIF-1α stabilization was accompanied by an increase in autophagy along with the morphological transformation of mitochondria from an elongated network to shorter disc-like forms. On the other hand, HIF-1α attenuation caused an induction in the expression of the apoptotic markers, cleaved caspase 3 and cleaved PARP, as well as the restoration of the normoxic morphology. The exposure of MCF-7 cells to 1 μM doxorubicin for 12 hours produced a differential effect in the bioreductive MTT assay between normoxic and hypoxic conditions (42.97 ± 3.095% vs. normoxic dox, p<0.01), while stimulating the apoptotic and autophagic pathways. Compared to the control, a significant expression of phospho-AMPK became evident at 21% O2, while the levels remained stable at ~0.1% O2 after doxorubicin exposure. Furthermore, chemotherapeutic treatment caused the morphology of the mitochondria to appear dot-like. Although the combination of the two drugs removed the differential effect witnessed in the MTT assay, there was no significant change when compared to doxorubicin. Levels of apoptotic cell death decreased under both oxygen conditions. While HIF-1α and autophagy decreased under normoxia, they remained elevated under hypoxia. In the in vivo component of the study, the administration of doxorubicin and 2-methoxyestradiol, alone or in combination, did not affect the rate of tumour growth or induce systematic toxicity in any of the experimental mice. When drugs were administered separately, a decrease in apoptosis along with a concomitant increase in autophagy and p-AMPK expression became noticeable while neither treatment had any significant effect on the expression of HIF-1α. Adjuvant administration, however, was capable of attenuating HIF-1α along with autophagy. Discussion: By inducing (CoCl2) and inhibiting (2-methoxyestradiol; siRNA duplex) HIF-1α, it was established that the autophagic pathway in the in vitro experimental setting of this study was dependent on the expression of HIF-1α. The bioreductive MTT assay measures the metabolic state of a cell, which is an indirect indication of cellular viability. Based on this, hypoxia was shown to confer survival to neoplastic MCF-7 cells based on the differential effect witnessed after doxorubicin treatment. Apart from the induction of apoptosis and its associated mitochondrial fragmentation, the chemotherapeutic drug increased the activation of the metabolic sensor, AMPK, which upregulated autophagy during normoxia. While this autophagic process may assist in the killing mechanism, we speculate that the autophagy upregulated under hypoxia may be responsible for the survival effect and is most likely dependent on HIF-1α. In contrast to eliciting a synergistic cytotoxic effect, the combination of doxorubicin with 2-methoxyestradiol produced an antagonistic effect on cellular viability instead. We propose that under normoxia, the combined treatment may stimulate the MCF-7 neoplastic cells to enter a state of growth arrest, or senescence, since the results indicate that the decrease in HIF-1α-dependent autophagy did not significantly affect cellular viability. Under hypoxia, despite the incorporation of the pharmacological HIF-1α inhibitor (2-methoxyestradiol), the expression levels of HIF-1α remained unaffected. We speculate that this could be the result of a potentiated stabilization of HIF-1α caused by the build-up of ROS and TCA intermediates which may be the outcome of mitochondrial dysfunction inflicted upon adjuvant therapy under hypoxia. Furthermore, it is also likely that the slight mitogenic effect observed within the MTT assay may be caused by the conversion of 2-methoxyestradiol to a chemically-reactive estrogen derivative, possibly by the action of doxorubicin, and the fact that an ER-positive cancer cell line was employed in this study. With regards to the in vivo experimental model, we speculated that the failure of the molecular changes to manipulate the growth of the tumour could have been the result of an ineffective time- and/or dose regime. Conclusion: We therefore reject our hypothesis based on the fact that an antagonistic rather than synergistic effect was witnessed when the tumorigenic MCF-7 cell line was treated with adjuvant therapy. The results warrant the need for extensive testing on the pharmacodynamics of 2-methoxyestradiol, and more informative techniques to compliment the study. / AFRIKAANSE OPSOMMING: Inleiding: Borskanker is die mees algemeen gediagnoseerde kanker asook die hoof oorsaak van kanker-verwante sterftes in vrouens wêreldwyd. Konvensionele behandeling behels die toediening van doxorubicin, ‘n anti-kankermiddel wat aan die antrasiklien-familie van chemoterapeutiese middels behoort. Kankerselle begin egter toenemend weerstandbiedend raak teen doxorubicin behandeling. Daar is al bewys dat die voorkoms van hipoksiese sones, wat ‘n algemene eienskap van soliede tumore is, die seleksie vir weerstandbiedende klone van prolifererende kankerselle, veroorsaak. Neoplastiese selle kan hierdie hipoksiese toestande weerstaan en in hierdie ongunstige mikro-omgewing floreer deur sellulêre homeostase te modifiseer. Hierdie aanpassing staan bekend as die ‘hipoksiese respons’ en word bemiddel deur die aksies van die transkripsiefaktor reguleerder, HIF-1. Die verhoogde uitdrukking van HIF-1 in kankerweefsel word oor die algemeen geassosieer met ‘n swak prognose omdat dit die maligniteit vehoog. Hipotese en Doelwitte: Die hipotese van hierdie studie behels dus die volgende: Deur HIF-1α te inhibeer, sal die ‘beskermende’ hipoksiese respons wat in borskankerselle voorkom omseil kan word en sodoende die sitotoksisiteit van doxorubicin terapie verhoog. Die eerste doelwit van hierdie studie was dus om die hipoksiese kondisies te identifiseer waar MCF-7 selle ‘n doxorubicin-weerstandbiedende fenotipe vertoon. Daarna is die molekulêre paaie wat bydrae tot hierdie hipoksiese weerstand ondersoek asook hul moontlike verwantskap met die hipoksiese reguleerder, HIF-1α. Nadat die rol van HIF-1α bevestig is, was die volgende doelwit om te bepaal of die inhibisie van HIF-1α die weerstandbiedende fenotipe sal onderdruk en neoplastiese MCF-7 selle sal sensitiseer vir doxorubicin behandeling. Laastens is die herhaalbaarheid en effektiwiteit van behandeling in die in vitro eksperimente ook in ‘n diermodel getoets. 2-Methoxyestradiol is ‘n metaboliet van 17β-estradiol wat natuurlik in die liggaam voorkom. Dit is ook onlangs as ‘n anti-kanker middel geïdentifiseer as gevolg van die anti-verdelende en anti-angiogeniese eienskappe. Een van die eienskappe van 2-methoxyestradiol is dat dit ook die uitdrukking van HIF-1α kan onderdruk. Dit is dan ook vir hierdie rede dat 2-methoxyestradiol in hierdie studie as bykomende terapie saam met doxorubicin gebruik is. Metodes: Die in vitro eksperimentele model behels die gebruik van ‘n borsadenokarsinoom, estrogeenreseptor (ER)- positiewe sellyn, MCF-7. Hierdie neoplastiese selle is onder standaard weefselkultuur omstandighede gekweek totdat konfluensie van ~70-80% bereik is, waarna behandeling begin het. Die behandelingsprosedure behels ‘n 12 uur blootstelling aan doxorubicin (1 µM) chemoterapeutiese middel alleen of in kombinasie met die HIF-1α inhibitore, 2-methoxyestradiol (10 µM) of siRNA duplex (400 nM) in normoksiese (21% O2) en hipoksiese (~0.1% O2) toestande. Die selle is ook met CoCl2 behandel wat gedien het as ‘n positiewe kontrole vir HIF-1α uitdrukking. Molekulêre tegnieke wat tydens hierdie studie gebruik is, sluit die “Caspase-Glo® 3/7” bepaling in, asook die westelike kladtegniek en die MTT bepaling. Mitochondriale integriteit is bepaal deur middel van lewende sel afbeeldings/fluoresensie mikroskopie. Sellewensvatbaarheid is ten alle tye gemonitor. Hierdie eksperment is verder ook in ‘n pre-kliniese in vivo model uitgevoer waar C57BL/6 muise met E0771 xenografte (4 weke groei) geïnduseer is en in die volgende behandelingsgroepe verdeel is: (1) kontrole; (2) doxorubicin (5 mg.kg-1); (3) 2-methoxyestradiol (45 mg.kg-1); en (4) die kombinasie van laasgenoemde twee groepe. Die liggaamsgewig en die tempo van tumorgroei is tydens die hele eksperiment gemonitor. Resultate: CoCl2 behandeling het HIF-1α effektief gestabiliseer tydens normoksiese omstandighede. 2-Methoxyestradiol het HIF-1α uitdrukking tydens normoksiese en hipoksiese toestande onderdruk wanneer dit vergelyk is met siRNA transfeksie wat slegs tydens normoksiese toestande effektief was. HIF-1α stabilisering het gepaardgegaan met ‘n toename in autofagie asook morfologiese veranderinge in die mitochondria vanaf ‘n verlengde netwerk tot korter skyfagtige vorme. Aan die ander kant het HIF-1α onderdrukking ‘n toename in die apoptotiese merkers, nl kliewing in caspase-3 and PARP veroorsaak wat gepaard gegaan het met die herstel van die tubulêre mitochondriale netwerk. Die blootstelling van die MCF-7 selle aan 1 µM doxorubicin vir 12 ure het ‘n differensiële effek in die bioreduktiewe MTT bepaling tot gevolg gehad tussen normoksiese en hipoksiese toestande (42.97 ± 3.095%, p<0.1), terwyl die apoptotiese- en autofagiese paaie in beide toestande gestimuleer is. ‘n Insiggewende toename in fosfo-AMPK uitdrukking was sigbaar tydens normoksiese toestande van 21% O2, terwyl dit onveranderd gebly het tydens hipoksiese toestande van 0.1% ~O2 na doxorubicin behandeling. Die morfologie van die mitochondria het ‘n ‘kollerige’ voorkoms tydens doxorubicin behandeling gehad. Alhoewel die behandeling van die selle met beide middels gelyktydig, die differensiële effek soos weerspieël in die MTT bepaling ophef, is daar geen insiggewende verandering wanneer met doxorubicin behandeling vergelyk word nie. Apoptotiese seldood verminder met gelyktydige behandeling van biede middels tydens normoksiese en hipoksiese toestande. HIF1-α en autofagie het afgeneem tydens normoksiese toestande, maar bly vehoog tydens hipoksie. In die in vivo model, het die toediening van doxorubicin en 2-methoxyestradiol alleen en in kombinasie nie tumorgroei geaffekteer nie en ook nie sistemiese toksisiteit in enige van die eksperimentele muise tot gevolg gehad nie. Die afsonderlike toediening van die middels het ‘n afname in apoptose in ‘n toename in autofagie en p-AMPK uitdrukking tot gevolg gehad, terwyl afsonderlike toediening van die middels nie ‘n effek op HIF-1α uitdrukking gehad het nie. Die gelyktydige toediening van biede middels het egter ‘n onderdrukking van HIF1-α teweeggebring. Bespreking: Deur HIF-1α te induseer (CoCl2) en te inhibeer (2-methoxyestradiol en siRNA) in hierdie in vitro eksperimentele omstandighede, bevestig hierdie resultate dat autofagie afhanklik is van die uitdrukking van HIF-1α. Die bioreduktiewe MTT bepaling meet die metaboliese staat van die sel wat indirek sellewensvatbaarheid bepaal. Gebasseer op hierdie bepaling is bewys dat hipoksie ‘n weerstandbiedende fenotipe veroorsaak teen doxorubicin behandeling in neoplastiese MCF-7 selle. Doxorubicin veroorsaak ‘n toename in apoptose met geassosieerde mitochondriale fragmentering asook ‘n aktivering van die metaboliese sensor, AMPK, wat autofagie stimuleer in normoksiese omstandighede. Alhoewel ‘n toename in autofagie seldood kan stimuleer, spekuleer ons dat ‘n toename in autofagie tydens hipoksie verantwoordelik kan wees vir seloorlewing wat heel moontlik ook afhanklik van HIF-1α is. In kontras met die verwagting dat die kombinasie behandeling ‘n sinergistiese sitotoksiese effek sou teweegbring, dui ons resultate dat daar ‘n antagonistiese effek op sellewensvatbaarheid was. Ons stel voor dat die gekombineerde behandeling tydens normoksiese toestande MCF-7 neoplastiese selle stimuleer om in ‘n toestand van groeistaking in te gaan aangesien die resultate daarop dui dat ‘n afname in HIF-1α afhanklike autofagie nie sellulêre lewensvatbaarheid beïnvloed het nie. Tydens hipoksie, ten spyte van die bykomdende behandeling met die HIF-1α inhibitor (2-methoxyestradiol), het die vlakke van HIF-1α onveranderd gebly. Ons spekuleer dat dat dit die gevolg kan wees van die stabilisering van HIF-1α as gevolg van ‘n toename in ROS en TCA intermediate wat die gevolg van mitochondriale wanfunksie kan wees tydens bykomende terapie onder hipoksiese toestande. Dit is ook moontlik dat die mitogeniese effek wat waargeneem is met die MTT bepaling die gevolg kan wees van die omsetting van 2-methoxyestradiol na ‘n chemiese-reaktiewe estrogeen derivaat; moontlik as gevolg van die aksie van doxorubicin en die feit dat die sellyn wat in hierdie studie gebruik is, ‘n ER-positiewe kankersellyn is. Met verwysing na die in vivo eksperimentele model, spekuleer ons dat die molekulêre veranderinge wat nie in die tumorgroei weerspieël word nie, die resultaat van oneffektiewe tyds- en dosis behandelingswyses is, of foutiewe toediening van die middel kan wees. Gevolgtrekking: Ons verwerp dus ons hipotese gebaseer op die feit dat bykomende (adjuvante) behandeling eerder ‘n antogonistiese effek as ‘n sinergistiese effek op seldood in MCF-7 selle het. Hierdie resultate regverdig die nodigheid van intensiewe toetsing op die farmakodinamika van 2-methoxyestradiol asook die gebruik van meer informatiewe tegnieke om hierdie studie te komplimenteer. / CANSA and Marie Stander

Οι όγκοι εγκεφάλου αποτελούν μια ομάδα όγκων με ιδιαίτερα χαρακτηριστικά. Η ογκογένεση έχει συσχετισθεί σε αρκετούς όγκους, ανάμεσά τους και ορισμένοι όγκοι εγκεφάλου, με την πρωτείνη θερμικού σοκ 90 (Hsp90). Σε πρόσφατες μελέτες, έχει αποδειχθεί η η συμμετοχή της Hsp90 στην αποδόμηση της ογκοκατασταλτικής πρωτείνης pVHL, η οποία είναι απαραίτητητη για την αποδόμηση του μεταγραφικού παράγοντα που ενεργοποιείται στην υποξία (HIF-1α). Επιπλέον, έχει βρεθεί πως οι ανταγωνιστές της Hsp90 μειώνουν τα επίπεδα έκφρασης του VEGFR-3. Στόχος της παρούσας μελέτης ήταν η εκτίμηση των επιπέδων έκφρασης των μορίων Hsp90, pVHL, HIF-1α και VEGFR-3 στους όγκους εγκεφάλου και οι πιθανές συσχετίσεις μεταξύ τους. Εξετάσθηκαν συνολικά 89 óγκοι εγκεφάλου με την ανοσοϊστοχημική μέθοδο. Στους αστροκυτταρικούς όγκους, η Ηsp90 φαίνεται πως οδηγεί στη σταθεροποίηση του HIF-1α μέσω της σύνδεσής της με τη pVHL. Στα μυελοβλαστώματα από την άλλη, ο HIF-1α ρυθμίζεται από τη Hsp90 με τρόπο ανεξάρτητο από τη pVHL. Βρέθηκε συσχέτιση της έκφρασης του VEGFR-3 με τη Hsp90 αλλά και τον HIF-1α σε ομάδα όγκων του εγκεφάλου, η βιολογική συμπεριφορά των οποίων θα πρέπει να διερευνηθεί. / Brain tumors constitute a special group of tumors. In many tumors, including brain tumors, tumorigenesis has been associated with heat shock protein 90 (Hsp90). ). Recent studies have demonstrated that Hsp90 is essential for the degradation of tumor suppressor protein, pVHL, which is essential for the degradation of Hypoxia Induced Factor 1α (HIF-1α). In addition,it has been proved that Hsp90 antagonists, reduce the expression VEGFR-3. the aim of the current study was to estimate the levels of expression of Hsp90, pVHL, HIF-1α and VEGFR-3 in brain tumors and the possible correlations among them.89 human brain tumors were studied immunohistochemically. In astrocytic tumors, Hsp90 seems to stabilize HIF-1α, through binding to pVHL. On the other hand, in medulloblastomas, HIF-1α seems to be regulated by Hsp90, in a way that seems to be independent from pVHL. We found a correlation between the expression of VEGFR-3 and Hsp90 and HIF-1α in a group of brain tumors, the biological behavior of which must be studied.

Despite dramatic morphological differences between animals from different taxa, several important features in organization and sensory system processing are similar across animals. Because of this similarity, a number of different organisms including mammals, insects, and decapod crustaceans serve as valuable model systems for understanding general principles of environmental effects. This research examines intrinsic and extrinsic factors by behaviorally and physiologically means to identify the impact of environmental conditions on two distinct crayfish species- Procambarus clarkii (surface) and Orconectes australis packardi (cave). The research identified behavioral and physiological responses in these two morphological and genetically distinct species. The studies also examined multiple levels of complexity including social behavior, an autonomic response, chemosensory capabilities and neuronal communication, identified comparative similarities/differences, addressed learning and environmental influences on learning and examined behavioral and cellular responses to high levels of carbon dioxide. I found environmental factors directly influence crayfish behavior of social interactions. Interactions were more aggressive, more intense and more likely to end with a physical confrontation when they took place 'in water' than 'out of water'. The modified social interaction resulted in a altered fighting strategy. A study on motor task learning was undertaken which showed similar learning trends among these crayfish species despite their reliance on different sensory modalities. I also demonstrated learning was dependent on perceived stress by the organism. Previously trained crayfish inhibited from completing a task showed significant increase in an autonomic stress response. Studies on the behavioral and physiological responses to CO2 revealed that high [CO2] is a repellent in a concentration dependent manner. The autonomic responses in heart rate and an escape tailflip reflex shows complete cessation with high [CO2]. A mechanistic effect of CO2 is by blocking glutamate receptors at the neuromuscular junction and through inhibition of the motor nerve within the CNS.

The analysis of hemodynamic parameters and functional reactivity of cerebral capillaries is still controversial. The detailed mapping of tissue oxygen levels on the scale of micrometers cannot be obtained by means of an experimental approach, necessitating the use of theoretical methods in this investigating field. To assess the hemodynamics and oxygen transport in the cortical capillary network, 2D and 3D generic models are constructed (non-tree like) using random voronoi tessellation in which each edge represents a capillary segment. The modelling presented here is based on morphometric parameters extracted from physiological data of the cortex in which the spatial distribution of the diameter of the capillary is based on a Modified Murray method. This method led to a proper link between the diameter topology and flow pattern such that the maximum efficiency for flowing blood is concluded in the model of cortical capillary network. The approach is capable of creating an appropriate generic, realistic model of a cerebral capillary network relating to each part of the brain cortex because its geometrical density is able to vary the capillary density. The pertinent hemodynamic parameters are obtained by numerical simulation based on effective blood viscosity as a function of hematocrit and microvessel diameter, ESL (endothelial surface layer) effect, phase separation and plasma skimming effects. Using a solution method of the Green's function, the model is numerically developed to provide different simulations of oxygen transport for varying perfusion and metabolism in a mesoscale model of the cortical capillary network, bridging smaller and larger scale phenomena. The analysis of hemodynamic parameters (blood flow rate, velocity and hematocrit) demonstrates a consistency with the experimental observation. The distribution pattern of wall shear stress (WSS) in the network model supports the physiological data which in turn represents a proper matching between the hemodynamics and morphometrics in the cerebral capillary network. The distributions of blood flow throughout the 2D and 3D models seem to confirm the hypothesis in which all capillaries in a cortical network are recruited at rest (normal condition). The predictions showed a heterogeneous distribution in the flow pathways (aspect of length and inflow) and the pertinent transit time of red blood cell (RBC) in the network model which is dependent on varying perfusion rates. The analyses of oxygen transport in the model has demonstrated that oxygen levels in the tissue are sensitively dependent on the microvascular architecture and flow distribution. Unlike the homogeneous compartmental models, the mesoscale model presented in this study led to a prediction of tissue PO2 gradients throughout the tissue and a spatial distribution of tissue PO2 on the micron-scale for varying perfusion and metabolism. The predicted nonlinear changes in the oxygen extraction fraction (OEF) of the model as a function of the perfusion rate provide a basis for the quantitative interpretation of functional magnetic resonance imaging (fMRI) studies in terms of changes in local perfusion. The model is capable of predicting the brain oxygen metabolism under both normal and disease states, particularly, local hypoxia and local ischemia caused by misery perfusion syndrome. The hypoxic states for different perfusion rates and oxygen consumption rates demonstrated that in a significant decrease in brain perfusion (as can occur in stroke), the tissue hypoxia can be avoided by a moderate reduction in oxygen consumption rate. Increasing oxygen consumption rates (a realization of spatiotemporal stimulation of neural tissue) with respect to maintaining the tissue PO2 in the model led to a predicted flow-metabolism coupling in the model which supports the experimental studies of somatosensory and visual stimulation in humans by positron emission tomography (PET) and functional MRI (magnetic resonance imaging). A disproportionately large increase in blood supply is required for a small increase in the metabolic utilization (oxygen consumption rate) which in turn, is strongly dependent on the resting OEF such that the magnitude of the blood flow increases in the higher resting OEF.

Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases. Maintenance of vascular reactivity and endothelial viability is essential to preserving oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. These results suggest that hypoxic induction of MMP-2 activity potentiates contraction in systemic conduit and resistance arteries through proteolytic activation of big ET-1. The second part of the thesis investigated oxygen regulation of HO-2 protein and whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but not HO-1, protein level was maintained during hypoxia in human endothelial cells through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression increased the production of reactive oxygen species, decreased mitochondrial membrane potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but not during normoxia. These data indicate that HO-2 is translationally regulated and important in maintaining endothelial viability and function during hypoxia. In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in preserving vascular function during prolonged systemic hypoxia. These enzymatic pathways may, therefore, represent novel therapeutic targets that may be exploited to ameliorate the effects of hypoxia in patients with cardiopulmonary disease.

In this thesis I present a microfluidic platform developed to control dissolved gases and monitor dissolved oxygen concentrations within the microenvironment of isolated small blood vessels. Dissolved gas concentrations are controlled via permeation through the device substrate material using a 3D network of gas and liquid channels. Dissolved oxygen concentrations are measured on-chip via fluorescence quenching of an oxygen sensitive probe embedded in the device. Dissolved oxygen control was validated using the on-chip sensors as well as a 3D computational model. The platform was used in a series of preliminary experiments using olfactory resistance arteries from the mouse cerebral vascular bed. The presented platform provides the unique opportunity to control dissolved oxygen concentrations at high temporal resolutions (<1 min) and monitor dissolved oxygen concentrations in the microenvironment surrounding isolated blood vessels.

<p>The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited. </p><p>To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response. </p><p>Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.</p> / Dissertation

The hyperbolic relationship between power output and endurance time can be measured using all-out exercise. The aims of this thesis were to (i) assess whether the all-out test could be used under novel testing protocols to provide valid power-duration (P-D) parameter estimates; and (ii) attempt to elucidate the likely physiological composition of the P-D curvature constant. All-out tests were initiated from moderate-(M), heavy-(H) and severe-(S2 & S4) intensity ‘baselines’ (chapter 4). The work performed above end power (WEP) was not different to control under M or H conditions but was significantly, predictably reduced under the S2 & S4 conditions (control: 16.3 ± 2.2; M: 17.2 ± 2.4; H: 15.6 ± 2.3 kJ, P > 0.05; S2: 11.5 ± 2.5; S4: 8.9 ± 2.2 kJ, P < 0.05). The 3-min all-out test end power (EP) parameter was unaffected. Muscle glycogen may form part of the WEP. Type I (T1) and type II (T2) muscle fibres were depleted of their glycogen content prior to the all-out test (chapter 5). EP and WEP were unaffected by either T1 or T2 glycogen depletion. The all-out tests was conducted under hypoxic conditions alongside the criterion assessment of the P-D relationship (chapter 6). Normobaric moderate hypoxia caused a reduction in CP (control: 175 ± 25; hypoxia: 132 ± 17 W, P < 0.001) without affecting W′ (control: 13.2 ± 2.2; hypoxia: 12.3 ± 2.7 kJ, P > 0.05). The 3-min all-out test provided EP and WEP estimates, which did not differ to CP and W′ (control: EP 172 ± 30 W, WEP 12.0 ± 2.6 kJ; hypoxia EP 134 ± 23 W, WEP 12.5 ± 1.4 kJ, P > 0.05) providing the ergometer resistance was adjusted for the hypoxic conditions. Furthermore, a significant negative relationship was observed between %∆ ( O2peak – CP) and %∆W′ (r = -0.83, P < 0.001); thus, W′ may represent the relative ‘size’ of the severe-intensity domain. The all-out test was used to track training-induced changes in P-D parameters in response to 6-weeks of sprint or endurance training (chapter 7). EP & WEP were differently altered compared to CP and W′ following sprint training (CP 12 ± 9; EP -0 ± 9 % change; W′ -5 ± 25; WEP 11 ± 15 % change). The all-out test reliably tracked changes in CP and W′ following endurance training. In conclusion, the all-out test provides reliable EP and WEP values. Its validity is acceptable, but is perhaps affected by exercise training that is specific to the execution of the test. The W′ appears to be determined, to a large extent, by the relative size of the severe-intensity domain.

Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O-2, 60 min), hypoxia (Hx, 6% O-2, 60 min) or H/R (6% O-2, 60 min followed by 21% O-2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean +/- SD and analyzed via ANOVA followed by the post hoc Bonferroni t test. Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.