COPD is characterized by tissue inflammation and impaired remodeling that suggests fibroblast maintenance of structural homeostasis is dysregulated. Thus, we performed in vitro wound healing experiments on normal and diseased human lung fibroblasts and developed an ABM of fibroblasts closing a scratched monolayer using NetLogo to evaluate differences due to COPD or cigarette smoke condensate exposure. This ABM consists of a rule-set governing the healing response, accounting for cell migration, proliferation, death, activation and senescence rates; along with the effects of heterogeneous activation, phenotypic changes, serum deprivation and exposure to cigarette smoke condensate or bFGF. Simulations were performed to calibrate parameter-sets for each cell type using in vitro data of scratch-induced migration, viability, senescence-associated beta-galactosidase and alpha-smooth muscle actin expression. Parameter sensitivities around each calibrated parameter-set were analyzed. This model represents the prototype of a tool designed to explore fibroblast functions in the pathogenesis of COPD and evaluate potential therapies.

En raison d’une abondante matrice extracellulaire chondrogénique, peu vascularisée et hypoxique, le chondrosarcome, cancer du cartilage, est une tumeur chimio et radiorésistante. Une stratégie bispécifique s’appuyant sur deux caractéristiques spécifiques du chondrosarcome, une forte densité de charges négatives, ainsi qu’une hypoxie chronique sévère de la matrice extracellulaire, est actuellement développée au laboratoire et consiste à fonctionnaliser une prodrogue activée en hypoxie avec un ammonium quaternaire (AQ). Sur la base de résultats précliniques, une prodrogue, ICF05016, a été identifiée et fait actuellement l’objet d’une étude d’optimisation.Ce projet vise à identifier des relations structure-activité dans une série de moutardes phosphorodiamidates vectorisées par une fonction AQ. Dans une première partie, nous nous sommes intéressés à faire varier la nature des substituants de la fonction ammonium quaternaire ainsi que la longueur du bras espaceur séparant cette dernière de la moutarde. Ainsi, 27 dérivés ont été synthétisés par une séquence commune multi-étapes impliquant la phosphorylation d’un dérivé 2-nitro-5-hydroxyméthylimidazole.Un screening par résonance plasmonique de surface a permis de mettre en évidence 12 composés plus affins envers l’aggrécane que le composé de référence ICF05016. Cette étude a révélé une dépendance importante entre l’affinité à l’aggrécane et la nature de l’ammonium quaternaire, les composés benzyléspossédant tous des affinités supérieures. In vitrosur cultures cellulaires HEMC-SS, les composés ont affichés des ratios de cytotoxicité normoxie vs hypoxie (HCR)supérieurs à celui du lead ICF05016, et notamment le composé benzylé31f.Avec une constante de dissociation à l’aggrécane de 2,10 µM et un différentiel de cytotoxicité normoxie/hypoxie équivalent à celui de l’évosfosfamide en étude clinique, la composé 31f a été évalué en termes de stabilité et de clivage en conditions réductrices, mimant l’hypoxie tumorale. Démontrant une excellente stabilité sur 24 heures dans un tampon phosphate, le composé 31f s’est révélé être réduit par réduction chimique ou enzymatique via une nitroréductase, et ce avec une cinétique rapide. Ce dernier a été ainsi identifié au terme de l’évaluation in vitro et de cet axe de recherche comme un nouveau « lead ».Dans une seconde partie, nous avons choisi de moduler l’halogène et la position des bras alkylants portés par la moutarde phosphorodiamidate. Compte tenu de l’instabilité des composés de la série bromée, par rapport à la série chlorée, les analogues bromés des composés ICF05016 et 31f n’ont pu faire l’objet d’une évaluation in vitro. Pour les isomères de type moutarde ifosfamide, l’instabilité des précurseurs phosphoramidiques et les difficultés de synthèse inhérentes à l’introduction d’une fonction amine secondaire sur un chlorure de phosphoryle préalablement fonctionnalisé, nous ont contraints à réorienter nos travaux vers des structures issues d’une étape de chimie de click, permettant d’introduire le bras vecteur portant la fonction amine tertiaire indépendamment de l’étape de phosphorylation. Cette séquence réactionnelle pourrait être étendue à la synthèse de nouveaux analogues du composé 31f, identifié lors de ces travaux. / Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-resistant. A dual targeted therapy leveraging specific chondrosarcoma hallmarks, an extensive cartilaginous extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia,was investigatedby conjugation of a hypoxia-activated prodrug (HAP) to quaternary ammonium (QA) functions. Based on preclinical results, animidazole prodrug, ICF05016, was identified and provided the basis for a lead optimization study.This project was designed to identify structure-activity relationships in a series of QA-targeted phosphorodiamidate mustard conjugates. In a first part, a series of 27 conjugates, with different QA function and length of the alkyl linker, was synthesizedvia a common multi-step sequence involvingphosphorylation of a key 2-nitroimidazole alcohol intermediate. Then, biomolecular interactions between these QA derivatives and aggrecan were assessed using thesurface plasmon resonance technology. This screeningrevealed that the affinity depends more on the natureof the QA function, rather than on the linker length. The most promising results were obtainedwith QA bearing a benzyle group. Twelves compounds were then evaluated in terms of hypoxia selective cytotoxicityonthe HEMC-SS cell line. For all prodrugs, an overall improvement in hypoxic selectivity compared with the ICF05016 was obtained and the positive impact of thebenzyle QA function was again highlighted. With a dissociation constant of 2.10 µM in the SPR experiment and an attractive hypoxic selectivity, compound 31fwas further selected for a stability and reductive activation study. Activation of the prodrug and phosphoramide mustard release under reductive chemical conditions, and nitroreductase-based activation were demonstrated. From this study, compound 31f emerged as the most effective PG-targeted HAPs. In a second part, the nature of the halogen and the position of the alkylating arms carried by the phosphorodiamidic mustard were modified. Given the high instability of the final compounds of the brominated series, compared to the chlorinated series, brominated analogs of the compounds ICF05016 and 31fcould not be obtained with sufficient purity to be evaluated in vitro.For ifosfamide-like isomers, the design of the prodrugs was modified due to the instability of the phosphoramidic precursors or difficulties of introducing a secondary amine function on a phosphoryl chloride previously functionalized.A click chemistry approach was developed to tether the spacer arm carrying the tertiary amine function independently of the phosphorylation step and could be extended to the synthesis of triazoleanalogues of the new lead 31f.

Obstructive sleep apnea is a potentially life-threatening condition characterized by repetitive episodes of upper airway obstruction that occur during sleep, usually associated with a reduction in blood oxygen saturation. In US population, 9% of women, 24% of men, and 2% of children have been diagnosed with obstructive sleep apnea, suggesting that 18 million people may suffer from the consequences of nightly episodes of apnea. One of the most significant symptoms of obstructive sleep apnea is profound and repeated hypoxia. The analysis of the interaction between cardiovascular and respiratory signals has been a widely-explored area of research due to the significance of the results in describing a functional relationship between the underlying physiologic systems; however, statistical and analytical approaches to analyze the changes in these signals before and after hypoxia are still in their early stages of evolution. A major motivation for this research has been the lack of methodologies to detect mean and/or variance shifts and identify root sources of variation in time-frequency characteristics of multichannel data. The contributions of this thesis are twofold. First, multiscale energy distributions based on wavelet transformations of the analyzed physiological signs are analyzed. This is followed by the development of an online multichannel monitoring approach based on principal curves that detects changes in the wavelet coefficients extracted from the analyzed signals.

Sudden Infant Death Syndrome (SIDS) is the third leading cause of infant mortality between birth and the first year of life in the United States. Along with the identification of various maternal risk factors, the role of fetal hypoxia has been hypothesized to be one of many causal factors associated with SIDS. The purpose of this study was to develop a profile of the SIDS infant and assess whether six pregnancy complications consistent with fetal hypoxia were associated with the increased outcome of SIDS. The secondary data analysis of Florida linked birth to death certificate data specific to Hillsborough County and Duval County were analyzed retrospectively for the period of time between 1998 and 2000. Of the 86, 342 births, 69 SIDS cases were identified, 34 in Hillsborough County and 35 in Duval County. A majority of the infants were White males with an average age of death of 80 days. The Chi-Square test for Independence with Cramer's V, odds ratios and 95% confidence intervals were calculated to determine if an association existed between pregnancy complications, specific maternal risk factors and SIDS. Eclampsia was the only statistically significant prenatal complication found in this cohort (OR=4.67: 95% CI 1.49, 14.57). Maternal tobacco use (OR= 3.13: 95% CI 1.83, 5.36) and late initiation into prenatal care were also found to be significant in the SIDS cases, with the greatest risk occuring in women who did not receive prenatal care (OR=4.37: 95% CI 1.38, 13.89). These findings will assist with the development of a profile of infants who are at greater risk of dying of SIDS in Hillsborough County and Duval County as well as contribute to what is currently known about the association between fetal hypoxia and SIDS.

Cephalopods are an important component of many marine ecosystems and support large fisheries. Their active lifestyles and complex behaviors are thought to be driven in large part by competition with fishes. Although cephalopods appear to compete successfully with fishes, a number of their important physiological traits are arguably inferior, such as an inefficient mode of locomotion via jet propulsion and a phylogenetically limited means of blood-borne gas transport. In active shallow-water cephalopods, these traits result in an interesting combination of very high oxygen demand and limited oxygen supply. The ability to maintain active lifestyles despite these metabolic constraints makes cephalopods a fascinating subject for metabolic physiology. This dissertation focuses on the physiological adaptations that allow coleoid cephalopods to maintain a balance of oxygen supply and demand in a variety of environmental conditions. A critical component of understanding oxygen supply in any animal is knowing the means of oxygen delivery from the environment to the mitochondria. Squids are thought to obtain a fairly large portion of their oxygen via simple diffusion across the skin in addition to uptake at the gills. Although this hypothesis has support from indirect evidence and is widely accepted, no empirical examinations have been conducted to assess the validity of this hypothesis. In Chapter 2, I examined cutaneous respiration in two squid species, Doryteuthis pealeii and Lolliguncula brevis, by using a divided chamber to physically separate the mantle cavity and gills from the outer mantle surface. I measured the oxygen consumption rate in the two compartments and found that, at rest, squids only obtain enough oxygen cutaneously to meet demand of the skin tissue locally (12% of total). The majority of oxygen is obtained via the traditional branchial pathway. In light of these findings, I re-examine and discuss the indirect evidence that has supported the cutaneous respiration hypothesis. Ocean acidification is believed to limit the performance of squids due to their exceptional oxygen demand and pH-sensitivity of blood-oxygen binding, which may reduce oxygen supply in acidified waters. The critical oxygen partial pressure (Pcrit), defined as the PO2 below which oxygen supply cannot match basal demand, is a commonly reported index of hypoxia tolerance. Any CO2-induced reduction in oxygen supply should be apparent as an increase in Pcrit. In Chapter 3, I assessed the effects of CO2 (40 to 140 Pa) on the metabolic rate and Pcrit of two squid species: Dosidicus gigas and Doryteuthis pealeii. Carbon dioxide had no effect on metabolic rate or hypoxia tolerance in either species. Furthermore, considering oxygen transport parameters (e.g. Bohr coefficient, blood P50) and blood PCO2 values from the literature, I estimated an increase in seawater PCO2 to 100 Pa (≈1000 μatm/ppmv) would result in a maximum drop in hemocyanin-O2 saturation by 6% at normoxia and a Pcrit increase of ≈1 kPa (≈5% air saturation) in the absence of active extracellular pH compensation. Such changes are unlikely given the capacity for acid-base regulation in many cephalopods. Moreover, this estimated change is within the 95% confidence intervals of the Pcrit measurements reported here. Squid blood-O2 binding is more sensitive to pH than most other marine animals measured to date. Therefore, the lack of effect in squids suggests that ocean acidification is unlikely to have a limiting effect on blood-O2 supply in most marine animals. The pelagic octopod, Japetella diaphana, is known to inhabit meso- and bathypelagic depths worldwide. Across its range, individuals encounter oxygen levels ranging from nearly air-saturated to nearly anoxic. In Chapter 4, we assessed the physiological adaptations of individuals from the eastern tropical Pacific (ETP) where oxygen is extremely low. Ship-board measurements of metabolic rate and hypoxia tolerance were conducted and a metabolic index was constructed to model suitable habitat for aerobic metabolism. I found that animals from the ETP had a higher metabolic rate than animals from more oxygen-rich habitats. Despite their higher oxygen demand, they maintained better hypoxia tolerance than conspecifics from oxygen-rich Hawaiian waters. Furthermore, I found that hypoxia tolerance in Japetella has a reverse temperature dependence from most marine ectotherms, a characteristic that uniquely suits the physical characteristics of its oxygen-poor environment. Even with their high tolerance to hypoxia, the OMZ core likely has insufficient oxygen supply to meet the basal oxygen demand of Japetella. Despite the limited aerobic habitat in this region, species abundance was comparable to more oxygenated ocean regions, suggesting that physiological or behavioral plasticity such as altered hypoxia tolerance or hypoxic avoidance in this globally-distributed species is sufficient to maintain species fitness in this extreme environment. These findings contribute towards our understanding of the impacts of climate change on cephalopod physiology and biogeography. The study of environmental physiology provides a mechanistic basis for the understanding and prediction of ecological responses to climate change.

The physical tumor microenvironment contributes significantly to carcinogenesis, cancer progression and metastatic dissemination. Two main components of the tumor microenvironment, hypoxia and acidosis, are present in nearly every solid tumor and act as powerful selection forces against the tumor. Hypoxia and acidosis promote tumor heterogeneity and contribute to chemotherapy and radiotherapy resistance. This dissertation interrogates methods to target the tumor microenvironment including two novel studies describing mechanisms of buffer therapy resistance and targeting tumor hypoxia with vasodilators to enhance the efficacy of a hypoxia activated prodrug, TH-302. In the first study, mechanisms of buffer therapy resistance were identified and detailed. Many studies have shown that the acidity of solid tumors contributes to local invasion and metastasis. Oral pH buffers can specifically neutralize the acidic pH of tumors and reduce the incidence of local invasion and metastatic formation in multiple murine models. However, this effect is not universal as we have previously observed that metastasis is not inhibited by buffers in some tumor models, regardless of the buffer used. B16-F10 (murine melanoma), LL/2 (murine lung) and HCT116 (human colon) tumors are resistant to treatment with lysine buffer therapy, whereas metastasis is potently inhibited by lysine buffers in MDA-MB-231 (human breast) and PC3M (human prostate) tumors. In the current work, I confirmed that sensitive cells utilized a pH-dependent mechanism for successful metastasis supported by a highly glycolytic phenotype that acidifies the local tumor microenvironment resulting in morphological changes. In contrast, buffer-resistant cell lines exhibited a pH-independent metastatic mechanism involving constitutive secretion of matrix degrading proteases without elevated glycolysis. These results have identified two distinct mechanisms of experimental metastasis, one of which is pH-dependent (buffer therapy sensitive cells) and one which is pH-independent (buffer therapy resistant cells). Further characterization of these models has potential for therapeutic benefit. In the second study, improving the efficacy of hypoxia activated prodrug, TH-302, through induction of hypoxia was investigated. Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic tumors, both of which are associated with poor prognosis. Hypoxia activated prodrugs, such as TH-302, are specifically activated in hypoxic environments and are now in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the "Steal" phenomenon, where atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. The current study shows that MiaPaCa-2 tumors exhibit a "Steal" effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MiaPaCa-2 tumor volume growth after 18 days of treatment. Further optimization of hypoxia-inducing agents and dosing regimens may lead to increased TH-302 activity, potentially improving clinical outcome. The data presented here demonstrate methods to effectively target the tumor microenvironment for therapeutic benefit. Further investigation into mechanisms of action and biomarkers for therapy response may have important implications on clinical treatment regimens for cancer patients.

Abstract not available

Abstract not available

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietary selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.

Obstructive Sleep Apnoea (OSA) is a disease characterised by repetitive upper airway obstructions which are manifest by desaturation and arousal from sleep. It has been known for many years that this interruption to the normal architecture of sleep may present to the clinician as excessive daytime somnolence often with a complaint of difficulties with concentration and short term memory. Previous work had demonstrated a relationship between variables of cognitive dysfunction in patients with obstructive sleep apnoea, however, little was known about which components of the syndrome contributed to this outcome and whether specific clinical thresholds of sleep disordered breathing could be defined for the development of cognitive dysfunction. In the context of this body of work cognitive dysfunction is defined as: a level of cognitive performance below normal derived values for a given cognitive test, when the subjects performance is controlled for age, sex and level of education.