The purpose of this investigation was to examine selected biochemical mechanisms known to influence contractility and energy metabolism in the myocardium, with particular emphasis placed on the regulatory role of protein phosphorylation in the ventricular myocardium. The investigation was conducted in three phases; initially the cardiac contraction cycle was examined to determine whether reported fluctuations in myocardial cAMP levels were associated with other biochemical events known to be cAMP-dependent. The second phase involved the determination of specific kinase activities and endogenous substrates in a highly purified cardiac sarcolemmal preparation. In the final phase, ventricular myocytes were utilized to examine the ability of adenosinergic and muscarinic agonists to influence the isoproterenol-induced increases in protein phosphorylation. Studies in the first phase examined cyclic AMP levels and selected kinase activities in hearts frozen at various stages of the cardiac cycle. An automated clamping device, capable of freezing a perfused rat heart in less than 50 msec, was utilized to separate the cardiac cycle into various phases. Three different timing schemes were employed to divide the cycle into 2 to 4 segments. These different timing schemes revealed no significant differences in cAMP during the cardiac cycle. Myocardial cAMP values ranged from 2.5 to 4.1 pmol/min/mg protein in all phases. However, in one scheme there was a tendency for cAMP to be elevated in early systole, with minimal values occurring diastole. There were also no significant differences seen for either glycogen phosphorylase or cAMP-dependent protein kinase (PKA) activity between various phases of the cardiac cycle. Since no significant fluctuations were observed in the levels of cAMP or the activities of PKA or glycogen phosphorylase during a single cardiac contraction cycle, it would appear that these agents do not exert their effects on cardiac function on a beat to beat basis. The second phase of study examined the nature and function of individual protein kinases in the myocardium. Using a highly purified cardiac sarcolemmal preparation, kinase specific, synthetic substrates were employed to quantify the activities of cAMP-dependent (PKA), calcium/calmodulin-dependent (PKCM), calcium/phospholipid-dependent (PKC) and cGMP-dependent (PKG) protein kinases. Additionally, endogenous protein substrates were examined in this preparation to provide possible insight as to the function of these kinases in the heart. The activities of PKA, PKG, PKCM, and PKC in nmol 32P/min/μg protein were as follows: PKA, 1606; PKG, 35.7; PKCM, 353; and PKC, 13.2. Three endogenous protein substrates of apparent molecular weights of 15kD, 28kD and 92kD were phosphorylated. While no endogenous protein phosphorylation was detectable as a result of cG-PK activity, all of the substrates were phosphorylated, to varying degrees, by both PKA and CACM-PK. PKC phosphorylated only the 15kD substrate. Even though several endogenous kinases are evident in the sarcolemmal preparation, cAMP-dependent protein kinase demonstrates the greatest degree of activity. This kinase also appeared to be the most abundant; however, there is some concern as to the source of these kinases in the membrane preparation since endothelial membranes as well as cardiac membranes appeared to be present. Evidence for endothelial contamination was provided by the finding that the membrane preparation contained appreciable amounts of angiotensin converting enzyme (ACE) activity, an enzyme felt to reside in the vascular endothelium. Since studies with this preparation could not exclude contribution of nonmuscle cell membranes a model consisting solely of dispersed ventricular myocytes was developed. The third phase of these studies examined protein phosphorylation in primary cultures of ventricular myocytes. Specifically, these studies examined protein phosphorylation induced by exposure to isoproterenol (ISO), a catecholamine known to effect changes in the phosphorylation state of proteins in the heart by means of a β-adrenergic-mediated/cAMP-dependent mechanism was examined. Additionally, the effects of phenylisopropy-ladenosine (PIA) and carbamyl choline chloride (CARB) were examined with regard to their anti-adrenergic role(s) in this process. Adherent, collagenase-dispersed, radiolabelled (32p) ventricular myocytes exposed to ISO demonstrated a dose and time dependent increase in 32p incorporation into several endogenous protein substrates. When the myocytes were exposed (60 sec) to either PIA or CARB prior to the exposure to ISO, ISO-induced 32p incorporation into protein substrates of apparent molecular weight of 6kD, 31kD and 155kD was reduced up to 67% when compared to the effects of ISO alone. Additionally, both PIA and CARB attenuated the ISO-induced increase in PKA activity in the myocyte, yet only CARB was seen to produce an inhibitory effect on the ISO-induced increase in cAMP levels in the myocytes. The effects of CARB were dose-dependent and inhibited the effects of ISO on 32p incorporation at all doses tested. PIA elicited biphasic effects: lower PIA concentrations were inhibitory in nature, while higher concentrations of PIA appeared to potentiate the increase in 32p incorporation induced by ISO. Based on electrophoretic mobilities (SDS/PAGE) of the 6kD and the 155kD substrates, these substrates have been tentatively identified as the monomeric form of the sarcoplasmic reticulum-associated protein, phospholamban, and the contractile filament-associated protein, C protein, respectively. The 31kD substrate has been identified, by means of immunoblot, as the contractile filament-associated protein, troponin I. The role of protein phosphorylation in the myocardium involves complex, inter-related mechanisms that encompass extracellular, transmembranal and cytoplasmic elements in the heart. It is well understood that certain mechanisms of the contraction cycle known to vary on a beat to beat basis, such as myosin ATPase, involve changes in protein phosphorylation. However, the nature of the various kinases and substrates examined in this study appear to influence longer-term events of myocardial contractility. Mechanisms coupled with hormone action, modulation of second messenger-dependent components, and factors associated with changes in contractility seen with aging and disease are more likely to exhibit changes similar to those described herein. A better understanding of the underlying biochemistry may provide greater insight into the importance of these metabolic changes.

Runx1 is a master regulator of hematopoiesis, required for the initiation of definitive hematopoiesis in the embryo and essential for appropriate differentiation of many hematopoietic lineages in the adult. The roles of Runx1 in normal hematopoiesis are juxtaposed with the high frequency of Runx1 mutations and translocations in leukemia. Leukemia associated Runx1 mutations that retain DNA-binding ability have truncations or frame shifts that lose C-terminal domains. These domains are important for subnuclear localization of Runx1 and protein interactions with co-factors. The majority of leukemia associated Runx1 translocations also replace the C-terminus of Runx1 with chimeric fusion proteins. The common loss of Runx1 C-terminal domains in hematopoietic diseases suggests a possible common mechanism. We developed a panel of mutations to test the functions of these domains in vitro, and then developed mouse models to examine the consequences of losing Runx1 C-terminal domains on hematopoietic development and leukemogenesis in vivo. We previously observed that overexpression of a subnuclear targeting defective mutant of Runx1 in a myeloid progenitor cell line blocks differentiation. Gene expression analysis before differentiation was initiated revealed that the mutant Runx1 was already deregulating genes important for maturation. Furthermore, promoters of the suppressed genes were enriched for binding sites of known Runx1 co-factors, indicating a non-DNA-binding role for the mutant Runx1. To investigate the in vivo function of Runx1 C-terminal domains, we generated two knock-in mouse models; a C-terminal truncation, Runx1Q307X, and a point mutant in the subnuclear targeting domain, Runx1 HTY350-352AAA . Embryos homozygous for Runx1 Q307X phenocopy a complete Runx1 null and die in utero from central nervous system hemorrhage and lack of definitive hematopoiesis. Embryos homozygous for the point mutation Runx1HTY350-352AAA bypass embryonic lethality, but have hypomorphic Runx1 function. Runx1HTY350-352AAA results in defective growth control of hematopoietic progenitors, deregulation of B-lymphoid and myeloid lineages, as well as maturation delays in megakaryocytic and erythroid development. Runx1 localizes to subnuclear domains to scaffold regulatory machinery for control of gene expression. This work supports the role of transcription factors interacting with nuclear architecture for greater biological control, and shows how even subtle alterations in that ability could have profound effects on normal biological function and gene regulation.

Erythropoietin (Epo) modulates red blood cell production (erythropoiesis) by binding to its receptor and activating STAT5, a Signal Transducer and Activator of Transcription (STAT) protein implicated in both basal and stress erythropoiesis. Epo concentration in serum changes over three orders of magnitude, as it regulates basal erythropoiesis and its acceleration during hypoxic stress. However, it is not known how STAT5 translates the changes in Epo concentration into the required erythropoietic rates. We addressed this question by studying STAT5 phosphorylation, at the single cell level, in developing erythroblasts. We divided erythroid progenitors in tissue into several flow-cytometric subsets and found that each of them exhibited distinct modes of Stat5 activation, based on their developmental stage. STAT5 activation is bistable in mature erythroblasts, resulting in a binary (or digital), low-intensity STAT5 phosphorylation signal (p-Stat5). In early erythroblasts, and in response to stress levels of Epo, the low intensity bistable p-Stat5 signal is superseded by a high-intensity graded, or analog, signal. The gradual shift from high-intensity graded signaling in early erythroblasts to low intensity binary signaling in mature erythroblasts is due to a decline in STAT5 expression with maturation. We were able to convert mature, digital transducing erythroblasts into analog transducers simply by expressing high levels of exogenous STAT5. We found that EpoR-HM mice, expressing a mutant EpoR that lacks STAT5 docking sites, generate the binary, but not the analog, STAT5 signal. Unlike Stat5-null mice, which die perinatally, the EpoR-HM mice are viable but deficient in their response to stress, demonstrating that while binary STAT5 signaling is sufficient to support basal erythropoiesis, analog signaling is required for the stress response. Bistable systems contain a positive loop, which is important for flipping the switch between the two stable ‘on’ or ‘off’ states. We show that bistable activation is the result of an autocatalytic loop in which active STAT5 promotes further STAT5 activation. The isolated STAT5 N-terminal domain, which is not required for STAT5 phosphorylation, enhanced autocatalysis, converting a high intensity graded signal into a high intensity binary response. The N-terminal domain is known to participate in a radical conformational reorientation of STAT5 dimers inherent in STAT5 activation. We propose that the N-terminal domains of active STAT5 dimers facilitate the conformational reorientation of inactive dimers, in a prion-like autocatalytic interaction that underlies bistability and binary signaling. Together, bistable STAT5 activation, combined with a graded response allow erythropoietic rate to faithfully reflect a wide Epo concentration range, while preventing aberrant signaling.

Gestational diabetes mellitus (GDM), a pregnancy-induced metabolic disorder that affects 2-10% of pregnancies poses future risk for diabetes mellitus (DM) and cardiovascular disease in mother and child. However, few prospective studies have examined the effect of GDM on altered maternal and cord blood lipids, specifically HDL, LDL, triglycerides, and total cholesterol, both during and after pregnancy. We have evaluated the association between GDM and lipid metabolism in pregnant mothers and their infants using data from a prospective cohort study conducted at Baystate Medical Center’s Wesson Women and Infant’s Unit. GDM was assessed prenatally by 3-hr GTT blood samples and was confirmed by obstetrician review. Lipids were assessed via fasting and non-fasting blood samples obtained during 3-hr GTTs performed at 24-28 weeks of gestation and 6-8 weeks post-partum. Data for covariates were collected via an interview form administered at the time of recruitment. We used multivariable linear regression to evaluate the association between GDM status and maternal lipids during and after pregnancy as well as cord lipids. These study results inform future research on GDM as a risk factor for future metabolic disorders in mother and child.

Circulating angiogenic cells (CACs) are early predictors of cardiovascular health and are inversely proportional to related outcomes. Increased number and function of CACs is seen in healthy individuals compared with individuals with cardiovascular disease (CVD). Exercise increases CAC number and function in CVD populations, through a nitric oxide-mediated mechanism. Inactivity is a growing concern in industrialized nations; it is an independent risk factor for CVD and is linked to increased mortality. The purpose of this study was to understand the effect of reduced physical activity (rPA) on two CAC populations (CFU-Hill and CD34+) in highly active individuals. We examined the mechanisms underlying changes in CAC function as a result of rPA with maintained energy balance. The two sub-populations of CACs responded differently to rPA. CFU-Hill CACs, decreased in number and amount of intracellular nitric oxide while CD34+ cells, did not change. Gene expression analyses indicated that oxidative stress- related genes did not change in CFU-Hill cells with rPA. However, correlations between CFU-Hill cell numbers, intracellular nitric oxide, and genes that are related to nitric oxide were observed. We concluded that rPA caused the observed decrease in CFU-Hill number and intracellular nitric oxide through a decrease in nitric oxide cellular availability, not oxidative stress.

<p>Various studies have shown an association between neutrophilic airway inflammation and severe asthma, but have failed to establish a causal relationship. In these studies airway neutrophilia could be due to high steroid doses, an airway infection, an epiphenomenon of severe asthma or a combination of these. We have examined the role of neutrophils in severe asthma in patients on optimum steroid doses with controlled eosinophilic airway inflammation and chemotactic activity of IL-17 as one potential mechanism of neutrophil recruitment to the airway.</p> <p>We examined the number, viability and activity of neutrophils in blood and sputum of three groups of asthma subjects divided on the basis of asthma severity. We also compared direct migration of blood neutrophils towards IL-17 between non-asthmatics and severe asthma subjects.</p> <p>Viability and survival at 24 hours was measured by examining apoptotic and non-apoptotic cells. Activation was examined by measuring the production of hydrogen peroxide and the expression of primary and secondary granule proteins. In migration study, migration of neutrophils towards IL-17 was measured.</p> <p>Blood neutrophils were increased in severe asthma subjects as compared to moderate and mild asthma subjects. There was no difference in sputum neutrophil numbers. There was no difference in viability, although blood neutrophil 24 hour survival was increased in severe asthma subjects as compared to moderate asthma subjects. There was no difference in the level of activation amongst the three groups.</p> <p>IL-17 was not a chemotactic stimulus for neutrophils. The study results show that sputum neutrophil numbers and activation are not increased in severe asthma as compared to less severe asthma. Therefore, the study results do not support a causal relationship between airway neutrophilia and severe asthma.</p> <p>Airway neutrophilia observed in previous studies might be due to airway infections or high doses of steroids taken by study subjects.</p> / Master of Health Sciences (MSc)

<h4> </h4> / <p>Asthma is an inflammatory disorder of the airways, and there has been growing insight into the cellular and molecular mechanisms underlying the inflammatory basis of this disease. Research into the inflammatory mechanisms of asthma has progressively shifted focus from downstream effectors, such as mast cells and eosinophils, up to Th2 lymphocytes and their proallergic cytokines. Even more upstream in the allergic cascade are dendritic cells (DCs), potent APCs that orchestrate immune responses. Evidence supporting a role of DCs in regulating airway allergic inflammation is derived mainly from animal studies. In animal models of asthma, myeloid DCs (mDCs) induce and maintain airway inflammation, while plasmacytoid DCs (pDCs) mediate tolerance and lung homeostasis. It remains uncertain, however, whether this concept of pro-allergic mDCs and anti-allergic pDCs translates from animal to human models. The overall objective of this thesis was to investigate the biology of DC subsets in allergen-induced asthma in asthmatic subjects. Initially, we demonstrate that both mDCs and pDCs increase in the airways of subjects with mild asthma after allergen inhalation. Next, we describe a distinct subpopulation of mDCs, called mDC2s, and demonstrate their association with allergy and asthma severity. Expanding on these findings, we show that mDC2s increase in the airways of mild asthmatics after allergen challenge. Lastly, we explore the potential of pharmacological therapies, anti-OX40L MAb and anti-TSLP MAb, to affect DCs in subjects with mild asthma, and demonstrate no effect of either drug on circulating DC subsets. The studies presented here provide evidence for multiple DC subtypes being involved in the regulation of allergen-induced inflammatory responses, and support continued investigations into the biology of different DC subsets in allergen-induced asthma.</p> / Doctor of Philosophy (Medical Science)

Les pathologies respiratoires obstructives de l’enfant (asthme et broncho-alvéolites) sont l’une des principales causes d’admission en réanimation pédiatrique. Depuis plusieurs années, des progrès ont été faits pour réduire l’invasivité des soins se traduisant par une réduction de la morbidité. L’objectif de ce travail de thèse est de s’appuyer sur des mécanismes physiopathologiques pour proposer des stratégies d’optimisation ventilatoire et non ventilatoire chez ces enfants. Nous avons évalué l’impact du décubitus ventral couplé à la ventilation non invasive chez les nourrissons atteints de bronchiolite grave. Le décubitus ventral permet de réduire significativement l’effort inspiratoire et d’améliorer le couplage électromécanique du diaphragme. Ensuite nous avons évalué la « neurally adjusted ventilatory assist » (NAVA) qui est un mode ventilatoire proportionnel basé sur l’activité électrique du diaphragme. Nous avons démontré que la NAVA améliorait la synchronisation patient-respirateur et réduisait le travail respiratoire en comparaison à la « nasal continuous positive airway pressure » (nCPAP). Enfin, dans la pathologie asthmatique nous avons également décrit la faisabilité du haut débit nasal dans cette population. Ces stratégies nécessitent maintenant d’être validées sur des critères cliniques et feront l’objet de deux études multicentriques randomisées / Obstructive lung disease in children (asthma and bronchiolitis) are one of the main causes of admission to pediatric intensive care units. For several years, progress has been made to reduce the invasiveness of care resulting in a decrease in associated morbidity. The main objective of the thesis was to propose new ventilatory and non-ventilatory strategies based on physiopathology to optimize the care of such children.In children with severe bronchiolitis we evaluated the impact of prone position associated with non-invasive ventilation. The prone position decreases significantly the inspiratory work of breathing and improves the neuromechanical efficiency of the diaphragm. We also evaluated the effect of neurally adjusted ventilatory assist (NAVA) that is a proportional ventilatory mode based on the electrical activity of the diaphragm. We demonstrated that NAVA improved the patient-ventilator interactions and decrease the work of breathing in comparison with nasal continuous positive airway pressure (nCPAP). We also evaluated the feasibility of high flow nasal cannula as a respiratory support in children with severe asthma attack. These strategies need now to be validated on clinical outcomes and are the subject of two ongoing multicenter randomized trials

The effects of mechanical forces on endothelial cell function and behavior are well documented, but have not been fully characterized. Specifically, fluid pressure has been shown to elicit physical and chemical responses known to be involved in the initiation and progression of endothelial cell-mediated vascularization. Central to the process of vascularization is the formation of tube-like structures. This process—tubulogenesis—is essential to both the physiological and pathological growth of tissues. Given the known effects of pressure on endothelial cells and its ubiquitous presence in the vasculature, we investigated pressure as a magnitude-dependent parameter for the regulation of endothelial tubulogenic activity. To accomplish this, we exposed two- and three-dimensional bovine aortic endothelial cell (BAEC) cultures to static pressures of 0, 20, and 40 mmHg for 3 and 4 days. The most significant findings were: (1) cells in two-dimensional culture exposed to 20, but not 40, mmHg exhibited significantly (p < 0.05) increased expression of both VEGF-C and VEGFR-3, and (2) cells in three-dimensional culture exposed to 20, but not 40, mmHg exhibited significant (p > 0.05) increases in endothelial sprouting. These findings evidence the utility of pressure as a selective modulator of tissue microvascularization in vitro and implicates pressure as factor in pathological tubulogenesis in vivo.

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.