In an attempt to further our understanding of the relationship between the calpain system and postmortem tenderization, three muscles [biceps femoris (BF), infraspinatus (IS), and longissimus (LD)] from normal (N = 6) and callipyge (N = 6) sheep were studied. Callipyge is a genetic phenomenon where carriers of the callipyge gene present a hypertrophy of pelvic and torso muscles, such that BF and LD are affected while IS is not. It has been observed characteristically that calpastatin and m-calpain activities are increased in muscles of animals affected by the callipyge phenotype. Soluble calpain and calpastatin, and myofibril-bound μ-calpain activities, and myofibrillar fragmentation index (MFI) were determined at death, 1d, 3d and 10d postmortem. Sarcomere length was determined at 1d and 10d postmortem. Shear force of the longissimus muscle was determined at 1d, 3d and 10d postmortem. Western blots for calpastatin, μ-calpain, desmin, nebulin, titin, troponin-T and α-actinin were performed to follow the degradation pattern of those proteins. The calpastatin and m-calpain activities were more than two-fold greater in BF and LD muscles from callipyge than in the same muscles from normal animals. Calpastatin activities in infraspinatus muscle from normal animals were higher than in the other two muscles of this phenotype. Soluble μ-calpain activities were higher at death for normal phenotype in BF and IS muscles and it decreased rapidly during postmortem storage. However, the rate of this decrease in that activity was faster in normal than in callipyge phenotype. Myofibrils contained calcium dependent protease activity and this activity was inhibited by cysteine proteases inhibitors and by calpastatin to some degree. There was no difference in the myofibril-calcium dependent protease activity between phenotypes at any time postmortem, presenting lower activity at death. The magnitude of protein degradation and tenderization were assessed by MFI and shear force, respectively. Neither the MFI nor shear force changed appreciably during storage of the callipyge affected muscles. Calpastatin level seems to determine the rate of postmortem tenderization.

The objective of these studies was to functionally characterize canine cytochromes P450 from subfamilies 2B and 3A. Studies of the canine hepatic 2B form PBD-2 had previously revealed that this enzyme exhibits a species specific ability to metabolize 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) and catalyze progesterone 21-hydroxylation. Expression of the putative PBD-2 cDNA, 2B11, in three different heterologous systems revealed that the recombinant enzymes' apparent substrate specificities varied based upon the expression system. 2B11 expressed in COS cells and yeast did not metabolize progesterone in a manner consistent with that of the purified hepatic enzyme, suggesting that 2B11 did not encode PBD-2. However, subsequent expression of 2B11 in E. coli confirmed that this recombinant enzyme did metabolize progesterone into 21- and 16α-hydroxyprogesterone. The structural determinants of 2B11-mediated progesterone 21-hydroxylation were then examined via site-directed mutagenesis of amino acid residues 114, 290, 363, and 365. Consistent with the importance of these residues in androstenedione and 245-HCB metabolism, amino acid alterations Val 114 $\to$ Ile, Asp-290 → Ile, and Ile-363 → Val each decreased 2B11 progesterone 21-hydroxylation. 2B11 mutants at position 365 differed in their regioselective metabolism of progesterone, whereas these mutations had little affect on the stereoselective metabolism of androstenedione. Cytochrome P450 3A forms are of intense interest due to the wide range of therapeutic compounds metabolized by these enzymes. To determine if canine liver contained multiple 3A forms which exhibit substrate specificities similar to those of other species, the canine 3A form PBD-1 (3A12) was expressed in E. coli. 3A12, rabbit 3A6 and human 3A4 metabolized steroids and macrolide antibiotics into the same products at comparable rates. A 3-fold difference in the rate of troleandomycin demethylation between liver microsomes and 3A12, the identification of a novel putative 3A hepatic protein via NH₂-terminal sequencing, and isolation of a distinct 3A cDNA provided evidence for 3A multiplicity in canine liver microsomes. The identification of functionally distinct canine 3A forms could provide the framework for structure-function analysis of these clinically important enzymes. Furthermore, 3A multiplicity and the conservation of substrate specificity between canine and human forms suggests that the canine may be an appropriate model of human 3A metabolism.

The goal of the research described in this thesis is to further our understanding of the motor control strategies that are available to the child when learning to produce meaningful interactions with environmental surfaces. The principal aim is to explore the analytical techniques that could be used to evaluate the range of neuromechanical responses for the lower limb that would provide stability at the limb/environment interface during growth and development. Five studies are presented that provide both experimental data and theoretical perspectives that were coalesced in the formulation of a general model for the control of stability at the limb/environment interface. The first study presents an analytical technique that was devised to classify the spatial-temporal organization of the surface myoelectric activity into one of three distinct patterns: Burst, Tonic, and Tonic Burst. This classification permits the matching between the pattern of muscle activity and the kinematic and kinetic control of the ground contact phase of landing. In the second, and companion paper, different distributions of the Burst, Tonic, and Tonic Burst patterns across the muscles of the lower limb were associated with three mechanical responses of the limb to ground contact. Mechanically, the three limb responses show a progression toward an effective control of stability at the limb/environment interface and it was therefore proposed that the distribution of activity patterns could reflect the priorities of system at different stages of growth and development. The results support our hypothesis that more complex movements can be executed when the limits of stability are maximized. Study three presents a formal model for the control of stability at the limb/environment interface. The model was further applied to propose new theoretical approaches that could be used in the clinical milieu, shifting the focus of the evaluation to the range of feasible movements that would be available

Patients with mitochondrial myopathies characteristically exhibit pronounced exercise intolerance, often associated with lactic acidosis, tachycardia and muscle weakness. These clinical features are attributable to impaired electron transport chain function in skeletal muscle. The usual etiology is a primary defect in mitochondrial DNA (mtDNA), where the severity of impairment is presumably linked to the ratio of mutant to wild-type mtDNA. This dissertation presents novel therapeutic approaches to these genetic defects, aimed at attenuating mitochondrial dysfunction and ameliorating the clinical condition by employing exercise training alone or in conjunction with pharmacological therapy. Dichloroacetate (DCA) was administered to augment mitochondrial capacity by activating pyruvate dehydrogenase, thereby decreasing lactic acidosis. Endurance and resistance training paradigms were employed to induce mitochondrial and satellite cell proliferation respectively. The goals were to augment respiratory chain function, increase levels of wild type mtDNA, and reverse effects of chronic inactivity. The effects of these treatments on functional and mitochondrial capacity were defined by changes in: (1) work capacity, oxygen utilization, and circulatory responses during maximal exercise; (2) heart rate and blood lactate during submaximal exercise; (3) recovery kinetics of phosphate-containing metabolites measured using phosphorus magnetic resonance spectroscopy ( 31P MRS); (4) scores on a quality of life questionnaire. The cellular correlates for these indices were defined by changes in: (1) mitochondrial volume, (2) respiratory chain enzyme activity, and (3) levels of mutant/wild-type mtDNA. Although DCA administration alone lowered blood lactate, endurance training was more effective in improving exercise capacity, heart rate and blood lactate, 31P MRS recovery kinetics, and quality of life. Increased mitochondrial volume and respiratory chain function were closely linked

A model was developed to account for the static elastic behaviour of the lung tissue strip in terms of distributions of collagen and elastin fibers. Distributions of collagen fiber lengths and elastin fiber stiffnesses were determined by fitting the model to data from dog lung tissue strips. These distributions followed 1/f power-laws for more than 95% of the data. Computer simulations of two dimensional tissue strip models with 1/f distributions of collagen fiber lengths also predicted realistic stress-strain curves. The simulations illustrated the gradual development of geometric and stress heterogeneity throughout the tissue as the collagen fibers were recruited during stretch. This model suggests a mechanistic basis for the shape of the pressure-volume curve of whole lung. It also indicates how this curve may be affected by changes in tissue collagen and elastin similar to the changes occurring in the diseases of pulmonary emphysema and fibrosis. Nonparametric block-structured nonlinear models for describing both the static and dynamic stress-strain behaviour of the lung were applied to dog lung tissue strips and to whole rat lungs in vivo. Both the Wiener and Hammerstein models accounted for more than 99% of the tissue strip data, although the Hammerstein model was more consistently accurate across a range of perturbation amplitudes and operating stresses. Plastic dissipation of energy within the lung tissue strip was estimated at less than 20% of the total dissipation during slow sinusoidal cycling. The Hammerstein model was also the best of those investigated for describing the rat lung data in vivo, although there were dependencies of the model parameters on perturbation amplitude and operating point that indicate that a more complicated model is required for the whole lung. Finally, construction of a fiber recruitment model for the dynamic mechanical behaviour of lung tissue strips was attempted. However accurate reproduction of measured behaviour was no

No description available.

The goal of this work is to study human whole-body gas exchange and cerebral autoregulation using a mathematical model. Previously, a human cardiopulmonary (CP) model [45, 47] was developed, which included heart, closed-loop blood circulation, gas exchange at lungs and baroreflex control of arterial pressure. In the current study, two major extensions to the model are made. First, a description of gas exchange in the peripheral tissues is added and is coupled with the lung gas exchanger via the circulatory loop with variable transport delays. A peripheral chemosensitive loop is also added to mimic the influence of blood gas composition on the heart and vasculature. The CP model is then used to predict the integrated cardiovascular and blood-tissue gas transport responses to pronounced changes in lung gas composition, and thus simulates changes encountered in apnea with and without passive oxygenation. The second extension of the CP model includes a more detailed description of cerebral circulation, cerebrospinal fluid (CSF) dynamics, brain gas exchange and cerebral blood flow (CBF) autoregulation. Two CBF regulatory mechanisms are described: autoregulation and CO2 reactivity. Central chemoreceptor control of ventilation is also added. This new model is subsequently used to study cerebral hemodynamic and brain gas exchange responses to test protocols commonly used in the assessment of CBF autoregulation (e.g., carotid artery compression and the thigh cuff test). The model closely mimics the experimental findings and provides biophysically based insights into the dynamics and interactions of the associated physiological systems. In summary, this work represents a bold effort in large-scale modeling of physiological systems. The presented model accurately describes the physiological systems and can explain how the cardiovascular, pulmonary and autonomic nervous systems interact in response to a variety of cardiopulmonary challenges, such as apnea, carotid artery compression and the thigh cuff test. With further refinement, the model may help investigators to better understand the complex biophysics of cardiopulmonary diseases such as sleep-related disorders of breathing (obstructive and central sleep apnea) and complications associated with head-injuries.

Arterial thrombosis is one of the important pathophysiological mechanisms that lead to cardiovascular diseases. In this thesis, we have made an attempt to better characterize the kinetic and molecular mechanisms that underlie the critical first step in arterial thrombosis, namely, the interaction between platelet glycoprotein (GP) Ib and von Willebrand factor (VWF). In the first part of the work, we evaluated the kinetics of interaction between platelet GP Ib-IX-V complex and VWF under arterial flow conditions. The GP Ibalpha subunit of GP Ib complex binds to VWF through the Al domain of VWF. Impaired GP Ib-VWF interaction due to GP Ibalpha mutations can result in bleeding abnormalities including platelet-type von Willebrand disease (ptVWD). We measured the cellular on- and off-rate constants of CHO cells expressing wild-type or gain- or loss-of-function mutant GP Ibalpha interacting with VWF-Al-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on VWF-Al surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of VWF-Al, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency was 3-fold more and 3-fold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GP Ibalpha affect both the association and dissociation kinetics of the GP Ibalpha-VWF-Al bond. In the second part of the work, we compared the interaction of unusually large multimers of VWF (ULVWF) and that of the normal plasma multimers of VWF (P-VWF) platelets. ULVWF multimers are implicated in the pathology of a thrombotic disorder, thrombotic thrombocytopenic purpura (TTP) due to their increased affinity for platelets. We found that the ULVWF multimers are more effective than the normal P-VWF multimers in mediating (a) platelet aggregation in solution at high shear stress; (b) ristocetin-modulated platelet agglutination and (c) platelet adhesion to immobilized VWF under arterial shear conditions.

Serving as the extra-thalamic relay from the ascending reticular activating system to the cerebral cortex, basal forebrain neurons and particularly the cholinergic neurons therein, are believed to play an important role in cortical activation that occurs during waking and paradoxical sleep (PS). However, basal forebrain neurons are also thought to be involved in slow wave sleep (SWS), and thus accordingly influence cortical activity in different ways across the sleep-waking cycle. The present research aimed to elucidate the way the basal forebrain modulates cortical activity and sleep-wake states in response to the different neurotransmitters contained in its brainstem afferents. Microinjections of chemicals into the basal forebrain of rats were performed using a remotely controlled apparatus that allowed study of electroencephalographic (EEG) and behavioral changes without disturbing the rat's normal sleep-waking cycle. In a first study using spectral analysis of the EEG, it was established that high frequency gamma (30--60 Hz) activity reflects cortical activation and behavioral arousal during waking and PS and that gamma varies reciprocally with delta (1--4 Hz) and positively with theta (4--8 Hz) across these states. Noradrenaline, which excites cholinergic neurons to discharge in a tonic mode and has differential effects on non-cholinergic neurons, increased gamma and decreased delta EEG activities while eliciting waking. The glutamate agonist, AMPA, produced similar effects, and NMDA, which is known to induce a bursting discharge in cholinergic neurons, additionally increased theta activity in association with an active waking state. Moreover, neurotensin, which acts selectively to excite cholinergic cells to discharge in a bursting mode, evoked gamma and theta while enhancing both waking and PS in the provoked absence of SWS. In contrast, serotonin, which inhibits cholinergic neurons and has minimal effects on non-cholinergic neurons, decreased gamma and th

Although leukotrienes (LTs) are implicated in the pathogenesis of asthma, it is still unclear which types of cells are of primary importance in their formation in asthmatic lungs. To elucidate the mechanism for the increased LT formation in asthma, we investigated the pulmonary cellular infiltration and LT synthesis by dispersed lung cells from BN rats following allergen challenge. / To enable us to do these studies we have developed improved HPLC methods for the analysis of complex mixtures of eicosanoids in biological samples using binary gradients containing trifluoroacetic acid. Samples containing PGB$ sb2$ (the internal standard), cysteinyl-LTs, LTB$ sb4,$ hydroxyeicosatetraenoic acids (HETEs) and the cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHTrE) can be analyzed in as little as 20 min. Mixtures which also contain more polar eicosanoids such as lipoxins and omega-oxidation products of LTB$ sb4$ can be analyzed in 40 min. / Ovalbumin (OVA) challenge of sensitized BN rats resulted in a significant influx of neutrophils into the lungs and a significant increase in the synthesis of 5-lipoxygenase products, in particular LTB$ sb4$, by lung cells 6 h after challenge. However, there was little change in the production of cysteinyl-LTs compared with saline challenge. There was a significant eosinophilic infiltration in the lungs 24 h after OVA challenge, but cysteinyl-LT production by lung cells was unaltered at this time suggesting that eosinophils from BN rats are unlikely to be a major site for the formation of these compounds. This was confirmed in experiments with partially purified eosinophils obtained from Sephadex-treated rats. In contrast, cysteinyl-LTs were synthesized in appreciable amounts by alveolar macrophages from BN rats. Administration of rabbit anti-rat PMNL serum abolished the influx of neutrophils into the lungs and appeared to reduce the LTB$ sb4$ synthesis by lung cells. There was a positive correlation between the numbers of neutrophils in the lung and LTB$ sb4$ production by lung cells. In contrast, macrophages were positively associated with cysteinyl-LT production. Exogenous LTB$ sb4$ and 5-oxo-ETE induced infiltration of eosinophils into the lungs and therefore may be responsible for the late phase eosinophilic infiltration in BN rat lungs following allergen challenge.