Thesis (doctoral)--University of London. / "Reprinted from the New phytologist, vols. XVI-XVIII, 1917-19." p. [xii]. Includes bibliographical references and index.

Thesis (doctoral)--University of London. / "Reprinted from the New phytologist, vols. XVI-XVIII, 1917-19." p. [xii]. Includes bibliographies and index.

This thesis summarizes a research programme on the role of hypoxia-inducible factor (HIF) and its 2alpha subunit in systemic human physiology. Experiments were performed to assess the role of HIF in the regulation of skeletal muscle metabolism, cardiac anatomy, function and energy metabolism, and in cardiopulmonary physiology. Patients with different genetic mutations affecting the HIF pathway were recruited for each main study. Chapter 1 presents an overview of human physiological responses to hypoxia in a historical perspective, with particular attention to the areas of human physiology that are relevant for the studies presented in the experimental chapters. Chapter 1 also presents a summary of the HIF pathway and the novel findings presented in this thesis. Chapter 2 illustrates the methods used to perform the experiments. Chapter 3 investigates skeletal muscle metabolism, cardiac anatomy, function and energy metabolism in patients with Chuvash polycythaemia, who have mildly elevated levels of HIF, associated with a mutation in von Hippel-Lindau gene, at whole body level. Chapter 3 shows major abnormalities associated with HIF pathway alterations in skeletal muscle energy metabolism, especially in conditions of metabolic stress such as during exercise and digestion of a meal. Chapter 4 shows that patients with Chuvash polycythaemia also have small hearts and reduced cardiac energy levels. Chapter 5 explores cardiopulmonary abnormalities in patients with gain-of-function mutations specifically in HIF-2alpha subunit; these patients are polycythaemic like patients with Chuvash polycythaemia. Observed abnormalities include pulmonary hypertension, elevated heart rate, cardiac output, ventilation, and the increment in pulmonary blood pressure in response to moderate hypoxia. Chapter 6 presents results from experiments in patients with classic von Hippel- Lindau disease, who are not usually polycythaemic. However, some degree of haploinsufficiency was observed in their neutrophils, suggesting a pseudo-hypoxic phenotype. Chapter 6 shows that von Hippel-Lindau disease is not associated with major cardiopulmonary abnormalities. Overall, the research reported in this thesis presents original experimental evidence for the effects of alterations in the HIF pathway on human physiology.

No description available.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kD cytoskeletal protein, dystrophin. Without dystrophin, the dystrophin-associated protein complex (DAPC) does not form and the plasma membrane is destabilised. There is no effective treatment and affected individuals die from respiratory failure and cardiomyopathy by age 30. This thesis describes experiments in which in vivo cardiac function was measured using non-invasive magnetic resonance imaging in a number of mouse models relevant to muscular dystrophy. As syncoilin forms a link from the DAPC to the cytoskeleton, it was postulated in Chapter 3 that the syncoilin knockout mouse would have cardiac defects similar to those caused by the loss of dystrophin. However, the loss of syncoilin did not alter the protein levels of its binding partners, measured by western blotting, and caused no defect in heart function or structure, measured using histological staining. Similarly, in Chapter 4, a mouse with a mutation in the transient receptor potential channel canonical type 3 (TRPC3), a receptor/stretch-activated cation channel thought to be involved in the pathogenesis of DMD, was found to have no functional or morphological cardiac defect. In the mdx mouse, a mouse model of DMD that lacks dystrophin, cardiomyopathy was prevented by either increasing levels of the dystrophin related protein, utrophin, or of dystrophin, in the diaphragm, which thereby restored diaphragm function. In Chapter 5 it was found that in a transgenic mdx mouse in which utrophin was over-expressed in skeletal muscle and diaphragm, but not in the heart, cardiac function was restored to wild-type levels. However, histologically the transgenic heart showed more fibrosis and immune cell infiltration than that of untreated mdx controls. In Chapter 6 it was found that in mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), that resulted in high dystrophin restoration in skeletal muscle and diaphragm only, cardiac function was also restored to wild-type levels. In Chapter 6 it was also found that in dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in skeletal muscle and diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. The novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice has implications for the study of heart function in the current DMD mouse models and suggests a new approach to treatment.

Traditional studies of simple perceptual choice tasks in vertebrates identified behavioural characteristics of deliberate decision-making that guided the development of general mathematical models, and the search for neurophysiological correlates. Current experimental and modelling efforts aim to uncover biophysical and circuit level mechanisms of decision-making processes. However, genetic manipulability constraints and lack of high-throughput assays make further progress in vertebrate studies a steep endeavour. In this thesis I studied decision-making in Drosophila melanogaster in trained two-alternative forced-choice olfactory tasks with varying stimulus contrast, using a high-resolution single fly behavioural assay. Analysing accuracy and reaction time as a function of task difficulty (i.e., stimulus contrast) showed that flies display behavioural characteristics of evidence accumulation processes, a signature of vertebrate decision-making: reaction times increased and perceptual accuracy declined as stimulus contrast decreased. Mutants for the gene encoding the transcription factor FoxP took longer than wild-type flies to form decisions of similar or reduced accuracy, especially in difficult tasks. Using the putative FoxP promoter to ascertain FoxP expression identified subsets of mushroom body intrinsic Kenyon cells, in αβ core and γ neurons, as potential sites of FoxP action. Disrupting FoxP expression or decreasing neuronal excitability specifically in αβ core neurons mimicked the phenotype observed in FoxP mutants. FoxP expression therefore affects the development or function of αβ core neurons in the progression of a decision process towards commitment. To identify molecular processes involved in evidence integration regulated by FoxP I further screened 2nd and 3rd chromosome deficiency lines in a sensitised FoxP mutant background, uncovering genomic regions of interest for further study. Finally, analysing naive performance in tasks of increasing difficulty showed that naive discriminations are faster and less accurate than trained ones, pointing to the existence of two decision-making systems. FoxP mutants appear to engage the slower, more accurate decision making system and the mushroom body seems to be involved in naive discriminations. The molecular and neuronal players involved in olfactory decision making in Drosophila melanogaster uncovered in this thesis will allow researching decision making systems to an unprecedented level of detail.

Breakdown of the disc extracellular matrix is thought to arise from increased activity of matrix metalloproteinases (MMPs). Aggrecan, one of the major disc matrix macromolecules, is degraded through action of MMPs and aggrecanases and its concentration falls early in the degeneration process. Loss of the constituent glycosaminoglycans (GAGs), which are osmotically active, leads to a decrease in osmotic pressure and subsequently to a fall in tissue hydration. Apart from the major biomechanical consequences, fall in extracellular osmolarity is known to influence cell function. Apart from aggrecan, other macromolecules such as fibronectin are also broken down by MMPs. Fibronectin fragments (Fnfs) have been identified in degenerate discs. Such Fnfs have been found to stimulate production and activity of MMPs in articular cartilage but little is known about the effect of these fragments on disc cells. The aim of the work was thus to determine whether Fnf stimulated expression of MMPs and hence induced aggrecan breakdown and loss in the disc and whether extracellular osmolarity influenced this potential response. NP cells or explants were harvested from adult bovine caudal discs. They were cultured in DMEM culture medium over a range of osmolarities with or without Fnf treatment. Profiles of gene expression of MMPs and their inhibitors and effect of changes in osmolarity on expression of selected MMPs were determined. The effect of Fnf on responses of cells and tissue explants from the central region of the disc, the nucleus pulposus (NP) and the role of changes in extracellular osmolarity in relation to GAG loss and expression of selected MMPs was then examined both at the protein level and by gene profiling using a microarray. My results showed that expression of MMPs by disc cells is regulated by extracellular osmolarity rather than the 30 kD Fnf, with the level of some MMPs secreted by disc cells and involved in degradation of disc matrix rising as osmolarity falls. These results could explain in part the finding that MMP expression increases with degree of disc degeneration i.e. with loss of aggrecan and fall in extracellular osmolarity. These also suggested that a fall in osmolarity could induce a degenerative cascade with proteolytic digestion of aggrecan leading to a fall in osmolarity and hence a further increase in proteinase expression and matrix degradation.

Calcium ions (Ca2+) are extremely important intracellular messengers, activating a plethora of cellular processes. Growing evidence now points to a major role for the local Ca2+ signal in driving specific cellular responses. The simplest and most fundamental local Ca2+ signal is the Ca2+ microdomain, which rapidly forms when Ca2+ permeable ion channels open. In non-excitable cells the dominant Ca2+ entry channels are store-operated Ca2+ channels (SOCCs). The best characterised is the Ca2+ release activated Ca2+ (CRAC) channel. How local Ca2+ entry through CRAC channels impacts on channel function however is unclear. I have investigated the interaction between the Ca2+ binding protein calmodulin and CRAC channel activity and subsequent agonist-induced Ca2+ signals. Furthermore, I have investigated a role for mitofusin 2 (a protein that is known to tether the ER and mitochondria) on these Ca2+ signals. Using three different calmodulin mutant constructs with alterations to their Ca2+ binding sensitivities, I have shown that calmodulin facilitates CRAC channel dependent Ca2+ entry and maintains agonist-induced cytosolic Ca2+ oscillations in a lobe-specific manner. Calmodulin has four Ca2+ binding sites, two on the N-lobe and two on the C-lobe. I found a dominant negative calmodulin mutant (CAM4M, where all four binding sites had been mutated), or one where the C-lobe could not bind Ca2+ (CAM2C), impaired both Ca2+ influx through CRAC channels and maintenance of cytosolic Ca2+ oscillations. In contrast, a Ca2+-insensitive N-lobe mutant had little effect, (CAM2N). Knockdown of the mitochondrial Ca2+ uniporter regulator (MICU1) or mitochondrial membrane depolarization had similar effects to those seen with CAM4M or CAM2C, suggesting that at least in part, the action of calmodulin was through regulation of mitochondrial Ca2+ dynamics. This was confirmed by directly measuring the mitochondrial matrix Ca2+ concentration in intact RBL-1 cells using the mitochondrial targeted, fluorescent protein, pericam. Both CAM4M and disruption of mitochondrial Ca2+ buffering impaired agonist-induced mitochondrial Ca2+ uptake, suggesting that the modulation of CRAC channels occurred through Ca2+-calmodulin facilitation of mitochondrial Ca2+ uptake. Using a mutant Orai1 (A73E) that cannot bind calmodulin, I have shown that calmodulin tethered to the CRAC channel provides a major source of calmodulin for effective mitochondrial Ca2+ uptake. Physiological relevance of my proposed pathway was provided from experiments where I showed knockdown of MICU1 impaired agonist-induced CRAC channel dependent NFAT-1-driven gene expression. In addition, I establish a crucial role for mitochondrial MFN2 and presumably its ability to properly link the mitochondria and ER in the control of CRAC channels and agonist-induced Ca2+ signals.

No description available.

Canids have long been considered to be conservative in their postcranial anatomy, so there are few studies examining individual canid taxa for locomotor adaptations. Canids are generally considered to be the most cursorial of the carnivorans. The limbs of large canids are generally adapted for rapid terrestrial locomotion, as these animals frequently rely on speed for prey capture. The prey animal is captured and killed using the jaws and teeth. Smaller canids, such as the red fox Vulpes and gray fox Urocyon, do not use their limbs primarily for fast running. The red fox appears to have many adaptations for running, including long slender legs, but these foxes do not run in their daily activities except when chased. The red fox uses its forelimbs to help in prey capture and its hind legs for leaping. The gray fox is an unusual canid since it regularly climbs trees. The limbs of the gray fox, especially the forelimb, are utilized in climbing. This dissertation contains a detailed description of the postcranial osteology and myology Vulpes and Urocyon cinercoargenteus and includes an analysis of these anatomical features in a functional framework. An examination of both the osteology and myology of the fore and hind limbs of these two foxes reveals that their behavior is reflected in a number of anatomical characters. Adaptations for leaping in the red fox include the presence of unusually long hind legs relative to the front legs, and an increase in the length of the distal bony limb elements relative to more proximal ones. In addition, the limb bones are very slender. Muscle bellies of tarsal and digital flexors and extensors are restricted to a proximal position on the limb, and muscles in general are emphasized that act along the long axis of the limbs. Adaptations of the gray fox for climbing include the presence of relatively short legs, a greater ability to rotate the radius on the ulna relative to other canids, and a relatively greater ability to abduct the hind limb. In addition, both red and gray foxes are able to retract their claws, an ability that is not generally associated with canids.