Studies have shown that long-term opioid agonist (such as morphine) treatment produces antinociceptive tolerance and increased pain sensitivity (hyperalgesia and/or allodynia), limiting the clinical efficacy of morphine. Prolonged opiate administration also upregulates spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) levels and enhances evoked CGRP release in the dorsal horn of rats. It was suggested that augmented spinal pain neurotransmission may contribute to paradoxical pain sensitization and antinociceptive tolerance. The cellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter are not fully clarified.Sustained morphine treatment was shown to augment the concentrations of inflammatory mediators, such as PGE2 in the spinal cord. Studies have shown that PGE2 stimulates cAMP formation and CGRP release by activation of Gs protein-coupled prostaglandin receptor types in primary sensory neurons. Interestingly, it was found earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). It is well demonstrated that cAMP activates cAMP-dependent protein kinase (PKA), which plays an important role in the modulation of presynaptic neurotransmitter release. Therefore, in this study, we investigate the physiological role of Raf-1 mediated AC superactivation and subsequent PKA activation in A. sustained morphine-mediated augmentation of basal or evoked pain neurotransmitter release in vitro, in cultured primary sensory neurons, and B. in vivo, in sustained morphine mediated paradoxical pain sensitization and antinociceptive tolerance in rats.Our data demonstrates that A. sustained morphine treatment augments both basal and capsaicin-evoked CGRP release from isolated primary sensory neurons in a PKA- and Raf-1- dependent manner. B. sustained morphine treatment- augments of PGE2-evoked CGRP release from these cells. C. selective knockdown of spinal PKA or Raf-1 protein levels by intrathecal PKA- or Raf-1-specific siRNA pretreatment completely attenuates sustained morphine-mediated thermal hyperalgesia, tactile allodynia and greatly reduces antinociceptive tolerance in rats.In conclusion, we suggest that Raf-1-mediated AC superactivation may have a crucial trigger role in sustained morphine-mediated compensatory adaptations in the nervous system. Thus, we expect that pharmacological attenuation of Raf-1-mediated AC superactivation may improve the clinical treatment of chronic and neuropathic pain.

Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP binding proteins found in archaea, plants, fungi and animals. Their function is poorly understood but they are implicated in cell division, proliferation, and growth, as well as several human medical conditions. The research reported here has utilised a variety of approaches including structural biology, biochemistry, expression profiling, and mutant analysis in order to investigate the cellular function of DRG proteins in plants. Recombinant, biologically active atDRG1 and atDRG2 protein from Arabidopsis thaliana was purified using in vitro refolding and was used in both structural studies and biochemical analysis. Crystallographic studies were carried out for both atDRG1 and atDRG2 across 3840 unique, independent crystallisation conditions for each protein. Heterogeneous nucleation was also used in a separate crystallography screen in order to induce nucleation and subsequent crystal growth however no diffraction quality protein crystal were produced in this study. The nucleotide binding and hydrolysis properties of recombinant atDRG1 and atDRG2 were measured in vitro, representing the first biochemical characterisation of DRG proteins. Both atDRG1 and atDRG2 were found to bind GDP and GTP in vitro without the assistance of exogenous exchange or activation factors. The Kcat for GTP hydrolysis by atDRG1 and atDRG2 was found to be 7.44 x 10-4 min-1 and 1.18 x 10-3 min-1 respectively which is consistent with proteins related to the DRG subfamily. An Arabidopsis thaliana atDRG2a knockout mutant was identified and characterised in this study as well representing the first DRG knockout mutant in a multicellular organism. We found that complete knockout of atDRG2a is not lethal in Arabidopsis and that the nearly identical atDRG2b protein is not upregulated in response to an absence of atDRG2a in the cell. The mutant did not display an obvious phenotype compared to wild-type. The expression profiles of the three Arabidopsis thaliana drg genes, drg1, drg2a, and drg2b, were characterised using drg promoter:GUS Arabidopsis transgenics and revealed several interesting features. Under normal conditions, drg1 and drg2a transcripts are present in all cells whilst drg2b transcripts are undetectable. When heat stress is applied, drg2b and drg1 are specifically up regulated and drg2a is not. During seed imbibition, drg2a and drg1 are specifically upregulated whilst drg2b is not. The expression pattern of the drg family closely mirrors that of chaperone/heat shock proteins and this would agree with previous research that suggests that DRG2a may perform a chaperone role. The ability of DRGs to bind nucleotides without assistance, their slow rate of GTP hydrolysis, heat stress activation, abundance in seeds, cytosolic localization, and domain conservation, all agree with the models proposed for spoOB associated G-protein (Obg) function, whereby Obgs stabilise or refold ribosomes or other proteins in response to stress. It is possible that DRGs perform a similar and complementary function to Obgs, specifically during heat stress, despite the low level of sequence conservation between Obgs and DRGs.

No description available.

No description available.

No description available.

No description available.

The thesis, Survey of the methods of costing of hospitalization, deals with the characteristics of the classification system DRG used for costing of hospitalization. The first half of the theoretical part of this work describes the Czech health care in general and specific ways of financing it. We focus on the costs related to the emergency care in hospitals which are classified by the DRG system. The second half of the theoretical part studies the actual principles of costing of hospitalization. The method chosen for this is called Activity Based Costing (ABC). It defines the procedures of costing which are being used in hospitals. At the end, we provide a description of the current and suggested procedures of costing of hospitalization and how they are utilized to set up parameters of the reimbursing mechanism. The practical part of this thesis maps the situation of hospitalization financing in Klatovska nemocnice, a. s. We suggest a new way of financing based on the methodology DRG Restart. Base on the data obtained in Klatovska nemocnice, a. s. we summarize and compare the results of the new method to the current one.

La douleur est perçue par des neurones spécialisés, les nocicepteurs, dont le corps cellulaire est localisé, au niveau du tronc, dans les ganglions de la racine dorsale (DRG). Ces neurones détectent les informations sensorielles en périphérie (peau, muscles ou viscères) et les transmettent aux neurones spinaux qu'ils connectent au niveau de la corne dorsale de la moelle épinière. D'un point de vue morphologique, anatomique, physiologique, mais également moléculaire, une hétérogénéité importante de ces neurones est observée. Le but principal du laboratoire est de trouver de nouvelles molécules impliquées dans les mécanismes moléculaires qui spécifient les différentes sous-populations neuronales des DRG. Dans ce cadre, il a été possible d'identifier et valider plusieurs gènes présentant un profil d'expression très particulier et spécifiant des populations neuronales bien distinctes au sein des DRG. Parmi ces gènes, tafa4 est principalement exprimé dans des neurones non-peptidergiques de type C. Tafa4 est une petite protéine sécrétée proche des chemokines de type CC dont la fonction est jusqu'à présent inconnue, et dont l'expression dans les DRG n'a encore jamais été décrite. Au cours de ce travail, j'ai pu identifier Tafa4 comme un nouveau marqueur d'une sous-population de neurones sensoriels des DRG : les C-LTMRs (C-Low Threshold MechanoReceptor). La génération d'une lignée de souris Tafa4 KO dans laquelle le gène tafa4 a été remplacé par la protéine fluorescente Vénus, nous a permis de mettre en évidence que la population de neurones tafa4+ projette en central dans la lamina II interne de la moelle épinière et en périphérique exclusivement au niveau de la peau poilue. / The perception of pain is initiated by the detection of noxious stimuli by the peripheral endings of primary nociceptive neurons. They are a specialized group of small-diameter pseudounipolar neurons with cell bodies in the dorsal roots ganglia (DRG). They give rise to thinly myelinated (Ad-fibers) or unmyelinated (C-fibers) afferent fibers, which convey the signal from the periphery to the dorsal horn of the spinal cord. Our laboratory is interested in molecular mechanisms which underlie the specification of somatic sensory neurons and their properties. In order to find novel molecular factors involved in this process, we identified several new nociceptor subtype specific genes by microarray experiments. Among these genes, tafa4 which encodes a small secreted protein distantly related to CC chemokine with unknown function, appears to have a DRG-specific expression from early developmental stages and becomes restricted to a subset of C-fibers non-peptidergic nociceptors in adult DRG. By using transgenic mice, we show that Tafa4 neurons specifically project to the dorsal horn lamina IIi and innervate the hairy skin. They have electrophysiological signature of C-Low-threshold mechanoreceptors (C-LTMRs), a population of sensory neurons implicated in the injury-induced mechanical hyper-sensitivity as well as in the affective component of touch. Mutant mice lacking Tafa4 do not present developmental defects and specify Tafa4 population correctly. However, despite no obvious molecular changes in Tafa4 mutants, these mice display significant increase in tissue injury induced hyper-sensitivity which could be reduced by intrathecally applied Tafa4 protein.

No description available.

Les canaux de type T Cav3.2 sont des canaux calciques activés pour de faibles dépolarisations membranaires. Ils ont un rôle important dans la régulation de l’excitabilité neuronale, particulièrement dans les neurones des ganglions rachidiens dorsaux (DRG) où ils sont impliqués dans la transmission de la douleur. Il est établi que les canaux Cav3.2, natifs et recombinants, sont inhibés par de faibles concentrations de métaux divalents tels que le zinc et le nickel et qu’ils sont modulés par des agents oxydo-réducteurs. In vitro, la mutation ponctuelle de l’histidine 191 en glutamine (H191Q) diminue fortement la sensibilité du canal Cav3.2 pour ces différents composés et il est proposé que cette régulation joue un rôle physiologique. L’objectif de ce travail de thèse a été d’étudier l’impact physiologique de cette modulation sur l’excitabilité neuronale et dans la perception de la douleur. Pour ce faire, nous avons généré une souris knock-in (KI) portant la mutation H191Q sur Cav3.2. L’étude électrophysiologique a été réalisée sur une population de neurones de DRG particulière : les cellules D-hair qui sont des mécanorécepteurs exprimant de grands courants Cav3.2. Nous avons validé que la sensibilité des canaux Cav3.2 neuronaux des souris KI est diminuée pour le zinc, le nickel et l’ascorbate. Nous montrons que cette régulation modifiée favorise une augmentation de l’excitabilité de ces neurones. Pour étudier l’impact de cette modulation in vivo, nous avons effectué des études comportementales. Les souris KI ne présentent pas de différence dans la perception de la douleur mécanique et thermique, ni dans l’hyperalgésie induite par l’inflammation et la neuropathie. Toutefois, dans le test à la formaline les souris KI montrent une réponse exacerbée dans la phase tardive. En résumé, nous décrivons ici un modèle animal original pour l’étude de la régulation metal/redox du canal Cav3.2 et identifions un rôle de cette modulation dans l’excitabilité des neurones D-Hair. Nos résultats obtenus in vivo indiquent cependant que cette modulation des canaux Cav3.2 aurait un impact limité dans les voies de la douleur. / Cav3.2 T-type channels are low-voltage activated calcium channels. They have an important role in the regulation of neuronal excitability, particularly in neurons of the dorsal root ganglia (DRG) where they are involved in pain transmission. It is established that Cav3.2 channels are inhibited by low concentrations of divalent metals such as zinc and nickel, and are modulated by redox agents. In vitro, the histidine191-to-glutamine mutation (H191Q) greatly reduces the Cav3.2 channel sensitivity to these compounds and it is proposed that this regulation plays a physiological role. The objective of this thesis was to study the physiological impact of this modulation on neuronal excitability and pain perception. To do this, we generated a knock-in (KI) mouse carrying the H191Q mutation on Cav3.2. Electrophysiological study was carried out on a particular population of DRG neurons, the D-hair cells, which are mechanoreceptors that express large Cav3.2 currents. We show that the sensitivity to zinc, nickel and ascorbate of the neuronal Cav3.2 channels is significantly reduced in the KI mouse. We also show that this modified regulation promotes an increase in the excitability of these neurons. To study the impact of this modulation in vivo, we performed behavioral studies. KI mice show no difference in the perception of mechanical and thermal pain, nor in hyperalgesia induced by inflammation and neuropathy. However, KI mice show an exaggerated response in the late phase in the formalin test. In summary, we describe here an original animal model to study the metal/redox regulation of Cav3.2 channel and identify a role of this modulation in the excitability of D-Hair neurons. Our results indicate, however, that this modulation of Cav3.2 channel may have a limited impact in the pain pathways.