Short-term memory of temporal aspects of noxious and innocuous thermal sensation : psychophysical and fMRI studies

La douleur peut être considérée comme un système de protection qui signale une menace et qui nous avertit des dégâts imminents aux tissus. En tant que mécanisme de défense, il nécessite l'apprentissage et la mémoire des expériences du passé pour la survie et les comportements liés à la douleur. Par conséquent, notre expérience de la douleur actuelle est fortement influencée par les expériences antérieures et l'apprentissage. Cependant, malgré son importance, notre compréhension actuelle de l'interaction entre le système de la douleur et le système de mémoire est très limitée. La mémoire de la douleur est un sujet de recherche très vaste. Il nécessite une compréhension des mécanismes impliqués à chaque étape du système de mémoire (mémoire immédiate, à court terme et à long terme) et l'interaction entre eux. Parmi les étapes multiples de la mémoire, la mémoire à court terme de la douleur est une zone qui est moins recherchée, alors qu'il existe une énorme quantité de recherche neuroscientifique dans la mémoire à court terme sur d'autres modalités, en particulier la vision. L'étude de la mémoire à court terme de la douleur est particulièrement importante car cette trace de la mémoire à court terme de la douleur est ensuite convertie en mémoire à long terme et affecte ensuite les expériences futures de la douleur. Cette thèse est largement axée sur la mémoire à court terme de la douleur. La complexité et la multi dimensionnalité de la douleur ajoutent encore un autre élément à la recherche sur la mémoire de la douleur. Par exemple, la trace de la mémoire de la douleur peut contenir des traces de mémoire de diverses composantes de la douleur telles que la réponse sensorielle affective, cognitive et motrice et l'interaction entre elles. Par conséquent, une première étape dans l'exploration neuroscientifique de la mémoire de la douleur nécessite la réduction de l'expérience de la douleur tout en englobant tous ces différents composants à un seul composant. Dans la recherche présentée ici, nous avons généralement examiné cela par des instructions d'attention ‘ top-down’ pour assister à la dimension sensorielle de la douleur. La recherche précédente sur la mémoire à court terme de la douleur a également porté principalement sur la dimension sensorielle de la douleur. Cependant, parmi les dimensions sensorielles de la douleur, la mémoire à court terme de l'intensité et de la dimension spatiale de la douleur a fait l'objet de recherches antérieures. Malgré son importance, la dimension temporelle de la douleur est restée complètement inexplorée dans la recherche sur la mémoire de la douleur. La recherche menée dans cette thèse est consacrée à l'exploration de la mémoire à court terme de la durée de la douleur. La durée de la douleur peut être suivie de manière indépendante, mais peut également être suivie conjointement avec la dimension d'intensité telle que le suivi dynamique de l'intensité de la douleur dans le temps. Les études menées dans cette thèse traitent spécifiquement du traitement isolé de la durée de la douleur ainsi que du traitement conjoint de la dimension durée / intensité de la douleur. La première étude psychophysique a exploré la nature de la représentation mentale du modèle de mémoire de la douleur thermique dynamique et a également été conçue pour aborder les différences de la dimension sensorielle et affective de la douleur thermique dans la mémoire à court terme. La deuxième étude psychophysique portait sur les propriétés de la mémoire à court terme de la sensation thermique non douloureux en comparant le suivi dynamique de la sensation et le suivi isolé de la durée d'un événement thermique non douloureux. La troisième étude poursuit l'exploration du traitement dynamique de la durée conjointement avec l'intensité par rapport au traitement isolé de la durée dans la mémoire à court terme en utilisant des stimuli thermiques douloureuse une résonance magnétique fonctionnelle (IRMF). Dans l'ensemble, les résultats des études psychophysiques ont montré une transformation significative de la durée et de la dynamique de la sensation thermique douloureux et non-douloureux dans la mémoire à court terme; comme la perte d'informations somatosensorielles temporelles en mémoire. Nous avons en outre montré une amélioration du rappel de la durée dans le suivi dynamique de la durée, en comparaison avec le suivi de la durée isolée. Nous avons également montré des différences dans les corrélats neuronaux de la mémoire à court terme de la durée de douleur par rapport à la dynamique de douleur. L'étude de l'IRMF a montré des similitudes frappantes dans les corrélats neuronaux sous-jacents à la mémoire à court terme de douleur et d'autres modalités telles que la contribution des coticés fronto-pariétales ainsi que les corticaux sensoriels impliqués dans le traitement perceptuel.Pain can be viewed as a protective system that signals threat and alerts us to impending tissue damage. As a defense mechanism, it necessitates the learning and memory of past painful experiences for survival and pain-related behavior. Therefore our current pain experience is heavily influenced by previous experiences and learning. However, despite its importance, our current understanding of the interaction between the pain system and the memory system is very limited. Pain memory is a very broad topic of research on its own. It requires an understanding of the mechanisms involved at each stage of the memory system (immediate, short-term, and long-term memory), and the interaction among them. Among the multiple stages of memory, the short-term memory of pain is an area that is less researched, while there are enormous amount of neuroscientific research in short-term memory of other modalities, particularly vision. Investigation of the short-term memory of pain is especially important as the short-term memory trace of pain is converted to long-term memory and subsequently affects future pain experiences. This thesis is broadly focused on the short-term memory of pain. The complexity and multi-dimensionality of pain adds yet another element to the research on pain memory. For example, the memory trace of pain may contain memory traces of various components of pain such as sensory, affective, cognitive, and motoric responses, and the interactions among them. Therefore, an initial step in the neuroscientific exploration of pain memory requires narrowing down the pain experience, which encompasses all of these various components, to one single component. In the research presented here, we achieved this using top-down attentional instructions to attend to the sensory component of pain. The previous research on short-term memory of pain also focused mainly on the sensory component of pain. However, within the sensory component of pain the short-term memory of intensity and spatial dimension of pain has been the focus of previous research. Despite its importance, the temporal dimension of pain remained completely unexplored in pain memory research. Thus, the research conducted in this thesis is devoted to the exploration of short-term memory of the duration of pain. Pain duration can be tracked independently, but it can also be tracked conjointly with intensity, such as in dynamic tracking of pain intensity over time. The studies addressed in this thesis examined the isolated processing of pain duration as well as conjoint processing of the duration and intensity of pain. The first psychophysical study explored the nature of the mental representation of the memory template of dynamic thermal pain sensation and, additionally, addressed the differences between the sensory versus affective dimensions of thermal pain sensation in short-term memory. The second psychophysical study focused on properties of the short-term memory of innocuous thermal sensation by comparing dynamic tracking of sensation versus isolated tracking of duration of an innocuous thermal event. The third study explored the dynamic processing of duration conjointly with intensity, versus the isolated processing of duration in short-term memory, using noxious thermal stimuli and functional magnetic resonance imaging (fMRI). Overall, the results of the psychophysical studies showed significant transformation of duration and dynamics information of noxious and innocuous thermal sensation in short-term memory, such as loss of temporal somatosensory information. Additionally, we showed improvement in duration recall during dynamic tracking versus isolated tracking of duration. The fMRI study revealed differences in neural correlates of short-term memory of pain duration versus pain dynamics. Importantly, it also showed striking similarities between neural correlates underlying the short-term memory of pain and those underlying other modalities, such as a contribution of fronto-parietal cortices as well as sensory cortices involved in perceptual processing

Intra- and inter-hemispheric interactions in somatosensory processing of pain : dynamical causal modeling analysis of fMRI data

La douleur est une expérience perceptive comportant de nombreuses dimensions. Ces dimensions de douleur sont inter-reliées et recrutent des réseaux neuronaux qui traitent les informations correspondantes. L’élucidation de l'architecture fonctionnelle qui supporte les différents aspects perceptifs de l'expérience est donc une étape fondamentale pour notre compréhension du rôle fonctionnel des différentes régions de la matrice cérébrale de la douleur dans les circuits corticaux qui sous tendent l'expérience subjective de la douleur. Parmi les diverses régions du cerveau impliquées dans le traitement de l'information nociceptive, le cortex somatosensoriel primaire et secondaire (S1 et S2) sont les principales régions généralement associées au traitement de l'aspect sensori-discriminatif de la douleur. Toutefois, l'organisation fonctionnelle dans ces régions somato-sensorielles n’est pas complètement claire et relativement peu d'études ont examiné directement l'intégration de l'information entre les régions somatiques sensorielles. Ainsi, plusieurs questions demeurent concernant la relation hiérarchique entre S1 et S2, ainsi que le rôle fonctionnel des connexions inter-hémisphériques des régions somatiques sensorielles homologues. De même, le traitement en série ou en parallèle au sein du système somatosensoriel constitue un autre élément de questionnement qui nécessite un examen plus approfondi. Le but de la présente étude était de tester un certain nombre d'hypothèses sur la causalité dans les interactions fonctionnelle entre S1 et S2, alors que les sujets recevaient des chocs électriques douloureux. Nous avons mis en place une méthode de modélisation de la connectivité, qui utilise une description de causalité de la dynamique du système, afin d'étudier les interactions entre les sites d'activation définie par un ensemble de données provenant d'une étude d'imagerie fonctionnelle. Notre paradigme est constitué de 3 session expérimentales en utilisant des chocs électriques à trois différents niveaux d’intensité, soit modérément douloureux (niveau 3), soit légèrement douloureux (niveau 2), soit complètement non douloureux (niveau 1). Par conséquent, notre paradigme nous a permis d'étudier comment l'intensité du stimulus est codé dans notre réseau d'intérêt, et comment la connectivité des différentes régions est modulée dans les conditions de stimulation différentes. Nos résultats sont en faveur du mode sériel de traitement de l’information somatosensorielle nociceptive avec un apport prédominant de la voie thalamocorticale vers S1 controlatérale au site de stimulation. Nos résultats impliquent que l'information se propage de S1 controlatéral à travers notre réseau d'intérêt composé des cortex S1 bilatéraux et S2. Notre analyse indique que la connexion S1→S2 est renforcée par la douleur, ce qui suggère que S2 est plus élevé dans la hiérarchie du traitement de la douleur que S1, conformément aux conclusions précédentes neurophysiologiques et de magnétoencéphalographie. Enfin, notre analyse fournit des preuves de l'entrée de l'information somatosensorielle dans l'hémisphère controlatéral au côté de stimulation, avec des connexions inter-hémisphériques responsable du transfert de l'information à l'hémisphère ipsilatéral.Pain is a perceptual experience comprising many dimensions. These pain dimensions interrelate with each other and recruit neuronal networks that process the corresponding information. Elucidating the functional architecture that supports different perceptual aspects of the experience is thus, a fundamental step to our understanding of the functional role of different regions in the cerebral pain matrix that are involved in the cortical circuitry underlying the subjective experience of pain. Among various brain regions involved in the processing of nociceptive information, primary and secondary somatosensory cortices (S1 and S2) are the main areas generally associated with the processing of sensory-discriminative aspect of pain. However the functional organization in these somatosensory areas is not completely clear and relatively few studies have directly examined the integration of information among somatic sensory regions. Thus, several questions remain regarding the hierarchical relationship between S1 and S2, as well as the functional role of the inter-hemispheric connections of the homologous somatic sensory areas. Likewise, the question of serial or parallel processing within the somatosensory system is another questionable issue that requires further investigation. The purpose of the present study was to test a number of causal hypotheses regarding the functional interactions between S1 and S2, while subjects were receiving painful electric shocks. We implemented a connectivity modeling approach, which utilizes a causal description of system dynamics, in order to study the interactions among activation sites defined by a data set derived from a functional imaging study. Our paradigm consists of 3 experimental scans using electric shock stimuli, with the stimulus intensity changing from moderately painful (level 3), to slightly painful (level 2), and to completely non-painful (level 1) during the final scan. Therefore our paradigm allowed us to investigate how stimulus intensity is encoded within our network of interest, and how the connectivity of the different regions is modulated across the different stimulus conditions. Our result is in favor of serial mode of somatosensory processing with thalamocortical input to S1 contralateral to stimulation site. Thus our results implicates that pain information is propogated from S1 contralateral through our network of interest comprising of bilateral S1 and S2. Our analysis indicates that S1→S2 connection is modulated by pain, which suggests that S2 is higher on the hierarchy of pain processing than S1, in accordance with previous neurophysiological and MEG findings. Lastly, our analysis provides evidence for the entrance of somatosensory information into the hemisphere contralateral to the stimulation side, with inter-hemispheric connections responsible for the transfer of information to the ipsilateral hemisphere

Feedforward and feedback pathways of nociceptive and tactile processing in human somatosensory system: A study of dynamic causal modeling of fMRI data

Nociceptive and tactile information is processed in the somatosensory system via reciprocal (i.e., feedforward and feedback) projections between the thalamus, the primary (S1) and secondary (S2) somatosensory cortices. The exact hierarchy of nociceptive and tactile information processing within this ‘thalamus-S1-S2’ network and whether the processing hierarchy differs between the two somatosensory submodalities remains unclear. In particular, two questions related to the ascending and descending pathways have not been addressed. For the ascending pathways, whether tactile or nociceptive information is processed in parallel (i.e., 'thalamus-S1′ and 'thalamus-S2′) or in serial (i.e., 'thalamus-S1-S2′) remains controversial. For the descending pathways, how corticothalamic feedback regulates nociceptive and tactile processing also remains elusive. Here, we aimed to investigate the hierarchical organization for the processing of nociceptive and tactile information in the ‘thalamus-S1-S2’ network using dynamic causal modeling (DCM) combined with high-temporal-resolution fMRI. We found that, for both nociceptive and tactile information processing, both S1 and S2 received inputs from thalamus, indicating a parallel structure of ascending pathways for nociceptive and tactile information processing. Furthermore, we observed distinct corticothalamic feedback regulations from S1 and S2, showing that S1 generally exerts inhibitory feedback regulation independent of external stimulation whereas S2 provides additional inhibition to the thalamic activity during nociceptive and tactile information processing in humans. These findings revealed that nociceptive and tactile information processing have similar hierarchical organization within the somatosensory system in the human brain

Synthesis and Evaluation of Influenza Virosome Gene Delivery and LCP-Based Hookworm Vaccine Delivery Systems

Delivery is very crucial in the development of therapeutic medicine. Therapeutic agents (genes, oligonucleotides, proteins, peptides, vaccines, drugs,…) are ineffective if they can not reach their desired site of action. Liposomes, polymers, micelles, and various micro- and nanoparticle delivery systems have been designed to try to overcome the delivery challenges. Here, two different delivery systems were pursued, one being modified influenza virosomes for gene delivery and the other a lipid-core-peptide (LCP) carrier system for hookworm vaccine delivery. Gene therapy has immense therapeutic potential, however, some of the obstacles holding back its clinical use include: low transfection efficiency, short-term gene expression, immunogenicity, and toxicity. Safe and efficient delivery system is required to overcome most of the complications. We decided to take advantage of the efficient delivery mechanisms of viruses and modify them to suit our requirements. Influenza (H1N1 A/NC/20/99) virosomes (empty viruses) were encapsulated with Nuclear Localization Signal (NLS) tetrameric peptide and plasmid DNA complexes. The virosomes were then coated with acid-labile distearoyl glycerol-orthoester-PEG(Polyethylene Glycol)5000-folate conjugates. The pH-sensitive Lipid-PEG-folate coating was designed to help evade the immune system and in the mean time to target it to the desired cells. The orthoester pH-sensitive bond undergoes hydrolysis in the acidic endosomal pH and dePEGylates to present the fusion proteins and allow for fusion and endosomal escape. The pH-sensitive Lipid-PEG-Folate was synthesized and characterized by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). The virosomes were characterized by protein and DNA quantitation, SDS-PAGE, and Transmission Electron Microscopy (TEM). The hydrolysis rate of the pH-sensitive conjugate was examined using RP-HPLC to determine the time required for dePEGylation at acidic pH. 60-70% degradation of the conjugate was observed in 15 min at pH 4 and pH 5. At pH 7.4 the conjugates were stable at 15 min, however, hydrolyzed in 30 min with up to 80% degradation. The hydrolysis rate was found to be much faster than expected. The virosomes with peptide/DNA complexes (charge ratio +/−1) were observed under TEM as heterogeneous mixture with most around 90nm in diameter. Confocal microscopy analysis was carried out to examine green fluorescent protein (GFP) expression of cells transfected using virosomes. It was determined that the virosomes had very strong fluorescence and transfection efficiency in HeLa cells. Histidine-rich peptide gene delivery is another interesting area of research that we also pursued. The synthesis and disulfide conjugation of histidine-rich peptide to Cys-Gly-PEG5kD-Folate was performed. The probable conjugate was observed by analytical RP-HPLC, however, characterization by MALDI was difficult. The transfection study showed that polyhistidine peptide/DNA complexes at charge ratio +/−10 were the most effective. Human hookworm infections are a major health burden affecting more than 700 million people. Currently, there are no human hookworm vaccines on the market. For our hookworm vaccine design we selected three helminth antigenic epitopes (Na-APR-1, Na-APR-2, and Sm-Cat-D). The three epitopes are the antibody binding regions in nematode hemoglobinases. The rational for the vaccine design was to generate antibodies to neutralize the nematode hemoglobinases and starve the hookworm to death. The self-adjuvanting Lipid-Core-Peptide (LCP) design was utilized for vaccine synthesis. Twelve vaccine constructs were synthesized: six peptides and six Lipid-Core Peptides (LCPs). The immunological studies are currently underway by our collaborator. The gene and vaccine delivery systems we have developed here both have great therapeutic potential. The modified influenza virosomes synthesized here have the potential for in vivo use for shielding the virosomes while dePEGylating at endosomal pH 4-5 in around 10-15 min. This is a very promising area of research that is emerging. There are so many various modifications that can be made to a whole range of virosomes and virus-like particles that could take gene therapy to new heights. Both the modified virosomes and LCP designs are potentially powerful delivery systems which could be great assets to the medical field

Mixed-cropping and Its Effects on Yield and Agronomical Traits of Barley (Hordeum vulgar L.) and Bersim Clover (Trifolium alexanderium L.)

Mixed-cropping of barley (Hordeum vulgar L.) and clover (Trifolium alexanderium L.) was studied in a randomized complete block design with 4 replications to detect their best planting patterns at Agriculture Research Center of Miandoab. The experiment comprised of 10 treatments: 9B:1C (nine rows of barley: one row of annual clover), 8B:2C, 7B:3C, 6B:4C, 5B:5C, 4B:6C, 3B:7C, 2B:8C, 1B:9C and sole croppings of each plants. The results indicated that intercropping as a whole had a significant effect on number of stem, plant height and yield of clover. On other hands, the highest clover yields (8.3 t/ha) was achieved in sole cropping and lowest (1.2 t/ha) in 10:90 (Barley: Clover) treatment. Results also showed that spike length, number of seed per spike, 1000-grain weight, protein percent, economical yield and biological yield were affected by mixed-cropping. Reducing planting rows of barley relative to clover caused 87% reduction in both economical and biological yields. 1000-grain weight also decreased from 45.82 to 39g. Total LER was 1 in all of patterns and in 40:60 (barley: clover) pattern was the highest. LER calculated based on forage in two crops was similar to LER which achieved on seed yield of barley and forage yield of clover. Decreasing in rows of barley diminished LER t

Modified Influenza Virosomes: Recent Advances and Potential in Gene Delivery

Influenza virosomes have proven to be effective vehicles for the delivery of antigens in the vaccination of humans against a number of pathogens. However, their potential as a means for gene delivery has yet to be realized. Chemical modification of viruses is emerging as a new strategy for production of safe and efficient gene delivery systems. Influenza virosomes exhibit many of the features of the virus, such as for cell binding, uptake and endosomal escape, which can be easily engineered into designer delivery vehicles capable of safe, efficient and cell-specific cargo delivery. This review focuses on the next generation of influenza virosomes and highlights aspects of their modification that may lead to simple but effective gene delivery vehicles

Lipid core peptide targeting the cathepsin D hemoglobinase of Schistosoma mansoni as a component of a schistosomiasis vaccine

The self-adjuvanting lipid core peptide (LCP) system offers a safe alternative vaccine delivery strategy, eliminating the need for additional adjuvants such as CpG Alum. In this study, we adopted the LCP as a scaffold for an epitope located on the surface of the cathepsin D hemoglobinase (Sm-catD) of the human blood fluke Schistosoma mansoni. Sm-catD plays a pivotal role in digestion of the fluke's bloodmeal and has been shown to be efficacious as a subunit vaccine in a murine model of human schistosomiasis. Using molecular modeling we showed that S. mansoni cathepsin D possesses a predicted surface exposed α-helix (A 263K) that corresponds to an immunodominant helix and target of enzyme-neutralizing antibodies against Necator americanus APR-1 (Na-APR-1), the orthologous protease and vaccine antigen from blood-feeding hookworms. The A 263K epitope was engineered as two peptide variants, one of which was flanked at both termini with a coil maintaining sequence, thereby promoting the helical characteristics of the native A 263K epitope. Some of the peptides were fused to a self-adjuvanting lipid core scaffold to generate LCPs. Mice were vaccinated with unadjuvanted peptides, peptides formulated with Freund's adjuvants, or LCPs. Antibodies generated to LCPs recognized native Sm-catD within a soluble adult schistosome extract, and almost completely abolished its enzymatic activity in vitro. Using immunohistochemistry we showed that anti-LCP antibodies bound to the native Sm-catD protein in the esophagus and anterior regions of the gastrodermis of adult flukes. Vaccines offer an alternative control strategy in the fight against schistosomiasis, and further development of LCPs containing multiple epitopes from this and other vaccine antigens should become a research priority