Phenotypic and molecular characterization of the onset of neuropathy in rodent models of diabetes mellitus

Abstract : The principal focus of this work was to study the molecular changes leading to the development of diabetic peripheral neuropathy (DPN). DPN is the most common complication associated with both type I and II diabetes mellitus (DM). This pathology is the leading cause of non-traumatic amputations. Even though the pathological and morphological changes underlying DPN are relatively well described, the implicated molecular mechanisms remain poorly understood. The following two approaches were developed to study the development of DPN in a rodent model of DM type I. As a first approach, we studied the implication of lipid metabolism in DPN phenotype, concentrating on Sterol Response Element Binding Protein (SREBP)-lc which is the key regulator of storage lipid metabolism. We showed that SREBP-1c was expressed in peripheral nerves and that its expression profile followed the expression of genes involved in storage lipid metabolism. In addition, the expression of SREBP-1c in the endoneurium of peripheral nerves was dependant upon nutritional status and this expression was also perturbed in type I diabetes. In line with this, we showed that insulin elevated the expression of SREBP-1c in primary cultured Schwann cells by activating the SREBP-1c promoter. Taken together, these findings reveal that SREBP-1c expression in Schwann cells responds to metabolic stimuli including insulin and that this response is affected in type I diabetes mellitus. This suggests that disturbed SREBP-1c regulated lipid metabolism may contribute to the pathophysiology of DPN. As a second approach, we performed a comprehensive analysis of the molecular changes associated with DPN in the Akital~1~+ mouse which is a model of spontaneous early-onset type I diabetes mellitus. This mouse expresses a mutated non-functional isoform of insulin, leading to hypoinsulinemia and hyperglycaemia. To determine the onset of DPN, weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Akital+/+ mice during the first three months of life. A decrease in MNCV was evident akeady one week after the onset of hyperglycemia. To explore the molecular changes associated with the development of DPN in these mice, we performed gene expression profiling using sciatic nerve endoneurium and dorsal root ganglia (DRG) isolated from early diabetic male Akita+/+ mice and sex-matched littermate controls. No major transcriptional changes were detected either in the DRG or in the sciatic nerve endoneurium. This experiment indicates that the phenotypic changes observed during the development of DPN are not correlated with major transcriptional alterations, but mainly with alterations at the protein level. Résumé Lors ce travail, nous nous sommes intéressés aux changements moléculaires aboutissant aux neuropathies périphériques dues au diabète (NPD). Les NPD sont la complication la plus commune du diabète de type I et de type II. Cette pathologie est une cause majeure d'amputations. Même si les changements pathologiques et morphologiques associés aux NPD sont relativement bien décrits, les mécanismes moléculaires provoquant cette pathologie sont mal connus. Deux approches ont principalement été utilisées pour étudier le développement des NPD dans des modèles murins du diabète de type I. Nous avons d'abord étudié l'impact du métabolisme des lipides sur le développement des NPD en nous concentrant sur Sterol Response Element Binding Protein (SREBP)-1 c qui est un régulateur clé des lipides de stockage. Nous avons montré que SREBP-1 c est exprimé dans les nerfs périphériques et que son profil d'expression suit celui de gènes impliqués dans le métabolisme des lipides de stockage. De plus, l'expression de SREBP-1c dans l'endoneurium des nerfs périphériques est dépendante du statut nutritionnel et est dérégulée lors de diabète de type I. Nous avons également pu montrer que l'insuline augmente l'expression de SREBP-1c dans des cultures primaires de cellules de Schwann en activant le promoteur de SREBP-1c. Ses résultats démontrent que l'expression de SREBP-1c dans les cellules de Schwann est contrôlée par des stimuli métaboliques comme l'insuline et que cette réponse est affectée dans le cas d'un diabète de type I. Ces données suggèrent que la dérégulation de l'expression de SREBP-1c lors du diabète pourrait affecter le métabolisme des lipides et ainsi contribuer à la pathophysiologie des NPD. Comme seconde approche, nous avons réalisé une analyse globale des changements moléculaires associés au développement des NPD chez les souris Akita+/+, un modèle de diabète de type I. Cette souris exprime une forme mutée et non fonctionnelle de l'insuline provoquant une hypoinsulinémie et une hyperglycémie. Afin de déterminer le début du développement de la NPD, le poids, le niveau de glucose sanguin et la vitesse de conduction nerveuse (VCN) ont été mesurés durant les 3 premiers mois de vie. Une diminution de la VCN a été détectée une semaine seulement après le développement de l'hyperglycémie. Pour explorer les changements moléculaires associés avec le développement des NPD, nous avons réalisé un profil d'expression de l'endoneurium du nerf sciatique et des ganglions spinaux isolés à partir de souris Akital+/+ et de souris contrôles Akita+/+. Aucune altération transcriptionnelle majeure n'a été détectée dans nos échantillons. Cette expérience suggère que les changements phénotypiques observés durant le développement des NPD ne sont pas corrélés avec des changements importants au niveau transcriptionnel, mais plutôt avec des altérations au niveau protéique. Résumé : Lors ce travail, nous nous sommes intéressés aux changements moléculaires aboutissant aux neuropathies périphériques dues au diabète (NPD). Les NPD sont la complication la plus commune du diabète de type I et de type II. Cette pathologie est une cause majeure d'amputations. Même si les changements pathologiques et morphologiques associés aux NPD sont relativement bien décrits, les mécanismes moléculaires provoquant cette pathologie sont mal connus. Deux approches ont principalement été utilisées pour étudier le développement des NPD dans des modèles murins du diabète de type I. Nous avons d'abord étudié l'impact du métabolisme des lipides sur le développement des NPD en nous concentrant sur Sterol Response Element Binding Protein (SREBP)-1c qui est un régulateur clé des lipides de stockage. Nous avons montré que SREBP-1 c est exprimé dans les nerfs périphériques et que son profil d'expression suit celui de gènes impliqués dans le métabolisme des lipides de stockage. De plus, l'expression de SREBP-1c dans l'endoneurium des nerfs périphériques est dépendante du statut nutritionnel et est dérégulée lors de diabète de type I. Nous avons également pu montrer que l'insuline augmente l'expression de SREBP-1c dans des cultures primaires de cellules de Schwann en activant le promoteur de SREBP-1c. Ses résultats démontrent que l'expression de SREBP-1c dans les cellules de Schwann est contrôlée par des stimuli métaboliques comme l'insuline et que cette réponse est affectée dans le cas d'un diabète de type I. Ces données suggèrent que la dérégulation de l'expression de SREBP-1c lors du diabète pourrait affecter le métabolisme des lipides et ainsi contribuer à la pathophysiologie des NPD. Comme seconde approche, nous avons réalisé une analyse globale des changements moléculaires associés au développement des NPD chez les souris Akita~~Z~+, un modèle de diabète de type I. Cette souris exprime une forme mutée et non fonctionnelle de l'insuline provoquant une hypoinsulinémie et une hyperglycémie. Afin de déterminer le début du développement de la NPD, le poids, le niveau de glucose sanguin et la vitesse de conduction nerveuse (VCN) ont été mesurés durant les 3 premiers mois de vie. Une diminution de la VCN a été détectée une semaine seulement après le développement de l'hyperglycémie. Pour explorer les changements moléculaires associés avec le développement des NPD, nous avons réalisé un profil d'expression de l'endoneurium du nerf sciatique et des ganglions spinaux isolés à partir de souris Akital+/+ et de souris contrôles Akita+/+. Aucune altération transcriptionnelle majeure n'a été détectée dans nos échantillons. Cette expérience suggère que les changements phénotypiques observés durant le développement des NPD ne sont pas corrélés avec des changements importants au niveau transcriptionnel, mais plutôt avec des altérations au niveau protéique.

Video Inspection of Highway Edgedrain Systems, HR-317, 1998

Minimizing infiltration of water in pavement structures has long been a priority of pavement designers. Incorporation of subsurface edgedrains is frequently an integral part of an pavement drainage system. In order for such a system to be effective however, it must be properly installed and maintained. With advances in video technology, inspection of edgedrain systems can now be conducted quite efficiently. This report documents the results of 287 video inspections of highway edgedrain systems in 29 states. These inspections were conducted to both demonstrate the capabilities of the technology as well as demonstrating some of the common problems associated with the performance of edgedrain systems. Findings indicated not only that the equipment was quite effective in identifying edgedrain performance concerns, but also how widespread the concerns of edgedrain performance are. Almost one third of the systems inspected had nonfunctional outlets, another third were either found to have non-functional mainlines or the mainlines could not be inspected due to physical obstructions. Only one third of the systems inspected were found to be performing as intended. Recommendations are provided for edgedrain design improvements to facilitate performance of the system and their inspections as well as recommendations to improve quality control during construction. Suggestions are also provided for maintenance procedures to address concerns identified in the inspection process. A Draft Guide Specification For Video Edgedrain Inspection and Acceptance is also provided as an Appendix.

Sensing pheromones : a role for the CNGA4 and TRPC2 proteins

Chez les mammifères, les phéromones sont des molécules clés dans la régulation des comportements sociaux au sein d'une espèce. Chez la souris, la détection de ces molécules se fait dans l'organe voméronasal (VNO] et implique le canal TRPC2 afin de dépolariser les neurones. Des différences de comportement entre des souris Trpc2-/- et des souris sans VNO suggèrent l'implication d'une autre protéine effectrice dans la voie de signalisation des phéromones. L'hypothèse étant que cette protéine formerait un canal hétéromérique avec TRPC2. CNGA4 est une protéine sans fonction connue dans le VNO des rongeurs. Elle appartient à la famille des protéines CNG qui joue un rôle important dans différentes voies de signalisation comme la vision ou l'olfaction. Etant donné sa présence dans le VNO, son rôle inconnu dans cet organe et son rôle important dans de nombreuses voies de signalisation, nous avons décidé d'étudier CNGA4 afin de connaître sa localisation, ses propriétés ou encore sa structure. Nous avons découvert que CNGA4 est exprimée dans les axons, les neurones immatures ainsi que sur les microvillosités des neurones de VNO. A l'aide de souris portant une version non fonctionnelle de CNGA4, nous avons pu montrer que cette protéine joue un rôle majeur dans la voie de signalisation des phéromones. Ainsi, les neurones du VNO portant une version non fonctionnelle de CNGA4 répondent moins fréquemment aux phéromones et par conséquent les phéromones activent également moins de neurones dans le bulbe olfactif accessoire, premier relais du VNO avec le cortex. Cette détection défaillante se traduit par une absence d'agressivité des souris mutantes ainsi que par une incapacité de ces souris à discriminer le sexe de leur conspécifique. Etant donné les propriétés similaires de CNGA4 et de TRPC2, nous avons supposé que les deux protéines pourraient interagir. Cette hypothèse a été confortée par l'observation que CNGA4 n'est plus exprimée dans les microvillosités du VNO des souris Trpc2-/-. A l'aide d'expériences d'expression hétérologue, nous avons pu observer que les deux protéines interagissent et forment un canal activé par un analogue du diacylglycérol suggérant que ce canal est fonctionnel. Ces résultats indiquent que CNGA4 formerait un canal hétéromérique avec TRPC2 et aurait dans ce canal une fonction modulatrice. Des expériences complémentaires sont nécessaires afin de connaître le rôle de chacune de ces protéines dans la voie de signalisation des phéromones. Sensing pheromones: a role for the CNGA4 and TRPC2 proteins Mammalian pheromones are key chemical signals in the regulation of intraspecies social behaviors. Detection of these pheromones, which takes place in sensory neurons of the vomeronasal organ (VNO), implies the activation of the transient receptor potential canonical channel 2 (TRPC2) as the final effector. Interestingly, discrepancies between Trpc2 /- mice and mice lacking a VNO suggest the implication of another protein in the pheromone signaling pathway. This protein could either form a heteromeric channel with TRPC2 or a separate homomeric ion channel. The cyclic nucleotide-gated channel subunit CNGA4 is also expressed in the rodent VNO but its role and properties in this organ remain unknown. CNGA4 belongs to the CNG channel family which is playing an important role in different sensory pathways such as in light and odorant detection. We thus decided to study the role of the CNGA4 protein in the mouse VNO. We found CNGA4 to be expressed in axons, dendrites and in the sensory microvilli. Using mice bearing a non-functional form of CNGA4 we further demonstrated the importance of the CNGA4 protein for the pheromone signaling pathway as neurons from mutant mice were responding less frequently to chemosensory cues. As a result, mutant mice displayed a non-aggressive behavior and an impaired sexual discrimination ability. Based on the CNGA4 localization and its role in the pheromone signaling pathway we hypothesized a possible interaction between CNGA4 and TRPC2 forming a heteromeric channel. First evidences for this interaction came from the absence of CNGA4 expression in the sensory microvilli of Trpc2-/- mice. Second, using transfected HEK cells as an expression system we could observe that CNGA4 and TRPC2 interact and translocate to the plasma membrane. Perfusion of a DAG analogue on co-transfected HEK cells resulted in a strong calcium entry suggesting that the two proteins form a functional channel. These results might suggest a modulatory role for CNGA4 in a heteromeric TRPC2+CNGA4 ion channel. Further experiments will give more insights on the combined role of these transduction ion channels in pheromone detection.

Regulation of the activity of DnaA and its role during the cell cycle of caulobacter crescentus

The initiation of chromosomal replication must be tightly regulated so that the genome is replicated only once per cell cycle. In most bacteria, DnaA binds to the origin of replication and initiates chromosomal replication. DnaA is a dual-function protein that also acts as an important transcription factor that regulates the expression of many genes in bacteria. Thus, understanding how this protein is regulated during the bacterial cell cycle is of major importance. The α-proteobacterium Caulobacter crescentus is an excellent model to study the bacterial cell cycle, mainly because it is possible to isolate synchronized cell cultures and because it initiates the replication of its chromosome once per cell cycle and at a specific time of the cell cycle. This latest feature is of special interest for the major aim of my thesis work, which focused on the temporal and spatial regulation of the activity of the essential DnaA protein in C. crescentus. In Escherichia coli, the Hda protein converts ATP-DnaA into ADP- DnaA by stimulating the ATPase activity of DnaA, to prevent over-initiation of chromosome replication. We propose that there exists a similar mechanism in C. crescentus, which is not only involved in the temporal control of chromosome replication, but also in the control of gene expression. First, we provided evidences indicating that the hydrolysis of the ATP bound to DnaA is essential for the viability of C. crescentus. Our results suggest that ATP-DnaA promotes the initiation of chromosome replication, since we found that cells over-expressing a DnaA protein with a mutated ATPase domain, DnaA(R357A), over-initiated chromosome replication, unlike cells expressing the wild-type DnaA protein at similar levels. By contrast, the DnaA(R357A) protein was less active than DnaA in promoting the transcription of three essential genes, suggesting that these may be more efficiently activated by ADP-DnaA than ATP-DnaA. We propose that the ATP-DnaA to ADP-DnaA switch down-regulates the initiation of DNA replication while activating the transcription of several essential genes involved in subsequent cell cycle events. Second, we studied the role of the HdaA protein, homologous to Hda, in promoting the ATP- DnaA to ADP-DnaA switch in C. crescentus. HdaA is essential for viability and its depletion in the cell leads to an over-replication of the chromosome, indicating that HdaA is a negative regulator of DNA replication. HdaA dynamically co-localizes with the replisome. In this work, we identified DnaN, the β-clamp of the DNA polymerase, as the replisome component that interacts directly with HdaA and that recruits HdaA to the replisome in live C. crescentus cells. We also showed that a mutant HdaA protein that cannot interact or co-localize with DnaN is not functional, indicating that HdaA is probably activated by DnaN. However, we found that another non-functional HdaA protein, mutated in the conserved Arginine finger of its AAA+ domain, was able to localize at the replisome, suggesting that the AAA+ domain of HdaA exerts its essential function after the recruitment of HdaA to the replisome. We propose that HdaA stimulates the ATPase activity of DnaA once DNA replication is ongoing, via its interaction with DnaN and the activity of the two conserved R fingers of DnaA and HdaA. Finally, we created different strains in which HdaA, DnaN or DnaA were over-produced. We observed that the over-production of HdaA seems to lead to a delay in chromosome replication, while the over-production of DnaN had an opposite effect. Our results also indicate that the over-production of DnaA may intensify the over-initiation phenotype of cells depleted for HdaA. We conclude that the dynamic interplay of HdaA and DnaN in the cell contributes to regulating the ATP-DnaA/ADP-DnaA ratio in the cell, to ensure once per cell cycle initiation of chromosomal replication in C. crescentus. Altogether, our work provided important information on the regulation of the activity of DnaA in C. crescentus. Since DnaA, HdaA and DnaN are well-conserved proteins, most of our findings are useful to understand how chromosome replication and gene expression are controlled by DnaA in many other bacterial species. - L'initiation de la réplication des chromosomes doit être précisément régulée de telle sorte que le génome ne soit répliqué qu'une seule fois par cycle cellulaire. Chez la plupart des bactéries, DnaA se lie à l'origine de réplication du chromosome et en initie sa réplication. DnaA est aussi un facteur de transcription qui régule l'expression de nombreux gènes bactériens. De ce fait, il est très important de comprendre comment DnaA est régulée au cours du cycle cellulaire bactérien. L'a-protéobactérie Caulobacter crescentus est un excellent modèle pour étudier le cycle cellulaire bactérien, essentiellement parce qu'il est aisé d'isoler des populations de cellules synchronisées à la même étape du cycle cellulaire et parce que cette bactérie n'initie la réplication de son chromosome qu'une seule fois et à un moment précis de son cycle. Cette dernière caractéristique est particulièrement pertinente pour l'objectif de mon travail doctoral, qui consistait à comprendre comment l'activité de la protéine essentielle DnaA est régulée dans l'espace et dans le temps chez C. crescentus. Chez Escherichia coli, la protéine Hda convertie DnaA-ATP en DnaA-ADP en stimulant l'activité ATPasique de DnaA, ce qui empêche la sur-initiation de la réplication du chromosome. Nous proposons qu'un mécanisme similaire existe chez C. crescentus. Il serait non seulement nécessaire au contrôle de la réplication du chromosome, mais aussi au contrôle de l'expression de certains gènes. Dans un premier temps, nous avons mis en évidence le fait que l'hydrolyse de l'ATP lié à DnaA est un processus essentiel à la viabilité de C. crescentus. Nos résultats suggèrent que DnaA-ATP initie la réplication du chromosome, comme nous avons observé que des cellules qui sur-expriment une protéine DnaA(R357A) mutée sans domaine ATPasique fonctionnel, sur-initie la réplication de leur chromosome, contrairement aux cellules qui sur-expriment la protéine DnaA sauvage à des niveaux équivalents. Au contraire, la protéine DnaA(R357A) était moins active que la protéine DnaA sauvage pour promouvoir la transcription de trois gènes essentiels, ce qui suggère que ces derniers sont peut-être plus efficacement activés par DnaA-ADP que DnaA-ATP. Nous proposons que la conversion de DnaA-ATP en DnaA-ADP réprime l'initiation de la réplication, tandis qu'elle active la transcription de plusieurs gènes impliqués dans des étapes plus tardives du cycle cellulaire. Dans un deuxième temps, nous avons étudié le rôle de la protéine HdaA, homologue à Hda, dans la conversion de DnaA-ATP en DnaA-ADP chez C. crescentus. Cette protéine est essentielle à la viabilité de C. crescentus et sa déplétion donne des cellules qui sur-initient la réplication de leur chromosome, suggérant que HdaA est un répresseur de la réplication du chromosome. HdaA co-localise de manière dynamique avec le réplisome. Lors de mon travail doctoral, nous avons démontré que DnaN, le β-clamp de l'ADN polymérase, est l'élément qui recrute HdaA au réplisome in vivo. Nous avons aussi montré qu'une protéine HdaA mutante qui ne peut pas interagir ou co-localiser avec DnaN, n'est pas fonctionnelle, ce qui suggère que HdaA est activée par DnaN. Nous avons néanmoins aussi isolé une autre protéine HdaA non fonctionnelle, dont une arginine conservée de son domaine AAA+ était mutée, mais qui pouvait toujours co-localiser avec le réplisome, ce qui suggère que le domaine AAA+ de HdaA est nécessaire après le recrutement de HdaA au réplisome. Nous proposons que HdaA stimule l'activité ATPasique de DnaA qu'une fois que la réplication a commencé, grâce à son interaction avec DnaN et aux deux arginines conservées des protéines HdaA et DnaA. Finalement, nous avons construit différentes souches sur-exprimant HdaA, DnaN ou DnaA. Nous avons observé que la sur-production de HdaA retarde la réplication du chromosome, tandis que la sur-production de DnaN a un effet opposé. Nos observations suggèrent aussi que la sur-expression de DnaA dans des cellules déplétées pour HdaA aggrave leur phénotype de sur-initiation. Nous en concluons que HdaA et DnaN collaborent étroitement et de manière dynamique pour réguler le rapport DnaA-ATP/DnaA-ADP dans la cellule, pour s'assurer que la réplication du chromosome ne soit initiée qu'une seule fois par cycle cellulaire chez C. crescentus. Globalement, notre travail a mis en évidence des informations importantes sur la régulation de l'activité de DnaA chez C. crescentus. Comme DnaA, HdaA et DnaN sont des protéines très conservées, la plupart de nos découvertes sont utiles pour mieux comprendre comment la réplication du chromosome bactérien et l'expression des gènes sont contrôlées par DnaA chez de nombreuses autres espèces bactériennes.

Organisation of genetic variation in multinucleate arbuscular mycorrhizal fungi

Les Champignons Endomycorhiziens Arbusculaires (CEA) forment une symbiose racinaire avec environ 80% des espèces connues de plantes vasculaires. Ils occupent une position écologique très importante liée aux bénéfices qu'ils confèrent aux plantes. Des études moléculaires effectuées sur des gènes ribosomaux ont révélé un très grand polymorphisme, tant à l'intérieur des espèces qu'entre celles-ci. Ces champignons étant coenocytiques et multinucléés, l'organisation de cette variabilité génétique intraspécifique pourrait avoir différentes origines. Ce travail se propose d'examiner l'organisation et l'évolution de cette variabilité. Sur la base de fossiles, l'existence des CEA remonte à au moins 450 millions d'années. Cette symbiose peut donc être considérée comme ancienne. Les premières données moléculaires n'indiquant pas de reproduction sexuée, une hypothèse fut élaborée stipulant que les CEA seraient des asexués ancestraux. La première partie de cette thèse (chapitre 2) met en évidence l'existence de recombinaison dans différents CEA mais montre également que celle-ci est insuffisante pour purger les mutations accumulées. La reproduction étant essentiellement asexuée, on peut prédire que les nombreux noyaux ont probablement divergé génétiquement. En collaboration avec M. Hijri nous avons pu vérifier cette hypothèse (chapitre 2). Dans le chapitre 3 j'ai cherché à comprendre si le polymorphisme était également présent dans une population naturelle du CEA Glomus intraradices au niveau intraspécifique, ce qui n'avait encore jamais été examiné. En comparant les empreintes génétiques d'individus obtenus chacun à partir d'une spore mise en culture, j'ai clairement démontré que d'importantes différences génétiques existent entre ceux-ci. Un résultat similaire, portant sur des traits quantitatifs d'individus de la même population, a été trouvé par A. Koch. Les deux études en ensemble montre que le polymorphisme génétique dans cette population est suffisamment grand pour être important au niveau écologique. Dans le chapitre 4, j'ai cherché a examiner le polymorphisme des séquences du gène BiP au sein d'un individu. C'est la première étude qui examine la diversité génétique du génome de CEA avec un autre marqueur que l'ADN ribosomique. J'ai trouvé 31 types de séquences différentes du gène BiP issu d'un isolat de G. intraradices mis en culture à partir d'une seule spore. Cette variation n'était pas restreinte à des zones sélectivement neutres du BiP. Mes résultats montrent qu'il y a un grand nombre de variants non-fonctionnels, proportionnellement au faible nombre de copies attendues par noyau. Ceci va dans le sens d'une partition de l'information génétique entre les noyaux.<br/><br/>Arbuscular mycorrhizal fungi (AMF) are root symbionts with about 80% of all known species of vascular land plants. AMF are ecologically important because of the benefits that they confer to plants. Molecular studies on AMF showed that rDNA sequences were highly variable between species and within species. Because AMF are coenocytic and multinucleate there are several possibilities how this intraspecific genetic variation could be organized. Therefore, the organization and evolution of this variation in AMF were investigated in the present work. Based on fossil records the AMF symbiosis has existed for 450 Million years and is therefore considered ancient. First molecular data indicated no evident sexual reproduction and gave rise to the hypothesis that AMF might be ancient asexuals. The first part of this thesis (Chapter 2) shows evidence for recombination in different AMF but also indicates that it has not been frequent enough to purge accumulated mutations. Given asexual reproduction, it has been predicted that the many nuclei in AMF should diverge leading to genetically different nuclei. This hypothesis has been confirmed by an experiment of M. Hijri and is also included in chapter 2 as the results were published together. In chapter 3 I then investigated whether intraspecific genetic variation also exists in a field population of the AMF Glomus intraradices. Comparing genetic fingerprints of individuals derived from single spores I could clearly show that large genetic differences exist. A similar result, based on quantitative genetic traits, was found for the same population by A. Koch. The two studies taken together show that the genetic variation observed in the population is high enough to be of ecological relevance. Lastly, in chapter 4, I investigated within individual genetic variation among BiP gene sequences. It is the first study that has analyzed genetic diversity in the AMF genome in a region of DNA other than rDNA. I found 31 sequence variants of the BiP gene in one G. intraradices isolate that originated from one spore. Genetic variation was not only restricted to selectively neutral parts of BiP. A high number of predicted non-functional variants compared to a likely low number of copies per nucleus indicated that functional genetic information might even be partitioned among nuclei. The results of this work contribute to our understanding of potential evolutionary strategies of ancient asexuals, they also suggest that genetic differences in a population might be ecologically relevant and they show that this variation even occurs in functional regions of the AMF genome.

Messaging Application Engine for Symbian Platform

Tämä diplomityö kuvaa viestintä sovelluksen ytimen kehitystyön Symbian-alustalle. Koko sovelluksen vaatimuksena oli vastaamattomiin puheluihin vastaaminen ennalta määritellyillä tekstiviesteillä käyttäjän määrittelemien sääntöjen mukaisesti. Ei-toiminnallisia vaatimuksia olivat resurssien käytön vähentäminen ja uudelleenkäytön mahdollistaminen. Täten tämän työn tavoitteena oli kehittää ydin, joka kapseloi sovelluksen sellaisen toiminnallisuuden, joka on käyttöliittymästä riippumatonta ja uudelleenkäytettävää. Kehitystyössä ohjasi Unified Process, joka on iteroiva, käyttötapauksien ohjaama ja arkkitehtuurikeskeinen ohjelmistoprosessi. Se kannusti käyttämään myös muita teollisuudenalan vakiintuneita menetelmiä, kuten suunnittelumalleja ja visuaalista mallintamista käyttäen Unified Modelling Languagea. Suunnittelumalleja käytettiin kehitystyön aikana ja ohjelmisto mallinnettiin visuaalisesti suunnittelun edistämiseksi ja selkiyttämiseksi. Alustan palveluita käytettiin hyväksi kehitysajan ja resurssien käytön minimoimiseksi. Ytimen päätehtäviksi määrättiin viestien lähettäminen sekä sääntöjen talletus ja tarkistaminen. Sovelluksen eri alueet, eli sovelluspalvelin ja käyttöliittymää, pystyivät käyttämään ydintä ja sillä ei ollut riippuvuuksia käyttöliittymätasolle. Täten resurssien käyttö väheni ja uudelleenkäytettävyys lisääntyi. Viestien lähettäminen toteutettiin Symbian-alustan menetelmin. Sääntöjen tallettamiseen tehtiin tallennuskehys, joka eristää sääntöjen sisäisen ja ulkoisen muodon. Tässä tapauksessa ulkoiseksi tallennustavaksi valittiin relaatiotietokanta. Sääntöjen tarkastaminen toteutettiin tavanomaisella olioiden yhteistoiminnalla. Päätavoite saavutettiin. tämä ja muut hyviksi arvioidut lopputulokset, kuten uudelleenkäytettävyys ja vähentynyt resurssien käyttö, arveltiin juontuvan suunnittelumallien ja Unified Processin käytöstä. Kyseiset menetelmät osoittivat mukautuvansa pieniinkin projekteihin. Menetelmien todettiin myös tukevan ja kannustavan kehitystyön aikaista oppimista, mikä oli välttämätöntä tässä tapauksessa.

Differential Dimerization of Variants Linked to Enhanced S-Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand-Binding Domain.

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.

Developing a user-centric event management system

This thesis describes the development of a software requirements specification for a user-centric event management system. The system is set to satisfy three goals: adding value for the event attendees, adding value for the event organizer, and reducing the costs of arranging and running an event. The requirements are identified by researching the prescriptive traits of event business and the current state of the case company and its environment. First the professional and human needs for events are scrutinized. Second, some recent reports about the current trends in the event business are reviewed. Then the event life cycle is presented using the model of new service development, and online promotion of events and especially word-of-mouth marketing receive special attention. Events are also regarded from the perspective of social networks and social media. The case company’s current state and its competitors are reviewed to formulate the needs which the system should fulfil. Then the currently available solutions for social media oriented event management are reviewed. The result is a set of functional and non-functional requirements. The functional requirements are categorized into social media, social networking, event personalization, event management, and system administration features. The specified features and non-functional requirements satisfy the three goals set for the system.

Building object-oriented software with the D-Bus messaging system

Object-oriented programming is a widely adopted paradigm for desktop software development. This paradigm partitions software into separate entities, objects, which consist of data and related procedures used to modify and inspect it. The paradigm has evolved during the last few decades to emphasize decoupling between object implementations, via means such as explicit interface inheritance and event-based implicit invocation. Inter-process communication (IPC) technologies allow applications to interact with each other. This enables making software distributed across multiple processes, resulting in a modular architecture with benefits in resource sharing, robustness, code reuse and security. The support for object-oriented programming concepts varies between IPC systems. This thesis is focused on the D-Bus system, which has recently gained a lot of users, but is still scantily researched. D-Bus has support for asynchronous remote procedure calls with return values and a content-based publish/subscribe event delivery mechanism. In this thesis, several patterns for method invocation in D-Bus and similar systems are compared. The patterns that simulate synchronous local calls are shown to be dangerous. Later, we present a state-caching proxy construct, which avoids the complexity of properly asynchronous calls for object inspection. The proxy and certain supplementary constructs are presented conceptually as generic object-oriented design patterns. The e ect of these patterns on non-functional qualities of software, such as complexity, performance and power consumption, is reasoned about based on the properties of the D-Bus system. The use of the patterns reduces complexity, but maintains the other qualities at a good level. Finally, we present currently existing means of specifying D-Bus object interfaces for the purposes of code and documentation generation. The interface description language used by the Telepathy modular IM/VoIP framework is found to be an useful extension of the basic D-Bus introspection format.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Histological changes in banana explants, cv. Nanicão (Musa spp., Group AAA), submitted to different auxins for induction of somatic embryogenesis

The present work analyzes the behavior of banana explants, cv. Nanicão (Musa spp. Group AAA) regarding somatic embryogenesis induction treatments with several auxins. Longitudinal segments of shoot meristematic apices of micropropagated banana plantlets cultivated and rooted in vitro were introduced in culture medium containing dicamba, picloram, 2,4-D or NAA in different concentrations. Explant samples were collected at 0, 7 and 10 days and prepared for light microscopy. Histological sections were used for comparison of the histological changes occurring after induction treatment with different auxins. Embryogenic response was observed only in treatments with picloram or dicamba, with distinct embryogenic regions observed at 14 and 21 days in culture, respectively. Histological sections of embryogenic regions of the explant at 26 days in culture revealed the formation of meristematic regions, structures with multiple root meristems, and somatic embryos at early globular stages. Embryo-like structures morphologically similar to Musa balbisiana zygotic embryos were sectioned and showed a lack of apical meristems and absence of procambial differentiation. These results indicate the induction of non-functional somatic embryos and the need for more studies on developmental aspects and maturation treatments for optimization of the process.

A systematic mapping study of web services discovery mechanisms

Context: Web services have been gaining popularity due to the success of service oriented architecture and cloud computing. Web services offer tremendous opportunity for service developers to publish their services and applications over the boundaries of the organization or company. However, to fully exploit these opportunities it is necessary to find efficient discovery mechanism thus, Web services discovering mechanism has attracted a considerable attention in Semantic Web research, however, there have been no literature surveys that systematically map the present research result thus overall impact of these research efforts and level of maturity of their results are still unclear. This thesis aims at providing an overview of the current state of research into Web services discovering mechanism using systematic mapping. The work is based on the papers published 2004 to 2013, and attempts to elaborate various aspects of the analyzed literature including classifying them in terms of the architecture, frameworks and methods used for web services discovery mechanism. Objective: The objective if this work is to summarize the current knowledge that is available as regards to Web service discovery mechanisms as well as to systematically identify and analyze the current published research works in order to identify different approaches presented. Method: A systematic mapping study has been employed to assess the various Web Services discovery approaches presented in the literature. Systematic mapping studies are useful for categorizing and summarizing the level of maturity research area. Results: The result indicates that there are numerous approaches that are consistently being researched and published in this field. In terms of where these researches are published, conferences are major contributing publishing arena as 48% of the selected papers were conference published papers illustrating the level of maturity of the research topic. Additionally selected 52 papers are categorized into two broad segments namely functional and non-functional based approaches taking into consideration architectural aspects and information retrieval approaches, semantic matching, syntactic matching, behavior based matching as well as QOS and other constraints.

The terrestrial Gastropoda Megalobulimus abbreviatus as a useful model for nociceptive experiments: effects of morphine and naloxone on thermal avoidance behavior

We describe the behavior of the snail Megalobulimus abbreviatus upon receiving thermal stimuli and the effects of pretreatment with morphine and naloxone on behavior after a thermal stimulus, in order to establish a useful model for nociceptive experiments. Snails submitted to non-functional (22ºC) and non-thermal hot-plate stress (30ºC) only displayed exploratory behavior. However, the animals submitted to a thermal stimulus (50ºC) displayed biphasic avoidance behavior. Latency was measured from the time the animal was placed on the hot plate to the time when the animal lifted the head-foot complex 1 cm from the substrate, indicating aversive thermal behavior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal stimulus (50ºC; N = 9 in each group). The results (means ± SD) showed an extremely significant difference in response latency between the group treated with 20 mg/kg morphine (63.18 ± 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 ± 3.19 s), 5.0 mg/kg (11.53 ± 1.64 s) and 7.5 mg/kg naloxone (7.38 ± 1.6 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 ± 8.53 s) and saline groups (29.1 ± 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64% in the saline group and maximum 83.2% decrease in the morphine group). These results provide evidence of the involvement of endogenous opioid peptides in the control of thermal withdrawal behavior in this snail, and reveal a stereotyped and reproducible avoidance behavior for this snail species, which could be studied in other pharmacological and neurophysiological studies.

Green Service Level Agreement under Sustainability Lens in IT Industry

Nowadays, when most of the business are moving forward to sustainability by providing or getting different services from different vendors, Service Level Agreement (SLA) becomes very important for both the business providers/vendors and as well as for users/customers. There are many ways to inform users/customers about various services with its inherent execution functionalities and even non-functional/Quality of Services (QoS) aspects through negotiating, evaluating or monitoring SLAs. However, these traditional SLA actually do not cover eco-efficient green issues or IT ethics issues for sustainability. That is why green SLA (GSLA) should come into play. GSLA is a formal agreement incorporating all the traditional commitments as well as green issues and ethics issues in IT business sectors. GSLA research would survey on different traditional SLA parameters for various services like as network, compute, storage and multimedia in IT business areas. At the same time, this survey could focus on finding the gaps and incorporation of these traditional SLA parameters with green issues for all these mentioned services. This research is mainly points on integration of green parameters in existing SLAs, defining GSLA with new green performance indicators and their measurable units. Finally, a GSLA template could define compiling all the green indicators such as recycling, radio-wave, toxic material usage, obsolescence indication, ICT product life cycles, energy cost etc for sustainable development. Moreover, people’s interaction and IT ethics issues such as security and privacy, user satisfaction, intellectual property right, user reliability, confidentiality etc could also need to add for proposing a new GSLA. However, integration of new and existing performance indicators in the proposed GSLA for sustainable development could be difficult for ICT engineers. Therefore, this research also discovers the management complexity of proposed green SLA through designing a general informational model and analyses of all the relationships, dependencies and effects between various newly identified services under sustainability pillars. However, sustainability could only be achieved through proper implementation of newly proposed GSLA, which largely depends on monitoring the performance of the green indicators. Therefore, this research focuses on monitoring and evaluating phase of GSLA indicators through the interactions with traditional basic SLA indicators, which would help to achieve proper implementation of future GSLA. Finally, this newly proposed GSLA informational model and monitoring aspects could definitely help different service providers/vendors to design their future business strategy in this new transitional sustainable society.

Model-based testing of software systems : functionality and performance

Software is a key component in many of our devices and products that we use every day. Most customers demand not only that their devices should function as expected but also that the software should be of high quality, reliable, fault tolerant, efficient, etc. In short, it is not enough that a calculator gives the correct result of a calculation, we want the result instantly, in the right form, with minimal use of battery, etc. One of the key aspects for succeeding in today's industry is delivering high quality. In most software development projects, high-quality software is achieved by rigorous testing and good quality assurance practices. However, today, customers are asking for these high quality software products at an ever-increasing pace. This leaves the companies with less time for development. Software testing is an expensive activity, because it requires much manual work. Testing, debugging, and verification are estimated to consume 50 to 75 per cent of the total development cost of complex software projects. Further, the most expensive software defects are those which have to be fixed after the product is released. One of the main challenges in software development is reducing the associated cost and time of software testing without sacrificing the quality of the developed software. It is often not enough to only demonstrate that a piece of software is functioning correctly. Usually, many other aspects of the software, such as performance, security, scalability, usability, etc., need also to be verified. Testing these aspects of the software is traditionally referred to as nonfunctional testing. One of the major challenges with non-functional testing is that it is usually carried out at the end of the software development process when most of the functionality is implemented. This is due to the fact that non-functional aspects, such as performance or security, apply to the software as a whole. In this thesis, we study the use of model-based testing. We present approaches to automatically generate tests from behavioral models for solving some of these challenges. We show that model-based testing is not only applicable to functional testing but also to non-functional testing. In its simplest form, performance testing is performed by executing multiple test sequences at once while observing the software in terms of responsiveness and stability, rather than the output. The main contribution of the thesis is a coherent model-based testing approach for testing functional and performance related issues in software systems. We show how we go from system models, expressed in the Unified Modeling Language, to test cases and back to models again. The system requirements are traced throughout the entire testing process. Requirements traceability facilitates finding faults in the design and implementation of the software. In the research field of model-based testing, many new proposed approaches suffer from poor or the lack of tool support. Therefore, the second contribution of this thesis is proper tool support for the proposed approach that is integrated with leading industry tools. We o er independent tools, tools that are integrated with other industry leading tools, and complete tool-chains when necessary. Many model-based testing approaches proposed by the research community suffer from poor empirical validation in an industrial context. In order to demonstrate the applicability of our proposed approach, we apply our research to several systems, including industrial ones.