Les motoneurones tiennent une place remarquable dans l'organisme : ils constituent l'interface entre le système nerveux central et le système musculaire. Leur excitabilité est une caractéristique primordiale dans le comportement moteur puisqu'elle définit la force musculaire développée en réponse à la commande motrice. Chez la souris, la décharge des motoneurones est marquée par la présence d'oscillations de mode mixte (MMOs) entre les potentiels d'action. Ces MMOs permettent la décharge des motoneurones à basse fréquence et sont responsables d'un régime de décharge particulier nommé zone sous-Primaire, pendant lequel la fréquence de décharge est très variable et le gain de la relation courant-Fréquence élevé. Nous avons étudié les mécanismes responsables de l'apparition de ces MMOs à la fois de manière expérimentale, dans une préparation in vivo de souris anesthésié, incluant l'utilisation du Dynamic Clamp, et théorique, au moyen d'un modèle mono-Compartimental de motoneurone. Nos résultats ont montré que ces MMOs étaient causées par les courants sodiques et potassiques responsables des potentiels d'action et qu'elles émergeaient d'un état de faible excitabilité de la membrane, dû à l'inactivation lente des courants sodiques. Nous avons également montré que le courant de post-Hyperpolarisation pouvait paradoxalement augmenter l’excitabilité des motoneurones et réduire les MMOs en dé-Inactivant le courant sodique. La Sclérose Latérale Amyotrophique (SLA) conduit à la dégénérescence spécifique de ces motoneurones qui s'accompagne d'une vacuolisation de leur arborisation dendritique. L'augmentation précoce de l'excitabilité des motoneurones dans la maladie a largement été évoquée pour rendre compte de leur atteinte. Une hyperexcitabilité, aussi bien d'origine intrinsèque qu'extrinsèque pourrait en effet produire une excitotoxicité délétère pour la cellule. Si une telle modification de l'excitabilité est en cause dans la maladie, elle devrait persister jusqu'aux âges auxquels se produisent les premières dénervations des jonctions neuromusculaires. Nous avons enregistré les propriétés électrophysiologiques des motoneurones dans une préparation in vivo de souris adultes SOD1-G93A, modèle de la SLA. Nos résultats ont montré que leur conductance d'entrée était augmentée dans les jours qui précèdent les premières dénervations de leurs jonctions neuromusculaires. Malgré cela, leur excitabilité n'était pas modifiée. Loin d'être intrinsèquement hyperexcitables, une fraction d'entre eux perdaient même leur capacité à décharger de manière répétée. Nous avons finalement étudié la vacuolisation qui prend place dans les dendrites des motoneurones au cours de la maladie et son lien avec la couverture synaptique. Nous avons montré que la vacuolisation dendritique prenait place avant les dénervations et que la taille des vacuoles augmentait avec l'âge des souris SOD1-G93A. De manière intéressante, cette progression semblait plus rapide dans les motoneurones les plus sensibles à la maladie. Bien que la couverture synaptique n'était pas modifiée au cours de la maladie, nous avons mis en évidence une densité de synapses excitatrices et inhibitrices plus importante sur les régions dendritiques qui se vacuolisent. Ces résultats suggèrent un lien entre l'activité synaptique et la formation de vacuoles dans les motoneurones au cours de la SLA. Les motoneurones ne présentant pas d'hyperexcitabilité intrinsèque, une excitotoxicité d'origine synaptique pourrait alors être responsable de leur dégénérescence. / Motoneurones hold a remarkable position in the organism: they are the interface between the central nervous system and the muscular system. Their excitability is a crucial characteristic in motor behavior since it determines the muscular force produced in response to motor command. In mice, motoneurone discharge is marked by the presence of sub-Threshold oscillations between action potentials which create a behavior of mixed mode oscillations (MMOs). These MMOs allow the motoneurones to fire at low frequency and are responsible for a sub-Primary range of discharge during which the firing frequency is irregular and the slope of current-Frequency relation is steep. We investigated the mechanisms responsible for these MMOs by in vivo recordings in anesthetized mice, using Dynamic Clamp, and by theoretical modelization in a monocompartimental model of motoneurone. Our results showed that MMOs were caused by sodium and potasium currents responsible for action potentials and that they emerged from a state of low membrane excitability caused by a slow inactivation of the sodium current. Paradoxically, we also showed that the after-Hyperpolarization current was able to increase the membrane excitability and to reduce MMOs by de-Inactivating the sodium current. Amyotrophic Lateral Sclerosis (ALS) leads to the specific degeneration of these motoneurones and is accompanied by a vacuolation of their dendritic trees. An early increase in motoneurons excitability during the disease has been widely proposed to account for their degeneration. Indeed, a motoneuron hyperexcitability of intrinsic or extrinsic origin could produce a deleterious excitotoxicity. If such a change of excitability is involved in the disease, it should last until the ages where the first denervation of neuromuscular junctions occurs. We recorded the electrophysiological properties of motoneurones in an in vivo preparation of adult SOD1-G93A mice, model of ALS. Our results showed that their input conductance was increased before the first denervation of their neuromuscular junctions. Nevertheless, their excitability was not modified. Far from being intrinsically hyperexcitable, a fraction of them even lost their ability to discharge repeatedly. We finally studied the vacuolation that takes place in dendrites of motoneurones during the disease and its relation with synaptic coverage. We have shown that the dendritic vacuolation takes place before the denervation and that the size of these vacuoles increases with age in SOD1-G93A mice. Interestingly, this increase was faster in the most vulnerable motoneurones. Although synaptic coverage was not altered in the disease, we ¬revealed higher densities of excitatory and inhibitory synapses on dendritic regions that vacuolate. These results suggest a link between synaptic activity and vacuoles formation in motoneurones during ALS. Motoneurones were not intrinsically hyperexcitable, instead, an excitotoxicity from a synaptic origin may be responsible for their degeneration.

Protein misfolding and aggregation underlie several neurodegenerative proteinopathies including amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) was the first gene found to be associated with familial ALS. Overexpression of human mutant or wild type SOD1 in transgenic mouse models induces motor neuron (MN) degeneration and an ALS-like phenotype. SOD1 mutations, leading to the destabilization of the SOD1 protein is associated with ALS pathogenesis. However, how misfolded SOD1 toxicity specifically affects human MNs is not clear. The aim of this thesis was to develop patient-derived, cellular models of ALS to help understand the pathogenic mechanisms underlying SOD1. To understand which cellular pathways impact on the level of misfolded SOD1 in human cells, we established a model using patient-derived fibroblasts and quantified misfolded SOD1 in relation to disturbances in several ALS-related cellular pathways. Misfolded SOD1 levels did not change following reduction in autophagy, inhibition of the mitochondrial respiratory chain, or induction of endoplasmic reticulum (ER)-stress. However, inhibition of the ubiquitin-proteasome system (UPS) lead to a dramatic increase in misfolded SOD1 levels. Hence, an age-related decline in proteasome activity might underlie the late-life onset that is typically seen in SOD1 ALS. To address whether or not SOD1 misfolding is enhanced in human MNs, we used mixed MN/astrocyte cultures (MNCs) generated in vitro from patient-specific induced pluripotent stem cells (iPSCs). Levels of soluble misfolded SOD1 were increased in MNCs as well as in pure iPSC-derived astrocytes compared to other cell types, including sensory neuron cultures. Interestingly, this was the case for both mutant and wild type human SOD1, although the increase was enhanced in SOD1 FALS MNCs. Misfolded SOD1 was also found to exist in the same form as in mouse SOD1 overexpression models and was identified as a substrate for 20S proteasome degradation. Hence, the vulnerability of motor areas to ALS could be explained by increased SOD1 misfolding, specifically in MNs and astrocytes. To investigate factors that might promote SOD1 misfolding, we focussed on the stability of SOD1 mediated by a crucial, stabilizing C57-C146 disulphide bond and its redox status. Formation of disulphide bond is dependent on oxidation by O2 and catalysed by CCS. To investigate whether low O2 tension affects the stability of SOD1 in vitro we cultured fibroblasts and iPSC-derived MNCs under different oxygen tensions. Low oxygen tension promoted disulphide-reduction, SOD1 misfolding and aggregation. This response was much greater in MNCs compared to fibroblasts, suggesting that MNs may be especially sensitive to low oxygen tension and areas with low oxygen supply could serve as foci for ALS initiation. SOD1 truncation mutations often lack C146, and cannot adopt a native fold and are rapidly degraded. We characterized soluble misfolded and aggregated SOD1 in patient-derived cells carrying a novel SOD1 D96Mfs*8 mutation as well as in cells fom an unaffected mutation carrier. The truncated protein has a C-terminal fusion of seven non-native amino acids and was found to be extremely prone to aggregation in vitro. Since not all mutation carriers develop ALS, our results suggested this novel mutation is associated with reduced penetrance. In summary, patient derived cells are useful models to study factors affecting SOD1 misfolded and aggregation. We show for the first time that misfolding of a disordered and disease associated protein is enhanced in disease-related cell types. Showing that misfolded SOD1 exists in human cells in the same form as in transgenic mouse models strengthens the translatability of results obtained in the two species. Our results demonstrate disulphide-reduction and misfolding/aggregation of SOD1 and suggest that 20S proteasome could be an important therapeutic target for early stages of disease. This model provides a great opportunity to study pathogenic mechanisms of both familial and sporadic ALS in patient-derived models of ALS. / Varje år insjuknar omkring 5300 personer i världen i motorneuronsjukdomen Amyotrofisk lateralskleros (ALS). Sjukdomen kännetecknas av degeneration av motorneuron i hjärnan och ryggmärgen, de nervceller som styr kroppens muskler, vilket leder till musklerförtvining och gradvis förlamning. ALS-patienter avlider oftast till följd av andningssvikt när sjukdomen når andningsmuskulaturen. I de allra flesta fall uppkommer ALS sporadiskt (SALS), det vill säga utan känd genetisk orsak, medan ärftliga fall (FALS) drabbar omkring 10 % och beror på mutationer i ett antal kända gener. Upp till 6 % av alla ALS fall kan härledas till mutationer i genen superoxid dismutas 1 (SOD1). SOD1 är ett enzym som ansvarar för att omvandla och oskadliggöra fria syreradikaler som bildas vid normal ämnesomsättning. 206 olika SOD1 mutationer har identifierats, alla orsakar inte ALS men många leder till att den tredimensionella proteinstrukturen förändras, vilket ökar proteinets benägenhet att felveckas. Initialt trodde man att SOD1 mutationer förhindrade proteinets normalfunktion och följaktligen orsakade ALS. Studier har emellertid visat att den enzymatiska funktionen ofta bevaras, även hos muterade proteiner. Däremot kan små mängder felveckat SOD1 störa andra viktiga cellulära funktioner. Felveckat SOD1 har en benägenhet att klumpa ihop sig och bilda aggregat i det centrala nervsystemet (CNS). Dessa aggregat återfinns hos patienter med såväl FALS som SALS vilket tyder på att även vildtyps-SOD1 kan felveckas och vara involverat i sjukdomsutvecklingen. De flesta studier är baserade på transgena musmodeller som uttrycker extremt stora mängder av muterat humant SOD1. Det är dock oklart hur väl studier i möss överensstämmer med sjukdomsutvecklingen hos ALS-patienter, där mängden SOD1 är betydligt lägre. En central fråga som fortfarande står obesvarad är varför just motorneuron degenererar i ALS, trots att SOD1 uttrycks i alla kroppens celler. Det övergripande syftet med den här avhandlingen har varit att karakterisera felveckat SOD1 i patientceller för att studera dess roll i ALSrelaterade sjukdomsmekanismer med fysiologiskt relevanta nivåer av SOD1. Samtliga studier är gjorda in vitro med celler från friska donatorer med vildtyps-SOD1, celler från patienter med SOD1-FALS, FALS som bär andra ALS-associerade gener, samt SALS. I de allra flesta fallen har vi analyserat både lösligt felveckat SOD1 samt aggregerade former av SOD1 proteinet.

The onset of a progressive, fatal illness such as Motor Neuron Disease (MND) inevitably results in physical and communication disabilities that impinge on the individuals’ ability to remain functionally independent. The loss of speech as a result of dysarthria, a motor speech disorder, is one of the most profound changes that the person with MND will experience. The decline in the individuals’ speech intelligibility, that negatively influences communication effectiveness, implies that in 80% of cases alternative and augmentative communication (AAC) strategies are required to support the daily communication needs of individuals with MND. The dyadic nature of chronic illness implies that multiple aspects of one of the most important adult relationships, marriage, will be affected. Roles and responsibilities performed by each member of the couple will continually change as the disease progresses. The emotional trauma of adjusting to the unavoidable alteration in their relationship elicits strong emotions such as guilt, anger and frustration. Communication is one of the most constructive ways of dealing with these emotions. The ability of spouses to convey their innermost thoughts, feelings and intimacy through communicative interaction is vitally important in marital communication. The aim of this study was to compare how persons with MND and their spouses perceive changes in their marital communication in relation to the deteriorating speech of persons with MND. Fourteen couples divided into two participant groups, persons with MND and spouses, participated in this non-experimental correlational research study. Data was collected during three visits at six-monthly intervals over a 12 month period. At each of these visits both participant groups completed a variety of objective and subjective measures, of which twenty percent were interrated by independent raters. Results confirmed the inevitable decline in speech intelligibility of persons with MND across the disease progression. The persons with MND did not report a change in their perception of marital communication although their spouses indicated a statistically significant decrease between the first and last visits. Interestingly, there was no statistically significant relationship between the deteriorating speech of persons with MND and the couples’ perception of marital communication, confirming that marital communication was not influenced by decreased speech intelligibility. / Thesis (PhD)--University of Pretoria, 2010. / Centre for Augmentative and Alternative Communication (CAAC) / unrestricted

Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid produced by many cyanobacteria, and thought to induce neurotoxic effects through excitotoxicity, contributing to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism-dementia complex (ALS-PDC) and Alzheimer’s. The ubiquitous nature of cyanobacteria, and evidence of biomagnification through our food web, creates a dire need for the development of an analytical platform that will provide accurate identification and quantification of BMAA amounts in our ecosystem and potential food supply. The present study evaluated the ability of a MALDI-ToF-MS method to detect and quantify BMAA in a variety of biological matrices. Through validation procedures, it was demonstrated that this MALDI-ToF-MS method provided comparable data to currently accepted analytical methods, specifically LC-MS/MS. Further, the development of said method reduced sample preparation and data acquisition time (1-2 seconds per sample), while providing high throughput analysis and eliminating the need for derivatization, chromatographic separation, and modification of amino acids.

This thesis describes implementation process of issue tracking systém in company Audatex Systems s.r.o. An analysis of company's processes has been performed and ideal state after implementation is proposed. The implemented system is going to be used not only for software defect tracking, but also for company's other needs like project management and internal communication. Output of company's analysis is a list of requirements for said system and a tender is performed -- along with setting up of test environments. Process of implementation and configuration is described along with experience from implementation proces. Each of shotlisted applications is configured for company's specific needs and is evaluated with an evaluation systém, that has been created for this specific tender. Situation after implementation is descibed in conclusion of this thesis. Main benefit of this thesis lies in comparision of applications and presenting their configurations for company's needs. Two free solutions are compared along with a comercial one. In conclusion this thesis answers a question, whether a comercial solution is really needed for this specific case.

A lipoperoxidação gera diversas espécies carbonílicas altamente reativas dentre as quais se destacam acroleína (ACR), malondialdeído (MDA), 4-hidroxi-2-hexenal (HHE) e 4-hidroxi-2-nonenal (HNE). A principal via endógena de metabolização desses compostos é através de conjugação com glutationa por ação da glutationa-S-tranferase. Contudo, diversos trabalhos têm mostrado que dipeptídeos contendo histidina, tal como a carnosina (CAR), também podem formar conjugados com aldeídos e auxiliar na detoxificação desses compostos. Em nosso trabalho adutos de CAR com ACR, HHE, HHEd5, HNE e HNEd11 foram sintetizados, purificados e caracterizados. A reação da CAR com ACR foi estudada em detalhes. Resultados mostraram que a carnosina reage com acroleína formando 03 produtos principais: m/z = 265, m/z = 283 e m/z = 303, sendo este último mais estável e mais abundante. Dados de RMN H1, COSY e HSQC permitiram elucidar a estrutura dessa molécula (m/z = 303) e propor uma rota de reação. Em seguida, uma metodologia baseada em cromatografia líquida acoplada à espectrometria de massas do tipo \"Ion Trap\" (ESI+ HPLC/MS-MS) foi desenvolvida e validada para quantificação simultânea dos adutos sintetizados. Pelo método desenvolvido é possível quantificar com precisão 25 pmol de CAR-HHE, 1 pmol de CAR-ACR e 1 pmol de CAR-HNE com um coeficiente de variação de aproximadamente 10 % e acurácia de 98 % (HHEd5 e HNEd11 foram usados como padrão interno). Análise em urina de adultos não fumantes mostraram que os produtos sintetizados estão presentes na urina de humanos em concentrações de 3,6 ± 1,4; 2,3 ± 1,5 e 1,3 ± 0,5 nmol / mg de creatinina, respectivamente para CAR-ACR, CAR-HHE e CAR-HNE. Em ratos transgênicos SODG93A modelo para esclerose lateral amiotrófica (ELA), a suplementação da dieta dos animais com 35 ± 5 mg carnosina/animal/semana melhorou a manutenção do peso e a sobrevida dos animais. Análises dos adutos sintetizados em amostras de músculo sugerem que a metabolização de aldeídos esteja comprometida nesses animais e que a carnosina poderia funcionar como \"scavenger\" para esses compostos. Esses resultados comprovam que dipeptídeos de histidina atuam na detoxificação de compostos carbonílicos e participa de suas vias de excreção. Além disso, a caracterização da estrutura e desenvolvimento de método sensível de detecção abre a possibilidade de utilização desses adutos como biomarcadores de estresse redox e exposição a aldeídos. / Lipid peroxidation generates reactive carbonyl species, including 4-hydroxy-2-nonenal (HNE), acrolein (ACR), 4-hydroxy-2-hexenal (HHE) and malondialdehyde (MDA). One major pathway of aldehyde detoxification in vivo is through conjugation with glutathione catalyzed by glutathione-S-transferases or, alternatively, by conjugation with endogenous histidine containing dipeptides, such as carnosine (CAR). The reaction of CAR with ACR was investigated in an effort to assess its possible biological role. One stable adduct was isolated by reverse-phase HPLC and characterized on the basis of extensive spectroscopic measurements. The proposed reaction route for product formation involves the reaction of the CAR amino group with ACR via a Schiff base formation followed by dehydration and cyclization through Michael addition in the imidazole ring forming an instable compound with m/z = 265. The subsequent reaction with another molecule of ACR followed by cyclization gives rise to the final product with m/z = 303.A highly sensitive method involving HPLC-MS analysis was developed for the simultaneous accurate quantification of CAR- ACR, CAR-HHE and CAR-HNE adducts in human urinary samples from non-smoking adults. This methodology permits quantification of 10 pmol CAR-HHE and 1 pmol of CAR-ACR and CAR-HNE. Adduct levels in urine were 3.6 ± 1.4, 2.3 ± 1.5, 1.3 ± 0.5 nmol/mg of creatinine, respectively to CAR-ACR, CAR-HHE and CAR-HNE. In SODG93A transgenic rats model to amyotrophic lateral sclerosis (ALS), the food supplementation of the animals with 35 ± 5 mg carnosine/animal/week improve de body weight and the life span of the ALS treated group. Analysis of the synthesized adducts in muscle sample showed suggest than aldehyde metabolization is compromised in this animals and that may be carnosine work like a scavenger for these compounds. Our results indicate that carnosine adduction can be an important detoxification route of α,β -unsaturated aldehydes. Moreover, carnosine adducts quantification may be useful as redox stress indicator in vivo.

No description available.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative fatale caractérisée par une perte progressive et sélective des neurones moteurs. La SLA est incurable et il n’existe aucun traitement efficace pour les personnes atteintes de cette maladie. Environ 90% des cas sont sporadiques tandis que 10% sont familiaux, et les patients décèdent généralement deux à cinq ans après l'apparition des premiers symptômes. De nombreuses anomalies génétiques sont associées à la SLA, incluant des mutations dans les protéines FUS, C9orf72, SOD-1 et TDP-43. Le laboratoire a développé un modèle transgénique de Caenorhabditis elegans surexprimant la protéine humaine mutante TDP-43(Q331K) dans les neurones moteurs GABAergiques. Nous avons également obtenu par mutagénèse et CRISPR-Cas9 des modèles physiologiquement représentatifs du nématode basés sur des mutations dans tdp-1, l'orthologue de TARDBP chez le C. elegans. L'objectif est de caractériser ces modèles et de déterminer s'ils peuvent récapituler certains aspects phénotypiques clés de la SLA, tels que des déficits moteurs et une neurodégénérescence dépendante de l'âge générant une paralysie. L’hypothèse est que le modèle TDP-1 pourra refléter plus précisément l’expression physiologique du gène dans la maladie humaine grâce à la mutation dans un gène endogène, l’absence de surexpression et l’expression ubiquitaire de la protéine TDP-1. Les résultats montrent que les modèles TDP-43/TDP-1 ont des déficits moteurs, une transmission synaptique altérée et une neurodégénérescence liée à l’âge. Cependant, seule la mutation dans TDP-43 semble avoir un effet sur la durée de vie. Ces modèles procurent différentes expressions physiologiques des protéines mutantes et donc, des phénotypes de niveaux d'intensité variables. Ils constitueront des outils utiles pour élucider de nouveaux mécanismes pathogéniques de la SLA ainsi que de bons candidats pour le criblage de médicaments et le développement de stratégies thérapeutiques. / Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive and selective loss of motor neurons. ALS is incurable and there are no effective treatments available for people living with the disease. About 90% of the cases are sporadic whereas 10% are familial, and patients usually die two to five years after symptom onset. Many gene defects are associated with ALS, including mutations in genes encoding FUS, C9orf72, SOD-1 and TDP-43 proteins. We have developed a transgenic Caenorhabditis elegans model expressing human mutant TDP-43(Q331K) in GABAergic motor neurons. We have also obtained by mutagenesis and CRISPR-Cas9 physiologically accurate models based on mutations in tdp-1, the C. elegans ortholog of TARDBP. Our objective is to characterize these models and determine if they can recapitulate key aspects of the disease such as motor deficits and age-dependent neurodegeneration causing paralysis. We believe that the TDP-1 model will reflect more precisely the physiological expression of the gene in the human disease because of its mutation in an endogenous gene, the absence of overexpression and ubiquitous protein expression. Our results show that both TDP-43 and TDP-1 models have motor deficits, synaptic transmission impairments and age-dependent neurodegeneration. However, only the TDP-43 mutation seems to have an effect on lifespan. These models provide different physiological expression of mutant proteins and thus phenotypes of varying intensity levels. They will be useful tools to elucidate new pathogenic mechanisms of ALS as well as being good candidates for drug screening and developing therapeutic strategies.

Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate.

Ehrenamtliche Deutschvermittlungspraxis will sichtbar gemacht und mit professionellen Ansätzen, Theorien und Forschung in Dialog gebracht werden. Der Fachtag „Deutsch von Anfang an für Flüchtlinge fördern“ (DAFF) ist einer der ersten Schritte, den diesbezüglichen Austausch zwischen Theorie und Praxis anzustoßen und damit eine Grundlage für die Systematisierung und Fundierung ehrenamtlicher Angebote der Sprachbegleitung in den ersten Aufenthaltsmonaten und darüber hinaus, z.B. auf dem Weg in die Arbeitswelt, zu schaffen. Mit Unterstützung des Fachverbandes Deutsch als Fremd- und Zweitsprache (FaDaF) und in Kooperation mit der Initiative Deutschkurse Asyl Migration Flucht (DAMF), einer Projektgruppe des Ausländerrats Dresden, ist der Dialog im August 2016 am Zentrum für Integrationsstudien (ZfI) der TU Dresden initiiert worden. Die vorliegende Dokumentation lässt den ersten Fachtag Revue passieren, fasst die zentralen Ergebnisse zusammen und bietet die Grundlage für einen nachhaltigen Austausch – mit dem Ziel, eine Schnittstelle zwischen ehrenamtlichen und institutionellen Angeboten zu gestalten, Erfahrungs- und Forschungswissen aufeinander zu beziehen und so dazu beizutragen, dass Fachwissenschaftler*innen, professionelle Lehrkräfte und ehrenamtliche Sprachbegleiter*innen miteinander arbeiten und sprechen, anstatt lediglich übereinander zu reden.:Vorwort 5 Grußwort von Petra Köpping, Sächsische Staatsministerin für Gleichstellung und Integration 7 1. Einleitung 9 2. Sprachlehre und Sprachbegleitung zwischen Ehrenamt und Institution: Idee und Tagungskonzept 11 3. Perspektiven zum Thema 3.1 Das Eröffnungspanel 13 3.2 Spannungsverhältnisse und wissenschaftliche Reflexion Rebecca Zabel (Herder-Institut der Universität Leipzig) 14 3.3 Integration durch Sprache Dorothea Spaniel-Weise (Friedrich-Schiller-Universität Jena) 15 3.4 Lehrmaterialien in der Erstaufnahmeeinrichtung Christina Schanzleh (Langenscheidt Verlag) 17 3.5 Ehrenamtlicher Einsatz und Qualitätssicherung Rico Ehren (Initiative DAMF, Student der TU Dresden) 18 3.6 Arbeitsmarktzugang und Arbeitsmarktintegration Solveig Buder (Verein Jugend . Arbeit . Bildung e.V.) 20 4. Erfahrungsaustausch, Fragen und Lösungskonzepte 4.1 Poster und Verständigung 23 4.2 Disqspace – der Diskussion Raum geben 24 5. Abschlussplenum 27 6. Fazit und Ausblick 31 Anhang Qualitätsmanagement natürlich auch im Ehrenamt, Poster von Deutschkurse Asyl Migration Flucht (DAMF) 34 Deutschbuch und Deutschkurs der Heilsarmee Dresden, Poster der Heilsarmee Dresden 35 Das Wormser Modell, ein Weiterbildungskonzept für ehrenamtliche Deutschlernhelfer*innen, Poster von Sandra Sulzer (TU Darmstadt) 36 Serlo ABC: Eine intuitive Open-Source-App zur Alphabetisierung im Selbststudium, Poster der Organisation Serlo 37 Spracherwerb und Verständigung, Poster des Sächsischen Staatsministeriums für Soziales und Verbraucherschutz (SMS) 38 „Deutsch im Keller“, Poster des Netzwerks „Willkommen in Löbtau“ 39 Lernen – Lehren – Helfen (Kooperationsprojekt zwischen der Deutsch-Uni Online und dem Institut für Deutsch als Fremdsprache der Ludwig-Maximilians-Universität), Poster von Anne Biedermann 40