Faculty of Humanities; School of Human and Community development; MA Research Report / Social participation is one of the most debilitating effects of aphasia. Yet, to date no clear definitions or models of social participation have been developed that can be applied within aphasiology. In addition, generic stroke scales are still the outcome measures of choice within research. There is a need for patient-centred measures that accurately document and assess the experiences and perceptions of those with aphasia. Therefore, the current research aimed to investigate the social participation of adults with aphasia by extending patient-centred measures and encompassing the views of the families, particularly the spouses, of those with aphasia. Four crosssectional parallel single case studies were conducted that involved a protocol combining the quantitative measure of the ASHA FACS with the qualitative tools of semi-structured interviews and observations. In addition, social network analyses were completed for each participant. Overall, open coding of the individual participants’ results produced common themes among the people with aphasia and common themes among their spouses. Each participant reported significantly altered social participation, which permeated throughout the family unit and was felt considerably by the spouses of those with aphasia. The results are discussed in relation to current social models and approaches to intervention, while professional role expansion and the needs of the South African context are also considered. Furthermore, the concept of resilience and its implications for future research are discussed.

Un grand nombre de maladies cardiaques apparaissent à la suite de problèmes développementaux dus à des mutations dans des gènes très conservés durant l'évolution. Il est donc crucial d'identifier les acteurs intervenants dans la cardiogenèse. Durant ma thèse, j'ai étudié la différenciation cardiaque, en utilisant la Drosophile comme modèle, avec comme objectifs d'identifier des nouveaux intervenants grâce à l'acquisition de données globales par une approche génomique tissue spécifique. J'ai tout d'abord mis au point un protocole permettant d'acquérir des données génomique spécifiquement dans le tube cardiaque de la Drosophile lors de la différenciation des cardiomyocytes. Ce fut une étape primordiale pour le reste de ma thèse. Ce protocole me permet d'isoler spécifiquement les cellules du système cardiaque.A partir de là, j'ai réalisé une cinétique transcriptomique qui a permis de mettre en évidence environ 1000 gènes différenciellement exprimés au cours de la différenciation cardiaque. Ensuite, En collaboration avec Delphine Potier, une thésarde bio-informaticienne de l'équipe, des modules Cis-régulateur (CRM) pouvant conduire à l'expression dans le tube cardiaque ont été prédits, ainsi que des facteurs de transcription putatifs régulant ces CRM. Ces nouveaux acteurs du réseau de régulation génique cardiaque sont en cours de validation.Dans un second temps, je me suis intéressé à un facteur de transcription à homéoboîte clé de la cardiogenèse : Abdominal-A (AbdA). AbdA est essentiel pour la différenciation de la partie postérieure du tube cardiaque, le cœur proprement dit, et à l'acquisition de sa fonction. Cependant, ses cibles ainsi que son action tissue spécifique sont encore inconnu à ce jourAfin d'apporter un élément de réponse, j'ai analysé le transcriptome consécutif à un Gain de Fonction de AbdA spécifiquement dans le système cardiaque ce qui m'a permis de mettre en évidence plus de 1000 gènes dérégulés par AbdA lors de la différenciation cardiaque. / Cardiac diseases generally arise from developmental disorders caused by mutations in genes that are highly conserved during evolution. It is therefore of prime interest to identify actors which participate to cardiogenesis. During my thesis, I have analyzed cardiac differentiation, using Drosophila as a model. My objective was to identify news actors of the cardiac Gene Regulatory Network (GRN) using a genomic approach and starting from tissue specific whole data acquisition.First, I have set a protocol to allow tissue specific genomic data acquisition during cardiac differentiation. It was a critical step for my thesis. This protocol allowed me to isolate specifically cardiac system cells.Using this protocol, I analyzed the transcriptome dynamics and determined that 1000 genes are dynamically expressed during cardiac differentiation.Next, in collaboration with Delphine Potier, a PhD student in bio-informatic in the team, cis-regulatory modules (CRM), which can drive expression in the cardiac tube, have been predicted, and also putative transcription factors regulating these CRM. These new cardiac GRN actors are currently tested in vivo.In a second time, I have analyzed the function of Abdominal-A (AbdA) a homeobox transcription factor which plays a key role during cardiogenesis. :. AbdA is crucial for the differentiation of the posterior part of the organ, called heart My aim was to identify cardiac specific AbdA targets.

Ausgehend von einer potentiell anti-Tumor-aktiven B-Zellen des menschlichen Immunsystem haben sich durch die Untersuchung des peripheren Blutes auf das Vorliegen von lambda3r-positiven, CD19+B-Zellen bei Magenkarzinompatienten und Probanden unterschiedlichen Alters einige sehr interessante Ergebnisse im Bereich der B-Zellimmunität ergeben. Es scheint dabei eine Art B-Zell-Immunosurveillance in Form dieser B-Zellen, sowohl bei Karzinompatienten, als auch bei Gesunden von früher Kindheit an zu geben. Die relative Verteilung dieser Zellen ändert sich dabei im Laufe des Lebens ensprechend den Veränderungen des gesamten B-Zellkompartiments. Es findet eine Abnahme mit dem Alter statt. Im Falle des Auftretens eines Magenkarzinoms kommt es dann zu einer relativen Expansion der in dieser Arbeit beschriebenen lambda3r-positiven CD19+B-Zellen trotz einer gleichzeitig stattfindenden bisher nicht erklärlichen Involution des restlichen B-Zellsystems. Bei der relativen Zunahme dieser Zellen handelt es sich um eine Art Boosterung. Das expandierte Zellkompartiment zeigt dabei Reifungstendenzen, sichtbar im Verlust des Oberflächenmoleküls IgD sowie der Expression von CD27 und IgG. Dem Oberflächenmarker CD5 scheint im Gegensatz zur initialen Hypothese bei der Erst-beschreibung der SC-1-positiven B-Zelle keine zentrale Rolle zuzukommen. / This project could identify a subset of CD19-positive b-cells within humans that shows strong correlation to the event of stomach cancer. This subset was proven to be in peripheral blood of healthy donors from early ages on and seems to be part of a kind of immunosurveillance system. In case of stomach cancer there is a differentiation of these cells from naive into mature b-cells. The main characteristics of the analysed cells is the CD19 antigen and the expression of the light chain gene lambda 3r in about 90%. In this study we compared the frequency and subtypes of these cells in healthy donors and stomach cancer patients. This setting allowed us also the quantitative and qualitative comparison of tho whole b-cell compartment at different ages and in the case of malignancy.

The neural crest is a transient population of cells that forms a diverse range of derivatives in vertebrate embryos. Neural crest cells also migrate extensively throughout the embryo. The specification of a number of neural crest derivatives, including pigment cells and neurons and glia of the peripheral nervous system, is dependent on the transcription factor Sox10. In sox10 mutant zebrafish embryos, these neural crest derivatives fail to specify and subsequently the cell differentiation and migration fails leading to apoptosis. Sox10 mutant embryos also display an ear defect although the precise role of Sox10 in the ear is less well defined. Thus Sox10 controls an extensive gene regulatory network that drives the development of an important subset of neural crest derivatives and also functions during ear development. This gene regulatory network is currently poorly defined. The aim of this project was to identify genes that are both direct and indirect targets of Sox10 to further elucidate this gene regulatory network. To achieve this, a microarray approach was adopted. Initially, fluorescence activated cell sorting was employed to enrich for sox10 expressing cells from 24 hours post fertilization sox10:GFP transgenic embryos. The transcriptomes of WT and sox10 mutant cells were compared by microarray analysis to identify differentially regulated genes. A large number of target genes were identified by this method and by an unbiased in situ hybridization screen, 28 genes were validated. Of these, 23 genes were expressed in cells of the neural crest and down-regulated in sox10 mutant embryos. The majority of these genes were expressed in cells of the melanocyte and xanthophore lineages. 5 genes were expressed in the ear (otic vesicle) of which three otic vesicle genes were down-regulated while two otic vesicle genes were up-regulated in sox10 mutant embryos. Unfortunately due to time constraints, a study into the function of one of these target genes could not be completed. The series of validated genes identified during this project has opened new opportunities for research and has identified a number of highly expressed marker genes that will be useful in future studies. In addition, the microarray data presented will be a useful resource to aid the identification of further targets of Sox10.

De neutrofila granulocyterna ingår i det icke specifika immunförsvaret och fagocyterarmikroorganismer och rester från kroppens egna celler. Neutrofilen innehåller fyra olika typerav granula, som vid aktivering av cellen kan uppregleras till plasmamembranet. Proteinetdysferlin har bevisats förekomma i membranet i en del av dessa granuler. Det tros ha en roll imembran fusion och underhåll av membranet.Dysferlin är ett transmembrant protein av typ II, och proteinet saknas i funktionellt tillståndvid sjukdomstillstånden Limb-Girdle muskeldystrofi 2B och Miyoshi myopati. I arbetetkommer en metod att utvecklas som skall användas till att studera proteinet dysferlin hos deneutrofila granulocyterna vid olika funktionella tillstånd. Mängden dysferlin iplasmamembranet hos oaktiverade och aktiverade celler jämförs också i arbetet.Antikroppar har använts för att märka in dysferlinet. En primär antikropp som binder tillproteinet och en sekundär antikropp som binder till den primära. Den sekundära antikroppenhar en fluorescent färg konjugerad. För analyserna har en FACS som ger diagram överfluorescenssignalen använts och ett konfokalmikroskop som avbildar cellen i olika skikt, vilkakan användas för att ta fram en tredimensionell bild av cellen.Både intracellulär och extracellulär inmärkning av dysferlin har gjorts och olika parametrarhar varierats mellan försöken. FACS analyserna har visat på att dysferlin möjligtvis skullekunna finnas i små mängder i plasmamembranet hos oaktiverade celler. Mängden dysferlin iplasmamembranet ökar vid aktivering av cellen, till följd av uppreglering av granula. Studier ikonfokalmikroskopet har visat på att dysferlin finns i cellmembranet hos aktiverade celler ochatt det finns lokaliserat till cytoplasman hos oaktiverade celler.

Homeostatic plasticity is important in neurobiology for stabilising neuronal networks in the face of Hebbian forms of synaptic plasticity that are thought to mediate memory storage. Impairment of homeostatic plasticity has also been implicated in neurological diseases such as Rett syndrome and fragile X syndrome. Homeostatic plasticity can be achieved through scaling of the strength of synaptic connections between neurones or by changes in intrinsic excitability. While homeostatic plasticity has been studied mainly using in vitro preparations, it is for the most part not known whether changes of neural activity in vivo induce homeostatic changes. The molecular pathway responsible for homeostatic plasticity still remains unclear. In this thesis, I have used stereotaxic surgery to over express Kir2.1, an inwardly rectifying potassium channel, in vivo in the brains of adult mice. I show that the expression of Kir2.1 through adeno-associated virus (AAV) does not cause any adverse effects in the dentate gyrus nor the CA1 of the mouse hippocampus. I go on to use slice patch clamp methods to measure the change in electrical properties of granule cells in the dentate gyrus and pyramidal cells in CA1 caused by expression of Kir2.1. I show that the excitability of neurones expressing Kir2.1 was reduced compared to control neurones. By 2 weeks after virus injection the neurones showed homeostatic plasticity in response to Kir2.1 over expression. Interestingly, the mechanism of adaptation was different in different types of cells; dentate gyrus granule cells adapted through change in their intrinsic excitability, whereas CA1 pyramidal cells adapted by modifying the strength of their synaptic inputs. To establish whether induction of homeostatic plasticity is associated with changes in gene expression I used fluorescent activated cell sorting (FACs) to isolate pure population of neurones infected with viruses. I then sequenced RNA extracted from neurones expressing Kir2.1 and control neurones. Analysis of the RNAseq data revealed molecular candidates involved in homeostatic plasticity. In summary, I show that Kir2.1 over expression causes change in excitability and subsequent homeostatic plasticity in vivo. The mechanism of adaptation differs between cell types. RNAseq results identify novel candidates for future investigation.

Cartilage lesion is a fairly common problem in orthopaedic practise. It is often a consequence of traumas, inflammatory conditions, and biomechanics alterations. However, as an avascular and aneural tissue, articular cartilage has minimal healing ability. Over the past decades, surgeons and scientists have proposed a nubmer of treatment strategies to promote restoration of articular cartilage, like arthroscopic lavage, microfracture surgery, osteochoncral autografts and allografts, autologous chondrocyte implantation, and other cell-based repairs. Nevertheless, these solutions often result in fibrocartilage, which has inferior mechanical and biochemical properties, with increased susceptibility to injury, which usually ultimately leads to osteoarthritis (OA). Stem cell therapy techniques are widely applied in treating disease or injury. Many medical researchers have proposed stem cell transplantation treatment for enhancing cartilage repair by using mesenchymal stem cells (MSCs) along with biocompatible scaffolds. In addition to that, chondrogenic progenitor cells (CPCs) have also been discovered in OA patients and healthy articular cartilage. However, neither the method for isolating CPCs is not well established, nor the origin and function is not fully understood. Stem cells may be measured in CFUs (Colony-forming units). Ideally, adult stem cells should be clonogenic. In other words, a single adult stem cell should be able to generate a line of genetically identical cells. Fully characteraization of stem/progenitor cell potential requires purified population. Single-cell cloned population maybe serve as a convincing source for study of stem/progenitor cells. Therefore, a single cell clonogenecity screening system was developed to identify and isolate putative stem/progenitor cells from cartilage based on fluorescence-activated cell sorting (FACS). Also, genetical and functional characterization of isolated cells was taken.

3D animation has developed rapidly in the multimedia nowadays, in computer games, virtual reality and films. Therefore, how to make a 3D model which is really true to life, especially in the facial expressions, and can have vivid actions, is a significant issue. At the present time, the methods to construct 3D facial model are divided into two categories: one is based on computer graphic technology, like geometric function, polygon, or simple geometric shapes, the other one is using hardware to measure a real face by laser scanning system, and three-dimensional digitizer. Moreover, the method to acquire the 3D facial expression primarily are applied as following: keyframing, motion capture, and simulation. The research covers two areas: 1. Use two CCDs to digitalize the facial expressions of a real person simultaneously from both right and left side, and save the obtained standard image. Then, get the feature match points from the two standard images in the space domain, and by using the Stereo to attain the ¡§depth information¡¨ which helps to build 3D facial model. 2. Use one CCD to continuously digitalize two facial expressions and get the feature match points¡¦ coordinates in the time domain to calculate the motion vector. By combining the ¡§depth information¡¨ from space domain and the motion vector from the time domain, the 3D facial model¡¦s motion sequence can be therefore obtained. If sufficient digitalized facial expressions are processed by the 3D facial model¡¦s motion sequence, a database could be built. By matching the feature points between the 2D test image and 2D standard image in the database, the standard image¡¦s ¡§depth information¡¨ and motion vector can be used and turn the test image into 3D model which can also imitate the facial expressions of the standard images sequences. The method to match the feature points between the test image and standard images in the database can be entirely processed by computers, and as a result eliminate unnecessary human resources.

Engineering protease substrate specificity and selectivity has the potential to yield entirely new possibilities in the analytical, biotechnological, and therapeutic domains. For example, therapeutic applications can be envisioned in which engineered proteases could replace antibodies by irreversibly inactivating a large excess of disease-associated target proteins in a catalytic fashion. Technological advances in molecular biology have made laboratory-based evolution techniques for protein engineering readily accessible. However, the ability to interrogate the activities and substrate preference of large numbers of protease variants is predicated on the availability of quantitative high-throughput assays that maintain the essential link between genotype and phenotype. In this work we have investigated a variety of novel single cell fluorescence assays and selections for engineering protease substrate specificity and selectivity, and demonstrated the utility of some of these systems for the engineering of novel enzymes. The second chapter of this dissertation reports the isolation of a highly active ([chemical formula]) variant of the Escherichia coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine, sulfotyrosine, phosphotyrosine and phosphoserine. The isolation of protease variants that can discriminate between substrates based on the posttranslational modification of Tyr was made possible by implementing a multi-color flow cytometric assay using multiple simultaneous counter-selection substrates for the screening of large mutant libraries. While primary sequence recognition may suffice for proteomic applications, many therapeutic applications of engineered proteases will require the cleavage of folded protein targets. Unfortunately, we have found that engineered proteases that can cleave peptides very efficiently are often unable to digest the same sequences inserted into the loop regions of a folded protein. The logical conclusion, then, is that an entire target protein or at least a protein domain, rather than peptide segments, must be incorporated into protease engineering screening assays. As a critical first step toward the development of next generation, single cell screening systems for therapeutic protease engineering we have developed novel assays that exploit cell surface capture of exogenous protein substrates. One assay (Chapter 3) relies on an autoinhibited protein fusion that capitalizes on the p53 antagonist MDM2 as a detector of protease activity in addition to its utility as a counter-selection substrate. Using this system we successfully isolated OmpT variants that selectively cleave a designed site within our autoinhibited substrate. A second high-throughput screen (Chapter 4) monitors native protein cleavage. Target proteins are captured at the cell surface using a polycationic tail, incorporating counter-selection, and the proteolytic state of the substrate can be monitored using epitope tags fused to the N-and C-termini and fluorescently labeled anti-epitope tag antibodies. / text

Im Rahmen der vorliegenden Dissertation wurde mithilfe eines 4 stündigen Radmarathons die zeitabhängige Freisetzung von zirkulierenden Vorläuferzellen untersucht. Hierzu wurde den 17 gesunden, gut trainierten Probanden während der Prozedur zu festgelegten Zeitpunkten Blut aus einer Venenverweilkanüle entnommen. Daraus wurden mittels Dichtegradient die mononuklearen Zellen isoliert und im Anschluss mittels Durchflusszytometrie die Konzentration der zirkulierenden Progenitorzellen quantifiziert. Es ließ sich ein signifikanter zeitversetzter Anstieg der Konzentration hämatopoietischer Progenitorzellen (CD34pos und CD133pos) wie auch endothelialer Progenitorzellen (CD34/KDRpos und CD133/KDRpos) ermitteln. Die maximale Konzentrationsänderung wurde nach 210 Minuten erreicht. Als Freisetzungsmediatoren wurden in dieser Arbeit die Konzentration von VEGF und IL-6 bestimmt. Auch hier stellte sich ein signifikanter Anstieg der Spiegel dar. Die Konzentrationsänderungen der einzelnen Zellpopulationen waren spätestens 24 Stunden nach Beendigung des Radmarathons nicht mehr nachweisbar. Des Weiteren ging die Arbeit der Frage nach, wie sich die Konzentration von Mikropartkel und endothelialer Mikropartikel als Marker für einen Endothelzellschaden auswirkt. Ein Anstieg der Konzentration reifer (CD146pos) Endothelzellen wurde verzeichnet. Ausgangspunkt dieser Arbeit war die sehr beschränkte und kontroverse Datenlage bezüglich der Konzentrationen von Progenitorzellen freigesetzt durch sportliche Aktivität.