Le canal calcique de type L, Cav1.2, joue un rôle clé dans le couplage excitation-contraction des myocytes ventriculaires. Il a été montré que la sous-unité Cavα1 était sujette à l’épissage alternatif et que ce phénomène pouvait mener à une protéine tronquée en C-terminal au niveau de l’exon 45 (Liao, Yong et al. 2005). D’autres groupes ont étudié différentes délétions au niveau de l’extrémité C-terminale (De Jongh, Warner et al. 1991; Gao, Cuadra et al. 2001). Les courants mesurés dans la configuration cellule entière, était significativement plus grands que le canal « pleine longueur ». Nous avons décidé de tester certaines de ces délétions (ΔC2030, ΔC1935, ΔC1856, ΔC1733, ΔC1700) en présence ou en absence de la sous-unité auxiliaire Cavβ3, susceptible d’interagir avec l’extrémité C-terminale de la sous-unité Cavα1 par l’intermédiaire de son domaine SH3 (Lao, Kobrinsky et al. 2008). Les résultats obtenus dans les ovocytes de Xénope ont mis en évidence que les sous-unités Cavα1.2 tronquées montraient des courants globaux plus élevés que le canal « pleine longueur » en présence de la sous-unité auxiliaire Cavβ3 et que les sous-unités Cavα1.2 tronquées donnaient des courants en absence de la sous-unité Cavβ3 contrairement à la sous-unité Cavα1.2 « pleine longueur ». Afin de vérifier si l’augmentation des courants macroscopiques était le résultat d’une augmentation du nombre de sous-unités Cavα1.2 à la membrane, nous avons choisi de quantifier la fluorescence spécifiquement due à cette sous-unité en utilisant la méthode de cytométrie de flux (FACS : « Fluorescence Activated Cell Sorting »). L’épitope HA a été inséré dans une région extracellulaire de la sous-unité Cavα1 du canal calcique Cav1.2 et un anticorps anti-HA couplé au FITC (« Fluorescein IsoThioCyanate ») a été utilisé pour observer la fluorescence. Nos résultats confirment que la sous-unité Cavα1-HA du canal calcique Cav1.2, s’exprime à la membrane plasmique en présence de la sous-unité auxiliaire Cavβ3, et qu’en absence de celle-ci, ne s’exprime que peu ou pas à la membrane. Les mêmes résultats ont été obtenus pour les trois délétions testées dans les mêmes conditions soit Cavα1.2-HA ΔC1935, Cavα1.2-HA ΔC1856 et Cavα1.2-HA ΔC1733. Ensemble, ces résultats suggèrent que l’augmentation des courants macroscopiques observés après une délétion partielle du C-terminal n’est pas causée par une augmentation du nombre de protéines Cavα1.2 à la membrane. / The L-type calcium channel, Cav1.2, plays an important role in the excitation-contraction coupling of the ventricular myocytes. It has been shown that the alternative splicing of Cavα1.2 subunit could lead to a truncated protein in the C-terminus at exon 45 (Liao, Yong et al. 2005). Many groups have studied deletions in the C-terminus (De Jongh, Warner et al. 1991; Gao, Cuadra et al. 2001). The currents, measured in the whole cell configuration, were significantly higher with the full-length channel. We chose to test some of these deletions (ΔC2030, ΔC1935, ΔC1856, ΔC1733, ΔC1700) in the presence or absence of the Cavβ3 auxiliary subunit which is likely to interact with the C-terminus of the Cavα1.2 subunit through its SH3 domain (Lao, Kobrinsky et al. 2008). The truncated Cavα1.2 subunit, expressed in Xenopus Oocytes, showed macroscopic currents that were greater than those of the full length channel in presence of the Cavβ3 subunit. In addition, the truncated Cavα1.2 subunits displayed currents in the absence of the Cavβ3 subunit in contrast with the Cavα1.2 full length subunit. To investigate whether the larger macroscopic currents resulted in an increase in the number of Cavα1.2 subunits at the plasma membrane, we chose the FACS (« Fluorescence Activated Cell Sorting ») method. An HA-tag was inserted in an extracellular region of the Cavα1.2 subunit and a FITC (« Fluorescein IsoThioCyanate ») coupled anti-HA antibody was used to measure fluorescence. Our results showed that the Cavα1.2-HA subunit of L- type channel is expressed at the plasma membrane in the presence of the Cavβ3 subunit whereas the Cavα1.2-HA subunit is slightly or not expressed at the plasma membrane in its absence. The same results were obtained for the three C-terminal deletions tested under the same conditions (CaVα1.2-HA ΔC1935, CaVα1.2-HA ΔC1856 and CaVα1.2-HA ΔC1733). Taken together, these results suggest that the increased macroscopic currents observed after a partial deletion of the C-terminus is not caused by an increased number of Cavα1.2 proteins expressed at the plasma membrane. Keywords:

Le canal calcique de type L, Cav1.2, joue un rôle clé dans le couplage excitation-contraction des myocytes ventriculaires. Il a été montré que la sous-unité Cavα1 était sujette à l’épissage alternatif et que ce phénomène pouvait mener à une protéine tronquée en C-terminal au niveau de l’exon 45 (Liao, Yong et al. 2005). D’autres groupes ont étudié différentes délétions au niveau de l’extrémité C-terminale (De Jongh, Warner et al. 1991; Gao, Cuadra et al. 2001). Les courants mesurés dans la configuration cellule entière, était significativement plus grands que le canal « pleine longueur ». Nous avons décidé de tester certaines de ces délétions (ΔC2030, ΔC1935, ΔC1856, ΔC1733, ΔC1700) en présence ou en absence de la sous-unité auxiliaire Cavβ3, susceptible d’interagir avec l’extrémité C-terminale de la sous-unité Cavα1 par l’intermédiaire de son domaine SH3 (Lao, Kobrinsky et al. 2008). Les résultats obtenus dans les ovocytes de Xénope ont mis en évidence que les sous-unités Cavα1.2 tronquées montraient des courants globaux plus élevés que le canal « pleine longueur » en présence de la sous-unité auxiliaire Cavβ3 et que les sous-unités Cavα1.2 tronquées donnaient des courants en absence de la sous-unité Cavβ3 contrairement à la sous-unité Cavα1.2 « pleine longueur ». Afin de vérifier si l’augmentation des courants macroscopiques était le résultat d’une augmentation du nombre de sous-unités Cavα1.2 à la membrane, nous avons choisi de quantifier la fluorescence spécifiquement due à cette sous-unité en utilisant la méthode de cytométrie de flux (FACS : « Fluorescence Activated Cell Sorting »). L’épitope HA a été inséré dans une région extracellulaire de la sous-unité Cavα1 du canal calcique Cav1.2 et un anticorps anti-HA couplé au FITC (« Fluorescein IsoThioCyanate ») a été utilisé pour observer la fluorescence. Nos résultats confirment que la sous-unité Cavα1-HA du canal calcique Cav1.2, s’exprime à la membrane plasmique en présence de la sous-unité auxiliaire Cavβ3, et qu’en absence de celle-ci, ne s’exprime que peu ou pas à la membrane. Les mêmes résultats ont été obtenus pour les trois délétions testées dans les mêmes conditions soit Cavα1.2-HA ΔC1935, Cavα1.2-HA ΔC1856 et Cavα1.2-HA ΔC1733. Ensemble, ces résultats suggèrent que l’augmentation des courants macroscopiques observés après une délétion partielle du C-terminal n’est pas causée par une augmentation du nombre de protéines Cavα1.2 à la membrane. / The L-type calcium channel, Cav1.2, plays an important role in the excitation-contraction coupling of the ventricular myocytes. It has been shown that the alternative splicing of Cavα1.2 subunit could lead to a truncated protein in the C-terminus at exon 45 (Liao, Yong et al. 2005). Many groups have studied deletions in the C-terminus (De Jongh, Warner et al. 1991; Gao, Cuadra et al. 2001). The currents, measured in the whole cell configuration, were significantly higher with the full-length channel. We chose to test some of these deletions (ΔC2030, ΔC1935, ΔC1856, ΔC1733, ΔC1700) in the presence or absence of the Cavβ3 auxiliary subunit which is likely to interact with the C-terminus of the Cavα1.2 subunit through its SH3 domain (Lao, Kobrinsky et al. 2008). The truncated Cavα1.2 subunit, expressed in Xenopus Oocytes, showed macroscopic currents that were greater than those of the full length channel in presence of the Cavβ3 subunit. In addition, the truncated Cavα1.2 subunits displayed currents in the absence of the Cavβ3 subunit in contrast with the Cavα1.2 full length subunit. To investigate whether the larger macroscopic currents resulted in an increase in the number of Cavα1.2 subunits at the plasma membrane, we chose the FACS (« Fluorescence Activated Cell Sorting ») method. An HA-tag was inserted in an extracellular region of the Cavα1.2 subunit and a FITC (« Fluorescein IsoThioCyanate ») coupled anti-HA antibody was used to measure fluorescence. Our results showed that the Cavα1.2-HA subunit of L- type channel is expressed at the plasma membrane in the presence of the Cavβ3 subunit whereas the Cavα1.2-HA subunit is slightly or not expressed at the plasma membrane in its absence. The same results were obtained for the three C-terminal deletions tested under the same conditions (CaVα1.2-HA ΔC1935, CaVα1.2-HA ΔC1856 and CaVα1.2-HA ΔC1733). Taken together, these results suggest that the increased macroscopic currents observed after a partial deletion of the C-terminus is not caused by an increased number of Cavα1.2 proteins expressed at the plasma membrane. Keywords:

The ubiquitin-proteasomal system which is the major pathway for intracellular protein degradation is also involved in sperm quality control in the mammalian epididymis. Defective sperm become surface- ubiquitinated during epididymal passage. The level of sperm surface ubiquitination negatively correlates with their quality. Hypothetically it is possible that after fertilization, highly ubiquitinated sperm, naturally present in mammalian ejaculates, would be actively recognized by oocyte (probably via 26S proteasomal complex). Subsequent partial or total sperm degradation should negatively affect the development of the potentially defective embryo. In this study, we examined the effect of sperm ubiquitination on the early embryonic development in pig (Sus scrofa f. domestica) using the method of intracytoplasmic sperm injection (ICSI). In vitro embryonic development to the blastocyst stage after ICSI was comparable with other laboratories. In this study, no significant difference was observed in the formation of pronuclei between oocytes fertilized by lower and highly ubiquitinated sperm cells. On the other hand, significantly better embryonic development to the blastocyst stage was observed in oocytes fertilized by sperm with lower surface ubiquitination (17 %) compared with oocytes fertilized by highly...

The current welfare-to-work reform in Britain is activating lone parents with older children and marks a step-change in the treatment of lone parents. While there has been some support for using age of child as selection criterion for the activation of lone parents, it is not clear whether this equates to selecting by ‘ability to work’ if interpreted as ability to obtain a job. The commitment of the current government to evidence-based-policy-making and the large amount of research available in this area form the justification for carrying out a policy appraisal of this aspect of the current welfare-to-work reform. The potential and likelihood to make substantial progress towards the lone parent employment and the child poverty target of the selection criteria will be assessed and compared to alternative approaches. Five selection models are identified in the international policy review: selection by age of child, transition status, employability or by caseworkers and finally, a voluntary model. The analysis is based on a critical discussion of the available evidence, an international policy review and secondary analysis of the Families and Children Study. I argue that the current approach of selecting lone parents by the age of child is unlikely to result in substantial progress towards the lone parent employment target and instead likely to create a substantial group of long-term unemployed lone parents. Alternative approaches, such as using different selection criteria that take into account the employability of lone parents are more likely to make progress towards the employment and child poverty target.

Experimentelle FACS-Flow-Analysen im Kontext einer radiogenen Lungenfibrose zur Veränderung der Zellproliferation von Maus-Lungen-Fibroblasten unter dem Einfluss von Kulturüberständen bestrahlter Fibroblasten. Additiv einzelne Versuche mit Antikörperzugabe gegen TGF-beta zur Evaluation eines hemmenden Effektes auf eine postulierte Arretierung der Fibroblasten in der G1-Phase der Zellteilung durch die Zytokine der Kulturüberstände bestrahlter Maus-Lungen-Fibroblasten. / FACS-Flow-Analyses on changes of cell-proliferations of mouse-lung-fibroblasts under the influence of culture-supernatants of irradiated fibroblasts in the context of a radiogenic lung fibrosis. Additionally some studies on a postulated inhibitory effect of an tgf-beta-antibody on the g1-phase cell-cycle-arrest of mitosis through cytokines in the supernatants of the irradiated fibroblasts.

Det här examensarbetet undersöker om ett anatomiskt system framtaget för att kategorisera ansiktsrörelser inom psykologisk forskning kan anpassas på en ansiktsrigg för att effektivare kunna framställa ansiktsanimation för datorspel. Rapporten beskriver varför Facial Action Coding System skapades, hur det används samt tar upp olika sammanhang som systemet tidigare använts för. Rapporten beskriver det praktiska arbetet för att konstruera en ansiktsrigg ämnat för datorspel och diskuterar skillnader mellan att rigga för datorspel och film. Examensarbetet beskriver klassiska användbarhetsprinciper och vad man bör tänka på när produkten skall bli så användningsbar som möjligt. Ett animationsverktyg baserat på FACS och de ”Action Units” som systemet innehåller har tagits fram för att genomföra undersökningen på animatörerna från min praktikplats på Starbreeze Studios AB. Produkten uppskattades av dess användare och slutsatser gick att dras i förhållande till rapportens frågeställning. FACS-systemet utvecklades dock inte för datorspel, vilket märks eftersom funktioner såsom fonems dessvärre saknas bland de fördefinierade ”Action Units” som annars hade effektiviserat systemet ytterligare. 80 % av respondenterna skulle kunna tänka sig att använda det framtagna systemet för en spelproduktion vilket pekar på FACS potential inom Spelproduktion.

A fixed population of oocytes within primordial follicles, established prior to or just after birth has been firmly believed to support the postnatal mammalian ovary throughout an individual’s reproductive lifespan. However, the identification and isolation of cells from adult mammalian ovaries characterised by the expression of both germ and stem cell markers, suggest the presence of mitotically active cells, termed oogonial stem cells (OSCs) that may have the potential to produce new oocytes in the postnatal mammalian ovary. Putative OSCs have been isolated from adult tissues of several mammalian species, including rodents and humans. Upon reintroduction to the ovarian niche, human and rodent OSCs have generated new oocyte like structures which, at least in rodents, have generated functional oocytes capable of fertilisation and subsequent embryonic development to produce healthy offspring. We hypothesised that OSCs could be isolated from adult bovine ovaries and upon establishment within the appropriate ovarian niche could initiate successful oogenesis. To investigate this hypothesis, we have utilised fluorescently activated cell sorting (FACS) to isolate putative bovine OSCs (bOSCs) and an ovarian aggregate model, in vitro and in vivo to explore the oogenic potential of these cells. Putative bOSCs were successfully isolated by FACS based on the cell surface expression of germ cell marker DDX4 and established in culture. Pluripotency (LIN28 and OCT4) and germ (IFITM3, PRDM1, C-KIT and DAZL) cell associated genes were expressed in putative bOSCs established in culture. However, DDX4 transcripts were not consistently observed throughout bOSC culture. Aggregation of putative bOSCs with neonatal murine ovarian somatic cells to form chimeric ovarian aggregates, cultured in a hanging drop model for 7 days maintained germ cell phenotype, marked by DAZL expression. A subpopulation of putative bOSCs showed a spherical morphology, an increase in cell size and an association with neighbouring cells. Xenotransplantation of chimeric ovarian aggregates to the kidney capsule of immune deficient mice for 21 days generated multi-laminar follicles and structures with morphological similarities to primordial follicles (termed pre-primordial follicle-like structures). RNA Scope was unsuccessful in determining the origin of oocytes within chimeric ovarian aggregates. However, oocytes from pre-antral follicles in chimeric ovarian aggregates (n=6; 60.9± 3.6 μm, mean ± SEM) were significantly (P < 0.0001) larger than murine oocytes (n=38; 34.5± 1 μm, mean ± SEM) aggregated with murine ovarian somatic cells as positive controls, suggesting that these oocytes are undergoing different growth dynamics. This work has shown that putative bOSCs characterised by the expression of pluripotency and germ cell associated genes are present within adult bovine ovarian tissue and can be isolated using FACS and established in culture. These data also suggest that putative bOSCs may have the potential to undergo oogenesis and illustrate the potential use of these cells as a tool to investigate germ cell differentiation.

Philosophiae Doctor - PhD / The estimated 5 × 10³⁰ prokaryotic cells inhabiting our planet sequester some 350–550 Petagrams (1 Pg = 1015 g) of carbon, 85–130 Pg of nitrogen, and 9–14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio- rospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). The results obtained here show that the R. erythropolis cell-free system was over thirty-fold more effective than the E. coli based system based on the number of hits obtained per million double emulsions (dE) droplets screened. Six beta-xylosidase encoding genes were isolated and confirmed from twenty-eight positive dE droplets. Most of the droplets that were isolated from the same gate encoded the same enzyme, indicating that this technique is highly selective. A comparison of the hit rate of this screening approach with the traditional E. coli based fosmid library method shows that mIVC-FACS is at least 2.5 times more sensitive. Although only a few hits from the mIVC-FACS screening were selected for confirmation of beta-xylosidase activity, the proposed hit rate suggests that a significant number of positive hits are left un-accessed through the traditional clone library screening system. In addition, these results also suggest that E. coli expression system might be intrinsically sub-optimal for screening for hemicellulases from environmental genomes compared to R. erythropolis system. The workflow required for screening one million clones in a fosmid library was estimated to be about 320 hours compared to 144 hours required via the mIVC-FACS screening platform. Some of the gene products obtained in both screening platforms show multiple substrate activities, suggesting that the microbial consortia of composting material consist of microorganisms that produce enzymes with multiple lignocellulytic activities. While this platform still requires optimisation, we have demonstrated that this technique can be used to isolate genes encoding enzymes from mixed microbial genomes. mIVC-FACS is a promising technology with the potential to take metagenomic studies to the second generation of novel natural products bio-prospecting. The astonishing sensitivity and ultra-high throughput capacity of this technology offer numerous advantages in metagenomic bio-prospecting. / National Research Foundation (NRF)

Microexpression detection plays a vital role in applications such as lie detection and psychological consultations. Current research is progressing in the direction of automating microexpression recognition by aiming at classifying the microexpressions in terms of FACS Action Units. Although high detection rates are being achieved, the datasets used for evaluation of these systems are highly restricted. They are limited in size - usually still pictures or extremely short videos; motion constrained; containing only a single microexpression and do not contain negative cases where microexpressions are absent. Only a few of these systems run in real time and even fewer have been tested on real life videos. This work proposes a novel method for automated spotting of facial microexpressions as a preprocessing step to existing microexpression recognition systems. By identifying and rejecting sequences that do not contain microexpressions, longer sequences can be converted into shorter, constrained, relevant sequences which comprise of only single microexpressions, which can then be passed as input to existing systems, improving their performance and efficiency. This method utilizes the small temporal extent of microexpressions for their identification. The extent is determined by the period for which strain, due to the non-rigid motion caused during facial movement, is impacted on the facial skin. The subject's face is divided into sub-regions, and facial strain is calculated for each of these regions. The strain patterns in individual regions are used to identify subtle changes which facilitate the detection of microexpressions. The strain magnitude is calculated using the central difference method over the robust and dense optical flow field of each subject's face. The computed strain is then thresholded using a variable threshold. If the duration for which the strain is above the threshold corresponds to the duration of a microexpression, detection is reported. The datasets used for algorithm evaluation are comprised of a mix of natural and enacted microexpressions. The results were promising with up to 80% true detection rate. Increased false positive spots in the Canal 9 dataset can be attributed to talking by the subjects causing fine movements in the mouth region. Performing speech detection to identify sequences where the subject is talking and excluding the mouth region during those periods could help reduce the number of false positives.

RBMS3 belongs to the family of c-myc gene single-strand binding proteins (MSSPs) that play important roles in transcriptional regulation. Here, we show that RBMS3 functions as a tumor promoter in triple-negative breast cancer (TNBC), a highly aggressive BC subtype. Analysis of RBMS3 expression shows that RBMS3 is upregulated at both mRNA and protein levels in TNBC cells. Functionally, overexpression of RBMS3 increases cell migration, invasion and cancer stem cell (CSC) behaviors. Moreover, RBMS3 induces expression of epithelial-mesenchymal transition (EMT) and CSC markers. Conversely, loss of RBMS3 in TNBC BT549 cells inhibits cell proliferation, migration and mesenchymal phenotype. Correlation analysis shows RBMS3 is associated with TGF-β signaling. Mechanistically, RBMS3 interacts with Smad2, Smad3 and Smad4 mRNA and regulates the stability of these transcripts. Importantly, RBMS3 prevents TGF-β-induced cytostasis and apoptosis in premalignant cancer cells. Moreover, RBMS3 inversely correlates with expression of ESRPs, epithelial-specific splicing regulatory proteins that regulate morphogenesis-associated alternative splicing events. ESRPs appear to suppress EMT through distinct mechanisms: ESRP1 restricted cell migration, whereas ESRP2 prevented cell growth. RBMS3 significantly facilitates the EMT process when ESRPs are lost. Collectively, the studies within this dissertation identify RBMS3 as a positive regulator of EMT and breast cancer progression by regulating the TGF-β signaling pathway.