Cryo-negative staining: advantages and applications for three-dimensional electron microscopy of biological macromolecules

Les échantillons biologiques ne s?arrangent pas toujours en objets ordonnés (cristaux 2D ou hélices) nécessaires pour la microscopie électronique ni en cristaux 3D parfaitement ordonnés pour la cristallographie rayons X alors que de nombreux spécimens sont tout simplement trop << gros D pour la spectroscopie NMR. C?est pour ces raisons que l?analyse de particules isolées par la cryo-microscopie électronique est devenue une technique de plus en plus importante pour déterminer la structure de macromolécules. Néanmoins, le faible rapport signal-sur-bruit ainsi que la forte sensibilité des échantillons biologiques natifs face au faisceau électronique restent deux parmi les facteurs limitant la résolution. La cryo-coloration négative est une technique récemment développée permettant l?observation des échantillons biologiques avec le microscope électronique. Ils sont observés à l?état vitrifié et à basse température, en présence d?un colorant (molybdate d?ammonium). Les avantages de la cryo-coloration négative sont étudiés dans ce travail. Les résultats obtenus révèlent que les problèmes majeurs peuvent êtres évités par l?utilisation de cette nouvelle technique. Les échantillons sont représentés fidèlement avec un SNR 10 fois plus important que dans le cas des échantillons dans l?eau. De plus, la comparaison de données obtenues après de multiples expositions montre que les dégâts liés au faisceau électronique sont réduits considérablement. D?autre part, les résultats exposés mettent en évidence que la technique est idéale pour l?analyse à haute résolution de macromolécules biologiques. La solution vitrifiée de molybdate d?ammonium entourant l?échantillon n?empêche pas l?accès à la structure interne de la protéine. Finalement, plusieurs exemples d?application démontrent les avantages de cette technique nouvellement développée.<br/><br/>Many biological specimens do not arrange themselves in ordered assemblies (tubular or flat 2D crystals) suitable for electron crystallography, nor in perfectly ordered 3D crystals for X-ray diffraction; many other are simply too large to be approached by NMR spectroscopy. Therefore, single-particles analysis has become a progressively more important technique for structural determination of large isolated macromolecules by cryo-electron microscopy. Nevertheless, the low signal-to-noise ratio and the high electron-beam sensitivity of biological samples remain two main resolution-limiting factors, when the specimens are observed in their native state. Cryo-negative staining is a recently developed technique that allows the study of biological samples with the electron microscope. The samples are observed at low temperature, in the vitrified state, but in presence of a stain (ammonium molybdate). In the present work, the advantages of this novel technique are investigated: it is shown that cryo-negative staining can generally overcome most of the problems encountered with cryo-electron microscopy of vitrified native suspension of biological particles. The specimens are faithfully represented with a 10-times higher SNR than in the case of unstained samples. Beam-damage is found to be considerably reduced by comparison of multiple-exposure series of both stained and unstained samples. The present report also demonstrates that cryo-negative staining is capable of high- resolution analysis of biological macromolecules. The vitrified stain solution surrounding the sample does not forbid the access to the interna1 features (ie. the secondary structure) of a protein. This finding is of direct interest for the structural biologist trying to combine electron microscopy and X-ray data. developed electron microscopy technique. Finally, several application examples demonstrate the advantages of this newly

Aging preferentially affects molecular pathways implicated in development and disease of myelinating glial cells

The central and peripheral nervous systems are involved in multiple agedependent neurological deficits that are often attributed to alterations in function of myelinating glial cells. However, the molecular events that underlie the age-related decline of glial cell function are unknown. We used Schwann cells as a model to study biological processes affected in glial cells by aging. We comprehensively profiled gene expression of the Schwann cell-rich mouse sciatic nerve throughout life, from day of birth until senescence (840 days of age). We combined the aging data with the microarray transcriptional data obtained using nerves isolated from Schwann cell-specific neuropathy-inducing mutants MPZCre/þ/Lpin1fE2-3/fE2-3, MPZCre/þ/ScapfE1/fE1 and Pmp22-null mice. A majority of age related transcripts were also affected in the analyzed mouse models of neuropathy (54.4%) and in development (59.5%) indicating a high level of overlapping in implicated molecular pathways. We observed that compared to peripheral nerve development, dynamically changing expression profiles in aging have opposite (anticorrelated) orientation while they copy the orientation of transcriptional changes observed in analyzed neuropathy models. Subsequent clustering and biological annotation of dynamically changing transcripts revealed that the processes most significantly deregulated in aging include inflammatory/ immune response and lipid biosynthesis/metabolism. Importantly, the changes in these pathways were also observed in myelinated oligodendrocyte- rich optic nerves of aged mice, albeit with lower magnitude. This observation suggests that similar biological processes are affected in aging glial cells in central and peripheral nervous systems, however with different dynamics. Our data, which provide the first comprehensive comparison of molecular changes in glial cells in three distinct biological conditions comprising development, aging and disease, provide not only a new inside into the molecular alterations underlying neural system aging but also identify target pathways for potential therapeutical approaches to prevent or delay complications associated with age-related and inherited forms of neuropathies.

Development and application of computational methodologies in qualitative modeling

La présente thèse s'intitule "Développent et Application des Méthodologies Computationnelles pour la Modélisation Qualitative". Elle comprend tous les différents projets que j'ai entrepris en tant que doctorante. Plutôt qu'une mise en oeuvre systématique d'un cadre défini a priori, cette thèse devrait être considérée comme une exploration des méthodes qui peuvent nous aider à déduire le plan de processus regulatoires et de signalisation. Cette exploration a été mue par des questions biologiques concrètes, plutôt que par des investigations théoriques. Bien que tous les projets aient inclus des systèmes divergents (réseaux régulateurs de gènes du cycle cellulaire, réseaux de signalisation de cellules pulmonaires) ainsi que des organismes (levure à fission, levure bourgeonnante, rat, humain), nos objectifs étaient complémentaires et cohérents. Le projet principal de la thèse est la modélisation du réseau de l'initiation de septation (SIN) du S.pombe. La cytokinèse dans la levure à fission est contrôlée par le SIN, un réseau signalant de protéines kinases qui utilise le corps à pôle-fuseau comme échafaudage. Afin de décrire le comportement qualitatif du système et prédire des comportements mutants inconnus, nous avons décidé d'adopter l'approche de la modélisation booléenne. Dans cette thèse, nous présentons la construction d'un modèle booléen étendu du SIN, comprenant la plupart des composantes et des régulateurs du SIN en tant que noeuds individuels et testable expérimentalement. Ce modèle utilise des niveaux d'activité du CDK comme noeuds de contrôle pour la simulation d'évènements du SIN à différents stades du cycle cellulaire. Ce modèle a été optimisé en utilisant des expériences d'un seul "knock-out" avec des effets phénotypiques connus comme set d'entraînement. Il a permis de prédire correctement un set d'évaluation de "knock-out" doubles. De plus, le modèle a fait des prédictions in silico qui ont été validées in vivo, permettant d'obtenir de nouvelles idées de la régulation et l'organisation hiérarchique du SIN. Un autre projet concernant le cycle cellulaire qui fait partie de cette thèse a été la construction d'un modèle qualitatif et minimal de la réciprocité des cyclines dans la S.cerevisiae. Les protéines Clb dans la levure bourgeonnante présentent une activation et une dégradation caractéristique et séquentielle durant le cycle cellulaire, qu'on appelle communément les vagues des Clbs. Cet évènement est coordonné avec la courbe d'activation inverse du Sic1, qui a un rôle inhibitoire dans le système. Pour l'identification des modèles qualitatifs minimaux qui peuvent expliquer ce phénomène, nous avons sélectionné des expériences bien définies et construit tous les modèles minimaux possibles qui, une fois simulés, reproduisent les résultats attendus. Les modèles ont été filtrés en utilisant des simulations ODE qualitatives et standardisées; seules celles qui reproduisaient le phénotype des vagues ont été gardées. L'ensemble des modèles minimaux peut être utilisé pour suggérer des relations regulatoires entre les molécules participant qui peuvent ensuite être testées expérimentalement. Enfin, durant mon doctorat, j'ai participé au SBV Improver Challenge. Le but était de déduire des réseaux spécifiques à des espèces (humain et rat) en utilisant des données de phosphoprotéines, d'expressions des gènes et des cytokines, ainsi qu'un réseau de référence, qui était mis à disposition comme donnée préalable. Notre solution pour ce concours a pris la troisième place. L'approche utilisée est expliquée en détail dans le dernier chapitre de la thèse. -- The present dissertation is entitled "Development and Application of Computational Methodologies in Qualitative Modeling". It encompasses the diverse projects that were undertaken during my time as a PhD student. Instead of a systematic implementation of a framework defined a priori, this thesis should be considered as an exploration of the methods that can help us infer the blueprint of regulatory and signaling processes. This exploration was driven by concrete biological questions, rather than theoretical investigation. Even though the projects involved divergent systems (gene regulatory networks of cell cycle, signaling networks in lung cells), as well as organisms (fission yeast, budding yeast, rat, human), our goals were complementary and coherent. The main project of the thesis is the modeling of the Septation Initiation Network (SIN) in S.pombe. Cytokinesis in fission yeast is controlled by the SIN, a protein kinase signaling network that uses the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this thesis, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN. Another cell cycle related project that is part of this thesis was to create a qualitative, minimal model of cyclin interplay in S.cerevisiae. CLB proteins in budding yeast present a characteristic, sequential activation and decay during the cell cycle, commonly referred to as Clb waves. This event is coordinated with the inverse activation curve of Sic1, which has an inhibitory role in the system. To generate minimal qualitative models that can explain this phenomenon, we selected well-defined experiments and constructed all possible minimal models that, when simulated, reproduce the expected results. The models were filtered using standardized qualitative ODE simulations; only the ones reproducing the wave-like phenotype were kept. The set of minimal models can be used to suggest regulatory relations among the participating molecules, which will subsequently be tested experimentally. Finally, during my PhD I participated in the SBV Improver Challenge. The goal was to infer species-specific (human and rat) networks, using phosphoprotein, gene expression and cytokine data and a reference network provided as prior knowledge. Our solution to the challenge was selected as in the final chapter of the thesis.

The career engagement scale : Development and validation of a measure of proactive career behaviors

Careers today increasingly require engagement in proactive career behaviors; however, there is a lack of validated measures assessing the general degree to which somebody is engaged in such career behaviors. We describe the results of six studies with six independent samples of German university students (total N = 2,854), working professionals (total N = 561), and university graduates (N = 141) that report the development and validation of the Career Engagement Scale - a measure of the degree to which somebody is proactively developing her or his career as expressed by diverse career behaviors. The studies provide support for measurement invariance across gender and time. In support of convergent and discriminant validity, we find that career engagement is more prevalent among working professionals than among university students and that this scale has incremental validity above several specific career behaviors regarding its relation to vocational identity clarity and career self-efficacy beliefs among students and to job and career satisfaction among employees. In support of incremental predictive validity, beyond the effects of several more specific career behaviors, career engagement while at university predicts higher job and career satisfaction several months later after beginning work.

Molecular insight into heart development and congenital heart disease: An update review from the Arab countries.

Congenital heart defect (CHD) has a major influence on affected individuals as well as on the supportive and associated environment such as the immediate family. Unfortunately, CHD is common worldwide with an incidence of approximately 1% and consequently is a major health concern. The Arab population has a high rate of consanguinity, fertility, birth, and annual population growth, in addition to a high incidence of diabetes mellitus and obesity. All these factors may lead to a higher incidence and prevalence of CHD within the Arab population than in the rest of the world, making CHD of even greater concern. Sadly, most Arab countries lack appropriate public health measures directed toward the control and prevention of congenital malformations and so the importance of CHD within the population remains unknown but is thought to be high. In approximately 85% of CHD patients, the multifactorial theory is considered as the pathologic basis. The genetic risk factors for CHD can be attributed to large chromosomal aberrations, copy number variations (CNV) of particular regions in the chromosome, and gene mutations in specific nuclear transcription pathways and in the genes that are involved in cardiac structure and development. The application of modern molecular biology techniques such as high-throughput nucleotide sequencing and chromosomal array and methylation array all have the potential to reveal more genetic defects linked to CHD. Exploring the genetic defects in CHD pathology will improve our knowledge and understanding about the diverse pathways involved and also about the progression of this disease. Ultimately, this will link to more efficient genetic diagnosis and development of novel preventive therapeutic strategies, as well as gene-targeted clinical management. This review summarizes our current understanding of the molecular basis of normal heart development and the pathophysiology of a wide range of CHD. The risk factors that might account for the high prevalence of CHD within the Arab population and the measures required to be undertaken for conducting research into CHD in Arab countries will also be discussed.

Long noncoding RNAs in cardiac development and ageing.

A large part of the mammalian genome is transcribed into noncoding RNAs. Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators of gene expression. Distinct molecular mechanisms allow lncRNAs either to activate or to repress gene expression, thereby participating in the regulation of cellular and tissue function. LncRNAs, therefore, have important roles in healthy and diseased hearts, and might be targets for therapeutic intervention. In this Review, we summarize the current knowledge of the roles of lncRNAs in cardiac development and ageing. After describing the definition and classification of lncRNAs, we present an overview of the mechanisms by which lncRNAs regulate gene expression. We discuss the multiple roles of lncRNAs in the heart, and focus on the regulation of embryonic stem cell differentiation, cardiac cell fate and development, and cardiac ageing. We emphasize the importance of chromatin remodelling in this regulation. Finally, we discuss the therapeutic and biomarker potential of lncRNAs.

Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

The lymphatic system maintains tissue fluid balance, and dysfunction of lymphatic vessels and valves causes human lymphedema syndromes. Yet, our knowledge of the molecular mechanisms underlying lymphatic vessel development is still limited. Here, we show that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of lymphatic vessel development. Endothelial-specific Cdk5 knockdown causes congenital lymphatic dysfunction and lymphedema due to defective lymphatic vessel patterning and valve formation. We identify the transcription factor Foxc2 as a key substrate of Cdk5 in the lymphatic vasculature, mechanistically linking Cdk5 to lymphatic development and valve morphogenesis. Collectively, our findings show that Cdk5-Foxc2 interaction represents a critical regulator of lymphatic vessel development and the transcriptional network underlying lymphatic vascular remodeling.

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease.

The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.

GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.

Toward on-the-fly quantum mechanical/molecular mechanical (QM/MM) docking: development and benchmark of a scoring function.

We address the challenges of treating polarization and covalent interactions in docking by developing a hybrid quantum mechanical/molecular mechanical (QM/MM) scoring function based on the semiempirical self-consistent charge density functional tight-binding (SCC-DFTB) method and the CHARMM force field. To benchmark this scoring function within the EADock DSS docking algorithm, we created a publicly available dataset of high-quality X-ray structures of zinc metalloproteins ( http://www.molecular-modelling.ch/resources.php ). For zinc-bound ligands (226 complexes), the QM/MM scoring yielded a substantially improved success rate compared to the classical scoring function (77.0% vs 61.5%), while, for allosteric ligands (55 complexes), the success rate remained constant (49.1%). The QM/MM scoring significantly improved the detection of correct zinc-binding geometries and improved the docking success rate by more than 20% for several important drug targets. The performance of both the classical and the QM/MM scoring functions compare favorably to the performance of AutoDock4, AutoDock4Zn, and AutoDock Vina.

Guideline adaptation: a methodology to enhance efficiency in guideline development and improve utilization.

BACKGROUND: Developing and updating high-quality guidelines requires substantial time and resources. To reduce duplication of effort and enhance efficiency, we developed a process for guideline adaptation and assessed initial perceptions of its feasibility and usefulness. METHODS: Based on preliminary developments and empirical studies, a series of meetings with guideline experts were organised to define a process for guideline adaptation (ADAPTE) and to develop a manual and a toolkit made available on a website (http://www.adapte.org). Potential users, guideline developers and implementers, were invited to register and to complete a questionnaire evaluating their perception about the proposed process.

Peatlands and plantations in Sumatra, Indonesia : Complex realities for resource governance, rural development and climate change mitigation

Peatlands play a crucial role in Indonesia's economic development, and in its stated goal of reducing greenhouse gas emissions. Improved peatland management - including a national moratorium on the granting of any new conversion licenses - forms a cornerstone of Indonesia's climate change mitigation commitment. At the same time, rapid expansion of the plantation sector is driving wide-scale drainage and conversion of peat swamp ecosystems. The province of Riau, in central Sumatra, finds itself at the crossroads of these conflicting agendas. This essay presents a case study of three islands on Riau's east coast affected by industrial timber plantation concessions. It examines the divergent experiences, perceptions and responses of communities on the islands. A mix of dramatic protests, localised everyday actions and constructive dialogue has succeeded in delaying or perhaps halting one of the concessions, while negotiations and contestation with the other two continue. With the support of regional and national non-governmental organisations and local government, communities are pursuing alternative development strategies, including the cultivation of sago, which requires no peat drainage. While a powerful political economy of state and corporate actors shapes the contours of socio-environmental change, local social movements can alter trajectories of change, promoting incremental improvements and alternative pathways.