Vascular biology of magnesium and its transporters in hypertension

Magnesium may influence blood pressure by modulating vascular tone and structure through its effects on myriad biochemical reactions that control vascular contraction/dilation, growth/apoptosis, differentiation and inflammation. Magnesium acts as a calcium channel antagonist, it stimulates production of vasodilator prostacyclins and nitric oxide and it alters vascular responses to vasoconstrictor agents. Mammalian cells regulate Mg(2+) concentration through special transport systems that have only recently been characterized. Magnesium efflux occurs via Na(2+)-dependent and Na(2+)-independent pathways. Mg(2+) influx is controlled by recently cloned transporters including Mrs2p, SLC41A1, SLC41A2, ACDP2, MagT1, TRPM6 and TRPM7. Alterations in some of these systems may contribute to hypomagnesemia and intracellular Mg(2+) deficiency in hypertension and other cardiovascular pathologies. In particular, increased Mg(2+) efflux through dysregulation of the vascular Na(+)/Mg(2+) exchanger and decreased Mg(2+) influx due to defective vascular and renal TRPM6/7 expression/activity may be important in altered vasomotor tone and consequently in blood pressure regulation. The present review discusses the role of Mg(2+) in vascular biology and implications in hypertension and focuses on the putative transport systems that control magnesium homeostasis in the vascular system. Much research is still needed to clarify the exact mechanisms of cardiovascular Mg(2+) regulation and the implications of aberrant cellular Mg(2+) transport and altered cation status in the pathogenesis of hypertension and other cardiovascular diseases.

Establishing a cell biology platform: isolation and preservation of human blood products

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Systems biology approaches for the design of novel Saccharomyces cerevisiae winemaking strains for enhanced flavour compounds synthesis

Tese de Doutoramento em Biologia Ambiental e Molecular

Sistemática filogenética y biología reproductiva con énfasis en solanum y capsicum (solanaceae). Systematics, phylogeny and reproductive biology with emphasis in solanum and capsicum (solanaceae).

Muchas respuestas a preguntas básicas sobre relaciones evolutivas, ubicación sistemática y evolución de caracteres morfológicos y ecológicos pueden ser obtenidas a través de las reconstrucciones filogenéticas. Sobre este contexto se pretende encarar en este proyecto estudios de filogenia molecular, revisiones sistemáticas, biología reproductiva y citogenética en Solanáceas americanas. Se intentará resolver la delimitación específica de Solanum sect. Solanum y Geminata, y Capsicum, y establecer relaciones filogenéticas en estos grupos. Se harán revisiones analizándose caracteres vegetativos y reproductivos críticos para evaluar su variabilidad y definir su valor taxonómico; para los estudios moleculares se utilizarán los marcadores ndhF, trnT-L, trnL-F y waxy. En base a los resultados se propondrán agrupamientos y relaciones de parentesco. Además, se hará un estudio cariosistemático para caracterizar y circunscribir especies en Solanum y miembros de la tribu Physaleae, y hasta variedades y/o cultivares en Capsicum, mediante técnicas clásicas y de bandeos de fluorescencia y AgNOR e hibridación in situ fluorescente (FISH). A nivel reproductivo, se estudiará la ecofisiología en las estructuras masculinas y su incidencia en la fructificación en Capscium baccatum. El desarrollo de esta temática comprende experiencias in vivo (a campo y en laboratorio) así como estudios histológicos y químicos.Se espera avanzar en la resolución de algunos problemas: 1) la complicada delimitación de especies de los taxones en estudio; 2) las relaciones filogenéticas en algunos de ellos; 3) la falta de conocimiento de la organización genómica; 4) el origen de las especies cultivadas de Capsicum. En cuanto a la biología reproductiva, para C. baccatum se pretende avanzar en el conocimiento de variables de relevancia en la reproducción, en especial los efectos del ambiente.

Rol de las metaloproteasas y caspasas en la conducta biológica del carcinoma basocelular. Role of the metalloproteinases and caspasa Biology in the conduct of basal cell carcinoma.

El carcinoma basocelular (CBC) representa el tumor maligno más frecuente de la piel en personas de raza blanca. Incide en mayores de 50 años de edad e infiltra los tejidos, pero rara vez ocasiona metástasis. En trabajos anteriores, observamos imágenes de cuerpos apoptóticos con MOAR (microscopía óptica de alta resolución), en pieles portadoras de CBC. Todas las células eucariotas poseen una maquinaria enzimática que interviene en la apoptosis. Estas enzimas son las proteincinasas pertenecientes a la familia de las caspasas. Se sintetizan como procaspasas, las cuales una vez activadas, actúan sobre otra caspasa en una reacción secuencial en cadena. Se reconoce que la apoptosis juega un rol importante en el balance y mantenimiento de los tejidos. Hipótesis: -Investigar si la expresión de metaloproteasas y caspasas en las células del CBC promueven y contribuyen a la progresión y extensión tumoral. Objetivos:-Profundizar el conocimiento de la conducta biológica del CBC, en referencia a la relación de los patrones de crecimiento macro y microscópicos del tumor, enfatizando en la expresión de metaloproteasas (colagenasa-3 y streptomelisina), CD-31, Ki-67, oncoproteína bcl-2 y caspasas, mediante la implementación de técnicas de inmunohistoquímica. -Analizar a nivel morfológico la presencia de cuerpos apoptóticos por medio de MOAR y determinar si influyen en la conducta biológica del tumor. Materiales y Métodos: Se realizará un estudio retrospectivo y prospectivo de pacientes asistidos en el Servicio de Dermatología del Hospital Nacional de Clínicas, desde el año 2000. Se utilizarán tomas incisionales de piel, que se fijarán en formol neutro al 10 por ciento y luego serán procesadas e incluidas en parafina y coloreadas con H-E (hematoxilina-eosina). Especimenes seleccionados, se utilizarán para técnicas de MOAR e inmunocitoquímica. El material para MOAR, se fijará en Karnovsky enfriado a pH 7,2 y se incluirá en resinas epóxicas, seccionados con un ultramicrótomo Porter BluMT1 (1micra) y coloreados con P.A.S. azul de toluidina, fuscina básica y metenamina-plata. La aplicación de las técnicas inmunohistoquímicas, se utilizaran sobre cortes desparafinados con xilol, que permitirán investigar en forma retrospectiva y prospectiva la matriz extracelular (MEC) y los elementos neoplásicos. Los anticuerpos monoclonales a utilizar serán: Colageno tipo IV clone CIV 22 M0785 monoclonal antibody (DAKO), CD44 clone E29 M0613 monoclonal antibody (DAKO), Oncoproteina bcl-2 clone 124 M0887 monoclonal antibody (DAKO), CD31: clone 35 BH11 MO631 monoclonal antibody (DAKO) K-167 clone PC10 M0879 monoclonal antibody.(DAKO) bclx y Caspasa 8. Metodología Estadística Los resultados serán evaluados mediante: a) Varianza Anova; para datos con distribución gausiana como media, SD. b) Krusal Walles; para rasgos de distribución no gausiana. c) Chi2 para determinar la diferencia significativa para proporciones. Resultados esperados: Del análisis histomorfológico cualitativo y cuantitativo de biopsias de piel con CBC, aportar determinaciones que clarifiquen la conducta biológica de este tumor.- Importancia del Proyecto: El CBC es una entidad que está aumentando su incidencia en forma exponencial, debido sobre todo a pautas sociales. No sólo ocasiona trastornos en los pacientes, sino que también, representa gastos en el sistema de salud. Asimismo, significa un desafío para la investigación. Esto permite explorar aspectos de la biología molecular, a partir de una entidad que posee características únicas.

Molecular estimation of dispersal for ecology and population genetics

The dispersal process, by which individuals or other dispersing agents such as gametes or seeds move from birthplace to a new settlement locality, has important consequences for the dynamics of genes, individuals, and species. Many of the questions addressed by ecology and evolutionary biology require a good understanding of species' dispersal patterns. Much effort has thus been devoted to overcoming the difficulties associated with dispersal measurement. In this context, genetic tools have long been the focus of intensive research, providing a great variety of potential solutions to measuring dispersal. This methodological diversity is reviewed here to help (molecular) ecologists find their way toward dispersal inference and interpretation and to stimulate further developments.

'Hearts and bones': the ups and downs of 'plasticity' in stem cell biology.

More than a decade ago, 'plasticity' suddenly became a 'fashionable' topic with overemphasized implications for regenerative medicine. The concept of 'plasticity' is supported by old transplantation work, at least for embryonic cells, and metaplasia is a classic example of plasticity observed in patients. Nevertheless, the publication of a series of papers showing rare conversion of a given cell type into another unrelated cell raised the possibility of using any unaffected tissue to create at will new cells to replace a different failing tissue or organ. This resulted in disingenuous interpretations and a reason not to fund anymore research on embryonic stem cells (ESc). Moreover, many papers on plasticity were difficult to reproduce and thus questioned; raising issues about plasticity as a technical artefact or a consequence of rare spontaneous cells fusion. More recently, reprogramming adult differentiated cells to a pluripotent state (iPS) became possible, and later, one type of differentiated cell could be directly reprogrammed into another (e.g. fibroblasts into neurons) without reverting to pluripotency. Although the latter results from different and more robust experimental protocols, these phenomena also exemplify 'plasticity'. In this review, we want to place 'plasticity' in a historical perspective still taking into account ethical and political implications.

Omics meets hypothesis-driven research. Partnership for innovative discoveries in vascular biology and angiogenesis.

The emergence of omics technologies allowing the global analysis of a given biological or molecular system, rather than the study of its individual components, has revolutionized biomedical research, including cardiovascular medicine research in the past decade. These developments raised the prospect that classical, hypothesis-driven, single gene-based approaches may soon become obsolete. The experience accumulated so far, however, indicates that omic technologies only represent tools similar to those classically used by scientists in the past and nowadays, to make hypothesis and build models, with the main difference that they generate large amounts of unbiased information. Thus, omics and classical hypothesis-driven research are rather complementary approaches with the potential to effectively synergize to boost research in many fields, including cardiovascular medicine. In this article we discuss some general aspects of omics approaches, and review contributions in three areas of vascular biology, thrombosis and haemostasis, atherosclerosis and angiogenesis, in which omics approaches have already been applied (vasculomics).

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

"If you know the enemy and know yourself, you need not fear the result of a hundred battles. If you know yourself but not the enemy, for every victory gained you will also suffer a defeat" (SunTzu the Art of War, 544-496 BC). Although written for the managing of conflicts and winning clear victories, this basic guideline can be directly transferred to our battle against apicomplexan parasites and how to focus future basic research in order to transfer the gained knowledge to a therapeutic intervention stratey. Over the last two decades the establishment of several key-technologies, by different groups working on Toxoplasma gondii, made this important human pathogen accessible to modern approaches in molecular cell biology. In fact more and more researchers get attracted to this easy accessible model organism to study specific biological questions, unique to apicomplexans. This fascinating, unique biology might provide us with new therapeutic options in our battle against apicomplexan parasites by finding its Achilles' heel. In this article we argue that in the absence of a powerful high throughput technology for the characterisation of essential gene of interests a coordinated effort should be undertaken to convert our knowledge of the genome into one of the phenome.

Biology and pathogenicity of "Chlamydia"- related organisms

Abstract : This thesis investigates the pathogenicity and biology of Parachlamydia acanthamoebae and other obligate intracellular bacteria related to chlamydiae. All these Chlamydia-like organisms replicate in amoebae. Some evolved to resist to macrophages and represent possible new agents of respiratory tract infection. Using serological and molecular approaches, we showed that Parachlamydia acanthameobae likely plays a role as an etiological agent of pneumonia [1,2]. We also showed that Parachlamydia was able to enter and survive within pneumocytes and lung fibroblasts [3]. Moreover, we developed an animal model of lung infection in mice, which fulfilled the third and fourth Koch postulate [4]. Given the likely role of Parachlamydia in pneumonia, we studied its antibiotic susceptibility. We showed that Chlamydia-related organisms were resistant to quinolones, mainly due to mutations in the QRDR of gyrA [5]. To have tools to investigate the role of other Chlamydia-related bacteria in pneumonia, we developed immunofluorescence assays and assessed the rate of serological cross-reactivity between all these Chlamydia-related bacteria [6]. We also developed new diagnostic specific PCRs [2,7] and sequenced additional genes that are useful for both taxonomic and diagnostic purposes [8]. Then, we applied these serological and molecular approaches to patients with and without respiratory tract infections. This led to the identification of a possible role of Protochlamydia naegleriophila [7] and of Waddlia chondrophila in pneumonia [1]. A significant part of the thesis also investigated interactions of Parachlamydia with macrophages [9] and the host range of Chlamydia-related bacteria [10]. In conclusion, there are growing body of evidence supporting the role of Chlamydia-like organisms as agents of pneumonia. Further studies are needed to precise their pathogenic role in this setting. The diagnostic tools developed during this thesis will be useful to investigate the role of these strict intracellular bacteria in other diseases in both humans and animals [11,12]. Résumé : Le but de cette thèse est de déterminer le rôle pathogène de Parachlamydia et des bactéries apparentées aux Chlamydia ainsi que d'étudier leur biologie. Parachlamydia acanthamoebae est une bactérie intracellulaire apparentée aux Chlamydia, et qui est résistante non seulement aux amibes mais aussi aux macrophages. Par une approche sérologique et moléculaire, nous avons montré que les bactéries apparentées aux Chlamydia jouent probablement un rôle comme agent de pneumonie [1,2]. De plus, nous avons démontré que P. acanthameobae est capable d'entrer et de survivre dans les pneumocytes et fibroblastes pulmonaires [3]. Nous avons ensuite développé un modèle animal remplissant les troisième et quatrième postulats de Koch [4]. Nous avons aussi démontré que les bactéries apparentées aux Chlamydia sont résistantes aux quinolones, en raison de mutations dans la région QRDR de gyrA [5]. Afin de mieux déterminer le rôle pathogène de ces bactéries, nous avons mis au point des techniques d' immunofluorescence et déterminé la cross-réaction sérologique entre les différentes bactéries apparentées aux Chlamydia [6]. Différentes PCR diagnostiques ont aussi été développées [2,7] et des gènes supplémentaires ont été séquencés, qui seront utiles à la taxonomie ainsi qu'au développement de nouvelles méthodes diagnostiques [8]. Ces méthodes ont été appliquées à des échantillons provenant de patient avec ou sans pneumonie et ont permis l'identification du possible rôle pathogène de Protochlamydia naegleriophila [7] et de Waddlia chondrophila [1]. L'interaction de Parachlamydia avec les macrophages [9] et la permissivité de différentes cellules aux bactéries apparentées aux Chlamydia [10] ont également été étudiés dans le cadre de cette thèse. En conclusion, plusieurs nouveaux éléments viennent renforcer l'hypothèse que les bactéries apparentées aux Chlamydia sont des agents de pneumonies. Cependant, d'autres études doivent être menées pour confirmer leur rôle dans cette maladie. Les méthodes diagnostiques développées ici seront très utiles pour déterminer le rôle pathogène de ces bactéries chez les humains et animaux [11,12]

Genetics and molecular epidemiology of multiple myeloma: the rationale for the IMMEnSE consortium (review).

There is strong evidence suggesting the presence of a genetic component in the aetiology of multiple myeloma (MM). However no genetic risk factors have been unequivocally established so far. To further our understanding of the genetic determinants of MM risk, a promising strategy is to collect a large set of patients in a consortium, as successfully done for other cancers. In this article, we review the main findings in the genetic susceptibility and pharmacogenetics of MM and present the strategy of the IMMEnSE (International Multiple Myeloma rESEarch) consortium in contributing to determine the role of genetic variation in pharmacogenetics and in MM risk.

ESCMID postgraduate technical workshop on intracellular bacteria: from biology to clinic.

Infection by intracellular bacteria can lead to several diseases in both veterinary and human medicine. Unfortunately, the biology of these intracellular bacteria is highly complex due to their interactions with their host cells. Thus, it is very important to develop several tools in order to better understand the complex intracellular life of these pathogens, so allowing to improve the diagnosis options and the treatments of infectious diseases that they are causing. The workshop organised in Villars-sur-Ollon (Switzerland) by the ESCMID Study group on intracellular bacteria was a good opportunity to enhance our knowledge on these fastidious pathogens. During 5 days, 15 speakers gave 41 talks, covering all fields, from biology to clinic of different intracellular bacteria such as Bartonella, Chlamydia, Coxiella, Ehrlichia, Listeria, Parachlamydia, Rickettsia, and Waddlia. The format of this postgraduate course, which took place in the Swiss mountains, allowed interactive sessions and living discussions between the participants coming from all around the world. One of the major strength was to gather epidemiologists, clinical microbiologists, infectious diseases specialists, entomologists, veterinarians as well as bioinformaticians, biochemists and biologists to deliver a unique "one-health science" on intracellular bacteria. Here, we summarise the main take-home messages delivered during this meeting.

Hémato-oncologie pédiatrique: de la biologie cellulaire aux traitements ciblés et individualisés [Pediatric hemato-oncology: from cellular biology to individualized targeted therapies].

Progresses in pediatric oncology over the last decades have been dramatic and allow current cure rates above 80%. There are mainly due to multicentre clinical trials aiming at optimizing chemotherapy protocols as well as local therapies in a stepwise approach. Most of the new anticancer drugs currently in development are based on targeted therapies, directed to specific targets present only in or on tumor cells, like growth factor receptors, mechanisms involved in proliferation, DNA repair, apoptosis, tumor invasion or angiogenesis. Concerning bone marrow transplantation also, new strategic approaches are in advanced development. They aim at reducing treatment induced toxicity and enhancing efficacy at the same time. This short paper would like to point out these new technologies, which should be known by the general practitioner.

FIB-SEM tomography in biology.

Three-dimensional information is much easier to understand than a set of two-dimensional images. Therefore a layman is thrilled by the pseudo-3D image taken in a scanning electron microscope (SEM) while, when seeing a transmission electron micrograph, his imagination is challenged. First approaches to gain insight in the third dimension were to make serial microtome sections of a region of interest (ROI) and then building a model of the object. Serial microtome sectioning is a tedious and skill-demanding work and therefore seldom done. In the last two decades with the increase of computer power, sophisticated display options, and the development of new instruments, an SEM with a built-in microtome as well as a focused ion beam scanning electron microscope (FIB-SEM), serial sectioning, and 3D analysis has become far easier and faster.Due to the relief like topology of the microtome trimmed block face of resin-embedded tissue, the ROI can be searched in the secondary electron mode, and at the selected spot, the ROI is prepared with the ion beam for 3D analysis. For FIB-SEM tomography, a thin slice is removed with the ion beam and the newly exposed face is imaged with the electron beam, usually by recording the backscattered electrons. The process, also called "slice and view," is repeated until the desired volume is imaged.As FIB-SEM allows 3D imaging of biological fine structure at high resolution of only small volumes, it is crucial to perform slice and view at carefully selected spots. Finding the region of interest is therefore a prerequisite for meaningful imaging. Thin layer plastification of biofilms offers direct access to the original sample surface and allows the selection of an ROI for site-specific FIB-SEM tomography just by its pronounced topographic features.

Knowledge management for systems biology a general and visually driven framework applied to translational medicine

Background: To enhance our understanding of complex biological systems like diseases we need to put all of the available data into context and use this to detect relations, pattern and rules which allow predictive hypotheses to be defined. Life science has become a data rich science with information about the behaviour of millions of entities like genes, chemical compounds, diseases, cell types and organs, which are organised in many different databases and/or spread throughout the literature. Existing knowledge such as genotype - phenotype relations or signal transduction pathways must be semantically integrated and dynamically organised into structured networks that are connected with clinical and experimental data. Different approaches to this challenge exist but so far none has proven entirely satisfactory. Results: To address this challenge we previously developed a generic knowledge management framework, BioXM™, which allows the dynamic, graphic generation of domain specific knowledge representation models based on specific objects and their relations supporting annotations and ontologies. Here we demonstrate the utility of BioXM for knowledge management in systems biology as part of the EU FP6 BioBridge project on translational approaches to chronic diseases. From clinical and experimental data, text-mining results and public databases we generate a chronic obstructive pulmonary disease (COPD) knowledge base and demonstrate its use by mining specific molecular networks together with integrated clinical and experimental data. Conclusions: We generate the first semantically integrated COPD specific public knowledge base and find that for the integration of clinical and experimental data with pre-existing knowledge the configuration based set-up enabled by BioXM reduced implementation time and effort for the knowledge base compared to similar systems implemented as classical software development projects. The knowledgebase enables the retrieval of sub-networks including protein-protein interaction, pathway, gene - disease and gene - compound data which are used for subsequent data analysis, modelling and simulation. Pre-structured queries and reports enhance usability; establishing their use in everyday clinical settings requires further simplification with a browser based interface which is currently under development.