Structure function relationships of ENaC and its role in sodium handling.

The epithelial sodium channel (ENaC) in the apical membrane of polarized epithelial cells is the rate-limiting step for Na entry into the cell; in series with the basolateral Na pump, it allows the vectorial transepithelial transport of Na ions. ENaC is expressed in different epithelia like the distal nephron or colon, and the airways epithelium. In the lung ENaC controls the composition and the amount of pulmonary fluid, whereas in the distal nephron ENaC under the control of aldosterone and vasopressin, is essential to adapt the amount of Na+ reabsorbed with the daily sodium intake. Activating mutations of ENaC cause severe disturbances of Na+ homeostasis leading to hypertension in human and in mouse models. Functional expression of ENaC in different cell systems allowed the identification of structural domains of the protein that are essential for channel function and/or modulation of channel activity. Site-directed mutations in specific domains of the channel protein lead to channel hyperactivity or channel loss of function. Knowledge about ENaC structure-function relationships opens new opportunities for development of pharmacological tools for controlling ENaC activity, such as channel activators of potential benefit in the treatment of pulmonary edema, or highly potent ENaC blockers with natriuretic effects.

In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule.

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that inactivates 11β-hydroxy glucocorticoids. High expression levels of 11β-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11β-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11β-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.

An update of the mechanisms of resistance to EGFR-tyrosine kinase inhibitors in breast cancer: gefitinib (Iressatrade mark)-induced changes in the expression and nucleo-cytoplasmic trafficking of HER-ligands (Review)

Intrinsic resistance to the epidermal growth factor receptor (EGFR; HER1) tyrosine kinase inhibitor (TKI) gefitinib, and more generally to EGFR TKIs, is a common phenomenon in breast cancer. The availability of molecular criteria for predicting sensitivity to EGFR-TKIs is, therefore, the most relevant issue for their correct use and for planning future research. Though it appears that in non-small-cell lung cancer (NSCLC) response to gefitinib is directly related to the occurrence of specific mutations in the EGFR TK domain, breast cancer patients cannot be selected for treatment with gefitinib on the same basis as such EGFR mutations have beenreported neither in primary breast carcinomas nor in several breast cancer cell lines. Alternatively, there is a generalagreement on the hypothesis that the occurrence of molecular alterations that activate transduction pathways downstreamof EGFR (i.e., MEK1/MEK2 - ERK1/2 MAPK and PI-3'K - AKT growth/survival signaling cascades) significantly affect the response to EGFR TKIs in breast carcinomas. However,there are no studies so far addressing a role of EGF-related ligands as intrinsic breast cancer cell modulators of EGFR TKIefficacy. We recently monitored gene expression profiles andsub-cellular localization of HER-1/-2/-3/-4 related ligands (i.e., EGF, amphiregulin, transforming growth factor-α, ß-cellulin,epiregulin and neuregulins) prior to and after gefitinib treatment in a panel of human breast cancer cell lines. First, gefitinibinduced changes in the endogenous levels of EGF-related ligands correlated with the natural degree of breast cancer cellsensitivity to gefitinib. While breast cancer cells intrinsically resistant to gefitinib (IC50 ≥15 μM) markedly up-regulated(up to 600 times) the expression of genes codifying for HERspecific ligands, a significant down-regulation (up to 106 times)of HER ligand gene transcription was found in breast cancer cells intrinsically sensitive to gefitinib (IC50 ≤1 μM). Second,loss of HER1 function differentially regulated the nuclear trafficking of HER-related ligands. While gefitinib treatment induced an active import and nuclear accumulation of the HER ligand NRG in intrinsically gefitinib-resistant breastcancer cells, an active export and nuclear loss of NRG was observed in intrinsically gefitinib-sensitive breast cancer cells.In summary, through in vitro and pharmacodynamic studies we have learned that, besides mutations in the HER1 gene,oncogenic changes downstream of HER1 are the key players regulating gefitinib efficacy in breast cancer cells. It now appears that pharmacological inhibition of HER1 functionalso leads to striking changes in both the gene expression and the nucleo-cytoplasmic trafficking of HER-specific ligands,and that this response correlates with the intrinsic degree of breast cancer sensitivity to the EGFR TKI gefitinib. Therelevance of this previously unrecognized intracrine feedback to gefitinib warrants further studies as cancer cells could bypassthe antiproliferative effects of HER1-targeted therapeutics without a need for the overexpression and/or activation of other HER family members and/or the activation of HER-driven downstream signaling cascades

Parkinson"s disease DJ-1 L166P alters rRNA biogenesis by exclusion of TTRAP from the nucleolus and sequestration into cytoplasmic aggregates via TRAF6

Mutations in PARK7/DJ-1 gene are associated to autosomal recessive early onset forms of Parkinson"s disease (PD). Although large gene deletions have been linked to a loss-of-function phenotype, the pathogenic mechanism of missense mutations is less clear. The L166P mutation causes misfolding of DJ-1 protein and its degradation. L166P protein may also accumulate into insoluble cytoplasmic aggregates with a mechanism facilitated by the E3 ligase TNF receptor associated factor 6 (TRAF6). Upon proteasome impairment L166P activates the JNK/p38 MAPK apoptotic pathway by its interaction with TRAF and TNF Receptor Associated Protein (TTRAP). When proteasome activity is blocked in the presence of wild-type DJ-1, TTRAP forms aggregates that are localized to the cytoplasm or associated to nucleolar cavities, where it is required for a correct rRNA biogenesis. In this study we show that in post-mortem brains of sporadic PD patients TTRAP is associated to the nucleolus and to Lewy Bodies, cytoplasmic aggregates considered the hallmark of the disease. In SH-SY5Y neuroblastoma cells, misfolded mutant DJ-1 L166P alters rRNA biogenesis inhibiting TTRAP localization to the nucleolus and enhancing its recruitment into cytoplasmic aggregates with a mechanism that depends in part on TRAF6 activity. This work suggests that TTRAP plays a role in the molecular mechanisms of both sporadic and familial PD. Furthermore, it unveils the existence of an interplay between cytoplasmic and nucleolar aggregates that impacts rRNA biogenesis and involves TRAF6

Role of an LRR receptor-like kinase in the establishment of a functional root endodermal barrier

The endodermis is a highly conserved cell layer present in the root of all vascular plants, except Lycophytes. This tissue layer establishes a protective diffusion barrier surrounding the vasculature and is expected to prevent passive, uncontrolled flow of nutrients through the root. This barrier property is achieved by the production of Casparian strips (CS), a localized cell wall impregnation of lignin in the anticlinal walls of each endodermal cell, forming a belt-like structure sealing the extracellular space. The CS act as a selective barrier between the external cell layers and the vascular cylinder and are thought to be important in many aspects of root function. For instance, selective nutrient uptake and sequestration from the soil, resistance to different abiotic and biotic stresses are expected to involve functional CS. Although discovered 150 years ago, nothing was known about the genes involved in CS establishment until recently. The use of the model plant Arabidopsis thaliana together with both reverse and forward genetic approaches led to the discovery of an increasing number of genes involved in different steps of CS formation during the last few years. One of these genes encodes SCHENGEN3 (SGN3), a leucine-rich repeat receptor-like kinase (LRR-RLK). SGN3 was discovered first by reverse genetic due to its endodermis-enriched expression, and the corresponding mutant displays strong endodermal permeability of the apoplastic tracer Propidium Iodide (PI) indicative of defective CS. One aim of this thesis is to study the role of SGN3 at the molecular level in order to understand its involvement in establishing an impermeable CS. The endodermal permeability of sgn3 is shown to be the result of incorrect localization of key proteins involved in CS establishment (the "Casparian strip domain proteins", CASPs), leading to non-functional CS interrupted by discontinuities. CASPs localize in the plasma membrane domain subjacent to the CS, named the Casparian Strip membrane Domain (CSD). The CSD discontinuities in sgn3 together with SGN3 localization in close proximity to the CASPs lead to the assumption that SGN3 is involved in the formation of a continuous CSD. In addition, SGN3 might have a second role, acting as a kinase reporting CSD integrity leading to lignin and suberin production in CSD/CS defective plants. Up to now, sgn3 is the strongest and most specific CS mutant available, displaying tracer penetration along the whole length of the seedling root. For this reason, this mutant is well suited in order to characterize the physiological behaviour of CS affected plants. Due to the lack of such mutants in the past, it was not possible to test the presumed functions of CS by using plants lacking this structure. We decided to use sgn3 for this purpose. Surprisingly, sgn3 overall growth is only slightly affected. Nevertheless, processes expected to rely on functional CS, such as water transport through the root, nutrient homeostasis, salt tolerance and resistance to an excess of some nutrients are altered in this mutant. On the other hand, homeostasis for most elements and drought tolerance are not affected in sgn3. It is surprising to observe that homeostatic defects are specific, with a decrease in potassium and an increase in magnesium levels. It indicates a backup system, set up by the plant in order to counteract free diffusion of nutrients into the stele. For instance, potassium shortage in sgn3 upregulates the transcription of potassium influx transport proteins and genes known to be induced by potassium starvation. Moreover, sgn3 mutant is hypersensitive to low potassium conditions. Hopefully, these results about SGN3 will help our understanding of CS establishment at the molecular level. In addition, physiological experiments using sgn3 should give us a framework for future experiments and help us to understand the different roles of CS and their involvement during nutrient radial transport through the root. -- L'endoderme est un tissu présent dans les racines de toutes les plantes vasculaires à l'exception des Lycophytes. Ce tissu établit une barrière protectrice entourant les tissus vasculaires dans le but d'éviter la diffusion passive et incontrôlée des nutriments au travers de la racine. Cette propriété de barrière provient de la production des cadres de Caspary, une imprégnation localisée de lignine des parties anticlinales de la paroi de chaque cellule d'endoderme. Cela donne naissance à un anneau/cadre qui rend étanche l'espace extracellulaire. Les cadres de Caspary agissent comme une barrière sélective entre les couches externes de la racine et le cylindre central et sont supposés être importants dans beaucoup d'aspects du fonctionnement de la racine. Par exemple, l'absorption sélective de nutriments et leur séquestration à partir du sol ainsi que la résistance contre différents stress abiotiques et biotiques sont supposés impliquer des cadres de Caspary fonctionnels. Bien que découverts il y a 150 ans, rien n'était connu concernant les gènes impliqués dans Ja formation des cadres de Caspary jusqu'à récemment. Durant ces dernière années, l'utilisation de la plante modèle Arabidopsis thaliana ainsi que des approches de génétique inverse et classique ont permis la découverte d'un nombre croissant de gènes impliqués à différentes étapes de la formation de cette structure. Un des ces gènes code pour SCHENGEN3 (SGN3), un récepteur kinase "leucine-rich repeat receptor-like kinase" (LRR-RLK). SGN3 a été découvert en premier par génétique inverse grâce à son expression enrichie dans l'endoderme. Les cadres de Caspary ne sont pas fonctionnels dans le mutant correspondant, ce qui est visible à cause de la perméabilité de l'endoderme au traceur apoplastique Propidium Iodide (PI). Un des objectifs de cette thèse est d'étudier la fonction de SGN3 au niveau moléculaire dans le but de comprendre son rôle dans la formation des cadres de Caspary. J'ai pu démontrer que la perméabilité de l'endoderme du mutant sgn3 est le résultat de la localisation incorrecte de protéines impliquées dans la formation des cadres de Caspary, les "Casparian strip domain proteins" (CASPs). Cela induit des cadres de Caspary non fonctionnels, contenant de nombreuses interruptions. Les CASPs sont localisés à la membrane plasmique dans un domaine sous-jacent les cadres de Caspary appelé Casparian Strip membrane Domain (CSD). Les interruptions du CSD dans le mutant sgn3, ainsi que la localisation de SGN3 à proximité des CASPs nous font penser à un rôle de SGN3 dans l'élaboration d'un CSD ininterrompu. De plus, SGN3 pourrait avoir un second rôle, agissant en tant que kinase reportant l'intégrité du CSD et induisant la production de lignine et de subérine dans des plantes contenant des cadres de Caspary non fonctionnels. Jusqu'à ce jour, sgn3 est le mutant en notre possession le plus fort et le plus spécifique, ayant un endoderme perméable tout le long de la racine. Pour cette raison, ce mutant est adéquat dans le but de caractériser la physiologie de plantes ayant des cadres de Caspary affectés. De manière surprenante, la croissance de sgn3 est seulement peu affectée. Néanmoins, des processus censés nécessiter des cadres de Caspary fonctionnels, comme le transport de l'eau au travers de la racine, l'homéostasie des nutriments, la tolérance au sel et la résistance à l'excès de certains nutriments sont altérés dans ce mutant. Malgré tout, l'homéostasie de la plupart des nutriments ainsi que la résistance au stress hydrique ne sont pas affectés dans sgn3. De manière surprenante, les altérations de l'ionome de sgn3 sont spécifiques, avec une diminution de potassium et un excès de magnésium. Cela implique un système de compensation établi par la plante dans le but d'éviter la diffusion passive des nutriments en direction du cylindre central. Par exemple, le manque de potassium dans sgn3 augmente la transcription de transporteurs permettant l'absorption de cet élément. De plus, des gènes connus pour être induits en cas de carence en potassium sont surexprimés dans sgn3 et la croissance de ce mutant est sévèrement affectée dans un substrat pauvre en potassium. Ces résultats concernant SGN3 vont, espérons-le, aider à la compréhension du processus de formation des cadres de Caspary au niveau moléculaire. De plus, les expériences de physiologie utilisant sgn3 présentées dans cette thèse devraient nous donner une base pour des expériences futures et nous permettre de comprendre mieux le rôle des cadres de Caspary, et plus particulièrement leur implication dans le transport radial des nutriments au travers de la racine. -- Les plantes terrestres sont des organismes puisant l'eau et les nutriments dont elles ont besoin pour leur croissance dans le sol grâce à leurs racines. De par leur immobilité, elles doivent s'adapter à des sols contenant des quantités variables de nutriments et il leur est crucial de sélectionner ce dont elles ont besoin afin de ne pas s'intoxiquer. Cette sélection est faite grâce à un filtre formé d'un tissu racinaire interne appelé endoderme. L'endoderme fabrique une barrière imperméable entourant chaque cellule appelée "cadre de Caspary". Ces cadres de Caspary empêchent le libre passage des nutriments, permettant un contrôle précis de leur passage. De plus, ils sont censés permettre de résister contre différents stress environnementaux comme la sécheresse, la salinité du sol ou l'excès de nutriments. Bien que découverts il y a 150 ans, rien n'était connu concernant les gènes impliqués dans la formation des cadres de Caspary jusqu'à récemment. Durant ces dernière années, l'utilisation de la plante modèle Arabidopsis thaliana a permis la découverte d'un nombre croissant de gènes impliqués à différentes étapes de la formation de cette structure. Un de ces gènes code pour SCHENGEN3 (SGN3), un récepteur kinase "leucine-rich repeat receptor-like kinase" (LRR- RLK). Nous montrons dans cette étude que le gène SGN3 est impliqué dans la formation des cadres de Caspary, et que le mutant correspondant sgn3 a des cadres de Caspary interrompus. Ces interruptions rendent l'endoderme perméable, l'empêchant de bloquer le passage des molécules depuis le sol vers le centre de la racine. En utilisant ce mutant, nous avons pu caractériser la physiologie de plantes ayant des cadres de Caspary affectés. Cela a permis de découvrir que le transport de l'eau au travers de la racine était affecté dans le mutant sgn3. De plus, l'accumulation de certains éléments dans les feuilles de ce mutant est altérée. Nous avons également pu montrer une sensibilité de ce mutant à un excès de sel ou de certains nutriments comme le fer et le manganèse.

Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response.

Cysteine cathepsin protease activity is frequently dysregulated in the context of neoplastic transformation. Increased activity and aberrant localization of proteases within the tumour microenvironment have a potent role in driving cancer progression, proliferation, invasion and metastasis. Recent studies have also uncovered functions for cathepsins in the suppression of the response to therapeutic intervention in various malignancies. However, cathepsins can be either tumour promoting or tumour suppressive depending on the context, which emphasizes the importance of rigorous in vivo analyses to ascertain function. Here, we review the basic research and clinical findings that underlie the roles of cathepsins in cancer, and provide a roadmap for the rational integration of cathepsin-targeting agents into clinical treatment.

Identification of novel factors during the establishment of the Casparian strip in the endodermis

Multicellular organisms rely on specialized tissues that allow for the controlled exchange of matter with their surrounding. In order to function properly, these tissues need to establish a tight connection between the individual cells to prevent uncontrolled passive diffusion across the extracellular space. In animals, these connections are called tight and adherens junctions and are a critical feature of epithelia. These connections, however, rely on direct protein-protein interaction of plasma membrane proteins of adjacent cells. Such a mechanism is not possible in plants due to the cell wall, which encases the individual cells. In order to absorb nutrients, while simultaneously preventing uncontrolled diffusion between cells, land plants have evolved the root endodermis, which is functionally equivalent to animal epithelia. Its cells are surrounded by a precisely localized and aligned, ring-like lignin deposition, called the Casparian strip, and therefore tightly connected between each other. Very little was known about the development of the endodermis and the Casparian strip until recently. In the meantime, however, we have identified a family of endodermis- specific proteins, the CASPs, which recruits extracellular proteins the specific Casparian strip membrane domain (CSD) to locally synthesize lignin in the cell wall. Yet, we hardly knew any specifics on how the CSD is initially defined and how the critically important CASPs are being recruited to it. We therefore conducted a forward genetic screen on the localization of CASPI-GFP in order to identify novel mutants, which lack a defined CSD. We identified 48 mutants, which fell into 15 different complementation groups. While some of the isolated genes had previously been identified through different approaches, nine novel genes, which had never been implicated in CSD development and maintenance, were identified. One of them, LORD OF THE RINGS 2 (.LOTR2) is described to greater detail in this work. LOTR2 encodes for EX070A1, a protein of the evolutionary conserved exocyst complex. This complex has frequently been implicated in various secretory processes across kingdoms. In Arabidopsis, it transiently defines the positioning of CASPI-GFP. We have performed a detailed analysis of the dynamics of EX070A1 and CASPI-GFP, including studies with other markers and propose a mechanism, by which the cytosolic EX070A1 transiently defines a plasma membrane domain to recruit transmembrane proteins, which then recruit extracellular enzymes for localized cell wall modification. Considering the ubiquitous expression of EX070A1, we think that this mechanism is potentially of importance not only for the endodermis and the Casparian strip but also for many other tissues, in which the cell wall becomes locally modified. In fact, many other tissues with secondary cell wall modifications contain proteins very similar to the CASPs. It will be interesting to see to which degree this mechanism is employed in other tissues. As for the endodermis, we have now identified the first gene, which is not specific to the endodermis but shows endodermis-specific dynamics. This might give us a better insight on how the plant modulates this ubiquitously present factor in a cell- or tissue-type specific manner. Considering the knowledge, mutants and tools, which are available to us for investigating the endodermis, the Casparian strip, the exocyst complex and EX070A1 might be just the right experimental system to address these questions. -- Les organismes multicellulaires dépendent des tissues spécialisé pour l'échange contrôlé entre eux et leur environnement. Pour leur bon fonctionnement, les cellules de ces tissus ont besoin d'être très étroitement assemblés afin de prévenir la diffusion non-contrôlée à travers l'espace extracellulaire. Chez les animaux, ces connexions sont appelées jonctions serrées et jonctions adhérentes. Ces jonctions dépendent des interactions directes entre les protéines des cellules voisines. Ceci n'est pas possible chez les plantes à cause de la paroi cellulaire qui recouvre chaque cellule individuellement. Pour absorber les nutriments et en même temps empêcher la diffusion non-contrôlé entre cellules, les plantes ont évolué 1'endoderme dans la racine, qui est fonctionnellement équivalent aux épithéliums des animaux. Les cellules de l'endoderme sont ceinturées par une déposition de lignine très précisément localisées comme un anneau et alignées entre les cellules, et qui, donc, connecte étroitement les cellules avoisinante: Le cadre de Caspary. Peu était connu sur le développement de l'endoderme et le cadre de Caspaiy jusqu'à il y a quelques années. Récemment, pourtant, nous avons identifié une famille de protéines spécifiques à l'endoderme, les CASPs, qui définissent le domaine membranaire du cadre de Caspaiy (CSD). Les CASPs recrutent les protéines extracellulaires nécessaire à la synthèse du cadre de Caspary vers une région limité dans la paroi cellulaire. Pourtant, on connaît très peu les processus spécifiques concernant la définition initiale du CSD et comment les CASPs, qui ont une importance cruciale, sont recrutées vers ce domaine. Par conséquent nous avons mené un crible génétique sur la localisation du CASPI- GFP, qui sert comme marqueur pour le CSD. Notre but étant d'isoler de nouveaux mutants affectés dans l'établissement du CSD. Nous avons identifié 48 mutants, en 15 groupes de complémentation. Bien que certains des gènes isolés étaient déjà impliqué dans la formation du cadre de Caspary, neuf nouveaux gènes n'ayant jamais été impliqués dans le développement ou la maintenance du CSD ont pu être identifiés. Un de ces gènes, LORD OF THE RINGS2 (LOTR2) sera décrit plus en détail dans cette étude. LOTR2 code pour EX070A1, qui est une protéine, du complexe exocyste. Ce complexe de protéines a très bien été conservé au cours de l'évolution. Il était souvent impliqué dans plusieurs processus de sécrétion dans toutes les branches de la vie. Chez Arabidopsis, EX070A1 définit la position du CSD d'une façon transitoire et recrute CASP1- GFP. Nous avons mené une analyse détaillée des dynamiques d'EX070Al et CASPI-GFP ainsi que, des études avec des autres mutants. Nous proposons un mécanisme, d'après lequel EX070A1, recruté du cytosol, définit un domaine dans la membrane plasmique pour localiser des protéines transmembranaires, ces dernières ensuite recruteront des enzymes extracellulaires pour la modification locale de la paroi cellulaire. Vu qu'EX070A1 est exprimé dans toute dans la plante, nous pensons que ce mécanisme est potentiellement important non seulement pour l'endoderme et le cadre de Caspary, mais aussi pour les autres tissus où la paroi cellulaire doit être localement modifiée. En effet, plusieurs autres tissus contiennent des protéines très similaires aux CASPs. Il serait intéressant de voir à quelle dégrée ce mécanisme est également utilisé dans ces tissues. En ce qui concerne l'endoderme, nous avons maintenant identifié le premier gène qui n'est pas exprimé spécifiquement dans l'endoderme, mais qui montre tout de même une dynamique caractéristique dans ce tissu. Il serait intéressant de voir comment la plante peut moduler ce facteur omniprésent d'une façon spécifique. Vu les connaissances, les mutants et les outils qu'on a maintenant à notre disposition, l'endoderme et son cadre de Caspary, le complexe exocyste et EX070A1 sont probablement des bons systèmes expérimentaux pour étudier ces questions. -- Identification des nouveaux facteurs pendant l'établissement du cadre de Caspary dans l'endoderme. Lothar Kalmbach, Département de Biologie Moléculaire Végétale (DBMV), Université de Lausanne. Comme tous les autres organismes multicellulaires, les plantes terrestres dépendent de tissus spécialisés pour l'échange contrôlé avec leur environnement. Ces tissus sont importants pour l'absorption des nutriments mais également pour éviter l'influx de composés toxiques. Chez les plantes, ce tissu se trouve dans la racine. C'est l'endoderme. Grâce au cadre de Caspary, qui permet une forte connexion entre les cellules au niveau de leur paroi, l'endoderme empêche les éléments toxiques d'entrer dans le système vasculaire. Depuis quelques années, nous comprenons de plus en plus la nature et la biosynthèse, ainsi que les protéines impliquées dans l'ancrage des enzymes à la membrane plasmique. Nous n'avons eu, par contre, aucune idée sur le mécanisme qui d'abord définit cet endroit dans la membrane plasmique. Nous avons mené un crible génétique sur la localisation de CASPI-GFP, une protéine, qui recrute les enzymes extracellulaires pour la synthèse du cadre de Caspary. Nous avons identifié plusieurs nouveaux gènes qui sont impliqués dans l'intégrité du cadre de Caspary. L'un de ces gènes est EX070A1, qui est un facteur ayant un rôle important lors de la sécrétion des protéines dans tous les organismes eukaryotes. Ces mutants sont gravement affectés au niveau du cadre de Caspary, mais surtout ils ne sont plus capables de localiser CASPI-GFP. Nous avons suivi la dynamique d'EX070Al et de CASP1-GFP en combinaison avec d'autres marqueurs. Nous avons pu montrer que l'accumulation d'EX070Al est spécifique pour l'endoderme et essentielle pour bien localiser CASPI-GFP et donc, le cadre de Caspary. Ces résultats nous aident à mieux comprendre le développement de l'endoderme mais peuvent potentiellement aussi être utilisés pour étudier les modifications de la paroi cellulaire dans d'autres cellules de la plante.

Regulating Mechanisms of Gonadal and Pituitary Tumorigenesis in Mice Producing Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are structurally and functionally similar glycoprotein hormones acting through the same luteinizing hormone chorionic gonadotropin receptor (LHCGR). The functions of LH in reproduction and hCG in pregnancy are well known. Recently, the expression of LHCGR has been found in many nongonadal tissues and cancers, and this has raised the question of whether LH/hCG could affect the function or tumorigenesis of these nongonadal tissues. We have also previously generated an hCG expressing mouse model presenting nongonadal phenotypes. Using this model it is possible to improve our understanding of nongonadal action of highly elevated LH/hCG. In the current study, we analyzed the effect of moderately and highly elevated hCG levels on male reproductive development and function. The main finding was the appearance of fetal Leydig cell (FLC) adenomas in prepubertal males. However, the development and differentiation of FLCs were not significantly affected. We also show that the function of hCG is different in FLCs and in adult Leydig cells (ALC), because in the latter cells hCG was not able to induce tumorigenesis. In FLCs, LHCGR is not desensitized or downregulated upon ligand binding. In this study, we found that the testicular expression of two G protein-coupled receptor kinases responsible for receptor desensitization or downregulation is increased in adult testis. Results suggest that the lack of LHCGR desensitization or downregulation in FLCs protect testosterone (Te) synthesis, but also predispose FLCs for LH/hCG induced adenomas. However, all the hCG induced nongonadal changes observed in male mice were possible to explain by the elevated Te level found in these males. Our findings indicate that the direct nongonadal effects of elevated LH/hCG in males are not pathophysiologically significant. In female mice, we showed that an elevated hCG level was able to induce gonadal tumorigenesis. hCG also induced the formation of pituitary adenomas (PA), but the mechanism was indirect. Furthermore, we found two new potential risk factors and a novel hormonally induced mechanism for PAs. Increased progesterone (P) levels in the presence of physiological estradiol (E2) levels induced the formation of PAs in female mice. E2 and P induced the expression and nuclear localization of a known cell-cycle regulator, cyclin D1. A calorie restricted diet was also able to prevent the formation of PAs, suggesting that obesity is able to promote the formation of PAs. Hormone replacement therapy after gonadectomy and hormone antagonist therapy showed that the nongonadal phenotypes observed in hCG expressing female mice were due to ovarian hyperstimulation. A slight adrenal phenotype was evident even after gonadectomy in hCG expressing females, but E2 and P replacement was able to induce a similar phenotype in WT females without elevated LH/hCG action. In conclusion, we showed that the direct effects of elevated hCG/LH action are limited only to the gonads of both sexes. The nongonadal phenotypes observed in hCG expressing mice were due to the indirect, gonadal hormone mediated effects of elevated hCG. Therefore, the gonads are the only physiologically significant direct targets of LHCGR signalling.

Molecular genetics of the immotile short tail sperm defect

Hedelmättömyyttä aiheuttavan siittiöiden puolihäntävian molekyyligenetiikka Suomalaisissa Yorkshire karjuissa yleistyi 1990-luvun lopulla autosomaalisesti ja resessiivisesti periytyvä hedelmättömyyttä aiheuttava siittiöiden puolihäntävika (ISTS, immotile short tail sperm). Sairaus aiheuttaa normaalia lyhyemmän ja täysin liikkumattoman siittiön hännän muodostuksen. Muita oireita sairailla karjuilla ei ole havaittu ja emakot ovat oireettomia. Tämän tutkimuksen tarkoituksena oli kartoittaa siittiöiden puolihäntävian aiheuttava geenivirhe ja kehittää DNA-testi markkeri- ja geeniavusteiseen valintaan. Koko genomin kartoituksessa vian aiheuttava alue paikannettiin sian kromosomiin 16. Paikannuksen perusteella kahden geenimerkin haplotyyppi kehitettiin käytettäväksi markkeri-avusteisessa valinnassa. Sairauteen kytkeytyneen alueen hienokartoitusta jatkettiin geenitestin kehittämiseksi kantajadiagnostiikkaan. Vertailevalla kartoituksella oireeseen kytkeytynyt alue paikannettiin 2 cM:n alueelle ihmisen kromosomiin viisi (5p13.2). Tällä alueella sijaitsevia geenejä vastaavista sian sekvensseistä löydetyn muuntelun perusteella voitiin tarkentaa sairauteen kytkeytyneitä haplotyyppejä. Haplotyyppien perusteella puolihäntäoireeseen kytkeytynyt alue rajattiin kahdeksan geenin alueelle ihmisen geenikartalla. Alueelle paikannetun kandidaattigeenin (KPL2) sekvensointi paljasti introniin liittyneen liikkuvan DNA-sekvenssin, Line-1 retroposonin. Tämä retroposoni muuttaa geenin silmikointia siten, että sitä edeltävä eksoni jätetään pois tai myös osa introni- ja inserttisekvenssiä liitetään geenin mRNA tuotteeseen. Molemmissa tapauksissa tuloksena on lyhentynyt KPL2 proteiini. Tähän retroposoni-inserttiin perustuva geenitesti on ollut sianjalostajien käytössä vuodesta 2006. KPL2 geenin ilmenemisen tarkastelu sialla ja hiirellä paljasti useita kudosspesifisiä silmikointimuotoja. KPL2 geenin pitkä muoto ilmenee pääasiassa vain kiveksessä, mikä selittää geenivirheen aiheuttamat erityisesti siittiön kehitykseen liittyvät oireet. KPL2 proteiinin ilmeneminen hiiren siittiön hännän kehityksen aikana ja mahdollinen yhteistoiminta IFT20 proteiinin kanssa viittaavat tehtävään proteiinien kuljetuksessa siittiön häntään. Mahdollisen kuljetustehtävän lisäksi KPL2 saattaa toimia myös siittiön hännän rakenneosana, koska se paikannettiin valmiin siittiön hännän keskiosaan. Lisäksi KPL2 proteiini saattaa myös toimia Golgin laitteessa sekä Sertolin solujen ja spermatidien liitoksissa, mutta nämä havainnot kuitenkin vaativat lisätutkimuksia. Tämän tutkimuksen tulokset osoittavat, että KPL2 geeni on tärkeä siittiön hännän kehitykselle ja sen rakennemuutos aiheuttaa siittiöiden puolihäntäoireen suomalaisilla Yorkshire karjuilla. KPL2 proteiinin ilmeneminen ja paikannus siittiön kehityksen aikana antaa viitteitä proteiinin toiminnasta. Koska KPL2 geenisekvenssi on erittäin konservoitunut, nämä tulokset tuovat uutta tietoa kaikkien nisäkkäiden siittiöiden kehitykseen ja urosten hedelmättömyyteen syihin.

Identification and characterization of new genes and pathways regulating spermatogonial cell fate

Spermatogenesis, i.e sperm production in the seminiferous tubules of the testis, is a complex process that takes over one month to complete. Life-long ability of sperm production ultimately lies in a small population of undifferentiated cells, called spermatogonial stem cells (SSCs). These cells give rise to differentiating spermatogonia, which are committed to mature into spermatozoa. SSCs represent a heterogeneous population of cells and many aspects of their basic biology are still unknown. Understanding the mechanisms behind the cell fate decision of these cells is important to gain more insights into the causes of infertility and testis cancer. In addition, an interesting new aspect is the use of testis-derived stem cells in regenerative medicine. Our data demonstrated that adult mouse testis houses a population of Nanog-expressing spermatogonia. Based on mRNA and protein analysis these cells are enriched in stage XII of the mouse seminiferous epithelial cycle. The cells derived from this stage have the highest capacity to give rise to ES cell-like cells which express Oct4 and Nanog. These cells are under tight non- GDNF regulation but their fate can be dictated by activating p21 signalling. Comparative studies suggested that these cells are regulated like ES cells. Taken together these data imply that pluripotent cells are present in the adult mammalian testis. CIP2A (cancerous inhibitor of PP2A) has been associated with tumour aggressiveness and poor prognosis. In the testis it is expressed by the descendants of stem cells, i.e. the spermatogonial progenitor cells. Our data suggest that CIP2A acts upstream of PLZF and is needed for quantitatively normal spermatogenesis. Classification of CIP2A as a cancer/testis gene makes it an attractive target for cancer therapy. Study on the CIP2A deficient mouse model demonstrates that systemic inhibition of CIP2A does not severely interfere with growth and development or tissue or organ function, except for the spermatogenic output. These data demonstrate that CIP2A is required for quantitatively normal spermatogenesis. Hedgehog (Hh) signalling is involved in the development and maintenance of many different tissues and organs. According to our data, Hh signalling is active at many different levels during rat spermatogenesis: in spermatogonia, spermatocytes and late elongating spermatids. Localization of Suppressor of Fused (SuFu), the negative regulator of the pathway, specifically in early elongating spermatids suggests that Hh signalling needs to be shut down in these cells. Introduction of Hh signalling inhibitor resulted in an increase in germ cell apoptosis. Follicle-stimulating hormone (FSH) and inhibition of receptor tyrosine kinases resulted in down-regulation of Hh signalling. These data show that Hh signalling is under endocrine and paracrine control and it promotes germ cell survival.

Two-dimensional atom localization and Raman cooling of tripod-type atoms

Both atom localization and Raman cooling, considered in the thesis, reflect recent progress in the area of all-optical methods. We focus on twodimensional (2D) case, using a four-level tripod-type atomic scheme for atom localization within the optical half-wavelength as well as for efficient subrecoil Raman cooling. In the first part, we discuss the principles of 1D atom localization, accompanying by an example of the measurement of a spontaneously-emitted photon. Modifying this example, one archives sub-wavelength localization of a three-level -type atom, measuring the population in its upper state. We go further and obtain 2D sub-wavelength localization for a four-level tripod-type atom. The upper-state population is classified according to the spatial distribution, which in turn forms such structures as spikes, craters and waves. The second part of the thesis is devoted to Raman cooling. The cooling process is controlled by a sequence of velocity-selective transfers from one to another ground state. So far, 1D deep subrecoil cooling has been carried out with the sequence of square or Blackman pulses, applied to -type atoms. In turn, we discuss the transfer of atoms by stimulated Raman adiabatic passage (STIRAP), which provides robustness against the pulse duration if the cooling time is not in any critical role. A tripod-type atomic scheme is used for the purpose of 2D Raman cooling, allowing one to increase the efficiency and simplify the realization of the cooling.

Towards understanding the kallikrein-kinin system: insights from measurement of kinin peptides

The kallikrein-kinin system is complex, with several bioactive peptides that are formed in many different compartments. Kinin peptides are implicated in many physiological and pathological processes including the regulation of blood pressure and sodium homeostasis, inflammatory processes, and the cardioprotective effects of preconditioning. We established a methodology for the measurement of individual kinin peptides in order to study the function of the kallikrein-kinin system. The levels of kinin peptides in tissues were higher than in blood, confirming the primary tissue localization of the kallikrein-kinin system. Moreover, the separate measurement of bradykinin and kallidin peptides in man demonstrated the differential regulation of the plasma and tissue kallikrein-kinin systems, respectively. Kinin peptide levels were increased in the heart of rats with myocardial infarction, in tissues of diabetic and spontaneously hypertensive rats, and in urine of patients with interstitial cystitis, suggesting a role for kinin peptides in the pathogenesis of these conditions. By contrast, blood levels of kallidin, but not bradykinin, peptides were suppressed in patients with severe cardiac failure, suggesting that the activity of the tissue kallikrein-kinin system may be suppressed in this condition. Both angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors increased bradykinin peptide levels. ACE and NEP inhibitors had different effects on kinin peptide levels in blood, urine, and tissues, which may be accounted for by the differential contributions of ACE and NEP to kinin peptide metabolism in the multiple compartments in which kinin peptide generation occurs. Measurement of the levels of individual kinin peptides has given important information about the operation of the kallikrein-kinin system and its role in physiology and disease states.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

Nitric oxide (NO) is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR) triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS) in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC) superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47% in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45% when compared to the contralateral SC of the same animals and by 64% when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.

Effects of intracerebroventricular bombesin administration on local cerebral glucose utilization in the restrained and unrestrained rat

Intracerebroventricular (ICV) administration of bombesin (BN) induces a syndrome characterized by stereotypic locomotion and grooming, hyperactivity and sleep elimination, hyperglycemia and hypothermia, hyperhemodynamics, feeding inhibition, and gastrointestinal function changes. Mammalian BN-like peptides (MBNs), e.g. gastrin-releasing peptide (GRP), Neuromedin C (NMC), and Neuromedin B (NMB), have been detected in the central nervous system. Radio-labeled BN binds to specific sites in discrete cerebral regions. Two specific BN receptor subtypes (GRP receptor and NMB receptor) have been identified in numerous brain regions. The quantitative 2-[14C]deoxyglucose ([14C]20G) autoradiographic method was used to map local cerebral glucose utilization (LCGU) in the rat brain following ICV injection of BN (vehicle, BN O.1Jlg, O.5Jlg). At each dose, experiments were conducted in freely moving or restrained conditions to determine whether alterations in cerebral function were the result of BN central administration, or were the result of BN-induced motor stereotypy. The anteroventral thalamic nucleus (AV) (p=O.029), especially its ventrolateral portion (AVVL) (pof metabolism under both restraint conditions. The effect was treatment dependent without interaction of the restraint conditions. Of all the regions that were reported to have high densities of BN receptors, the internal granular layer of the olfactory bulb (IGr) (p=O.028), and the suprachiasmatic nucleus (SCh) (p=O.003) exhibited BN treatment effects. BN effects on LCGU were also observed in the median eminence (ME) (p=O.011). Restraint, however, decreased LCGU in the lateral dorsal thalamic nucleus, ventrolateral and dorsomedial parts (LOVL and LOOM) (p=O.044, p=O.009), and the lateral geniculate (LG) (p=O.027). In sum, BN induced a marked and highly localized alteration in cerebral metabolism within parts of the anterior thalamus, which is the principle relay in the limbic circuitry. BN effects were also observed in IGr, Mi, SCh, and ME. Effects of restraint were found in LOVL, LOOM, and LG. It is suggested that increased LCGU in AV and AVVL may be the result of functional change in the limbic circuitry and the hypothalamus caused by BN receptor functional modification. In IGr, increased LCGU following BN administration is considered to be mainly the result the activation of NMB receptor, a subtype of BN receptors. In SCh, increased LCGU is believed to be caused both by BN effects on the thalamic, the hypothalamic, and the limbic functions and by activation of GRP receptor, another BN receptors subtype found in SCh. In ME, increased LCGU is suggested to be caused by BN effects on the hypothalamic functions, especially those related to the neuroendocrine functions. None of the alterations seen in these regions reflects the emission of stereotyped motor behaviors. Rather, they reflect a direct influence of BN central administration upon functioning of the cerebral regions influenced by BN administration. The restraint effects seen in LO, including LOOM and LOVL, are suggested to be the result of altered behavioral expression. The restraint effects seen in LG is suggested to be the result of reduced locomotion.