Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum

Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and ���c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis.

Charakterisierung von plasmafunktionalisierten Oberfl��chen mittels ToF-SIMS und multivariaten Analysemethoden

Die grundlegenden Prinzipien und M��glichkeiten der Oberfl��chencharakterisierung mittels ToF-SIMS (Flugzeit-Sekund��rionen Massenspektrometrie) werden an ausgew��hlten Beispielen aus einem aktuell laufenden und vom BMBF gef��rderten Verbundforschungsprojekt (Fkz: 03N8022A) zum Thema Nanofunktionalisierung von Grenzfl��chen vorgestellt. Ein Schwerpunkt innerhalb des Projekts stellen die nichtgeschlossenen Schichtsysteme dar, die entweder ��ber Dom��nenstrukturen oder einer definierten Einzelfunktionalisierung neuartige funktionelle Oberfl��chen bereitstellen. Mithilfe der sehr oberfl��chensensitiven ToF-SIMS Methode sowie der M��glichkeit einer graphischen Darstellung lateraler Molek��lionenverteilungen auf funktionalisierten Oberfl��chen k��nnen Informationen ��ber Struktur und Belegungsdichte der Funktionsschicht gewonnen werden. Die Kombination des ToF-SIMS Experimentes und eines multivariaten Algorithmus (partial least squares, PLS) liefert eine interessante M��glichkeit zur quantitativen und simultanen Bestimmung von Oberfl��cheneigenschaften (Element- und molekulare Konzentrationen sowie Kontaktwinkelwerte). Da das ToF-SIMS Spektrum einer plasmafunktionalisierten Oberfl��che im Allgemeinen eine Vielzahl unterschiedlicher Fragmentsignale enth��lt, l��sst eine einfache eindimensionale Korrelation (z.B. CF3 - Fragmentintensit��t ���� CF3-Konzentration) den gr����ten Teil der im Spektrum prinzipiell enthaltenen Information unber��cksichtigt. Aufgrund der gro��en Anzahl von atomaren und molekularen Signalen, die repr��sentativ f��r die chemische Struktur der analysierten Oberfl��chen sind, ist es sinnvoll, diese F��lle von Informationen zur Quantifizierung der Oberfl��cheneigenschaften (Elementkonzentrationen, Kontaktwinkel etc.) zu verwenden. Zus��tzlich erm��glicht diese Methode eine quantitative Bestimmung der Oberfl��cheneigenschaften auf nur ��m-gro��en Bereichen. Das ist vorteilhaft f��r Untersuchungen chemisch strukturierter Oberfl��chen, da die Gr����e der Strukturierung f��r viele Anwendungen in einem Bereich von mehreren ��m liegt. Anhand eines Beispieles aus dem biologisch-medizinischen Fachgebiet, soll der erfolgreiche Einsatz multivariater Modelle aufgezeigt werden. In diesem Experiment wurden menschlichen Bindegewebs- (Fibroblasten) und Pankreaszellen auf plasmafunktionalisiserten Oberfl��chen kultiviert, um die Beeinflussung der Funktionalisierung auf das Zellwachstum zu untersuchen. Die plasmabehandelten Oberfl��chen wurden durch die Verwendung von TEM-Gittern mit ��m-gro��en ��ffnungen chemisch strukturiert und das Wachstumsverhalten der Zellen beobachtet. Jedem dieser ��m-gro��en Bereiche k��nnen mithilfe der multivariaten Modelle quantitative Gr����en zugeordnet werden (Konzentrationen und Kontaktwinkelwerte), die zur Interpretation des Wachstumsverhaltens der Zellen beitragen.

Cell Host-Microbe Interactions: Turning Pathogen Mechanisms Into Cell's Advantages

Host-Pathogen Interaction is a very vast field of biological sciences, indeed every year many un- known pathogens are uncovered leading to an exponential growth of this field. The present work lyes between its boundaries, touching different aspects of host-pathogen interaction: We have evaluate the permissiveness of Mesenchimal Stem cell (FM-MSC from now on) to all known human affecting herpesvirus. Our study demonstrate that FM-MSC are full permissive to HSV1, HSV2, HCMV and VZV. On the other hand HHV6, HHV7, EBV and HHV8 are susceptible, but failed to activate a lytic infection program. FM-MSC are pluripotent stem cell and have been studied intensely in last decade. FM-MSC are employed in some clinical applications. For this reason it is important to known the degree of susceptibility to transmittable pathogens. Our atten- tion has then moved to bacterial pathogens: we have performed a proteome-wide in silico analy- sis of Chlamydiaceae family, searching for putative Nuclear localization Signal (NLS). Chlamy- diaceae are a family of obligate intracellular parasites. It���s reasonably to think that its members could delivered to nucleus effector proteins via NLS sequences: if that were the case the identifi- cation of NLS carrying proteins could open the way to therapeutic approaches. Our results strengthen this hypothesis: we have identified 72 protein bearing NLS, and verified their func- tionality with in vivo assays. Finally we have conceived a molecular scissor, creating a fusion protein between HIV-1 IN protein and FokI catalytic domain (a deoxyexonuclease domain). Our aim is to obtain chimeric enzyme (trojIN) which selectively identify IN naturally occurring target (HIV LTR sites) and cleaves subsequently LTR carrying DNA (for example integrated HIV1 DNA). Our preliminary results are promising since we have identified trojIN mutated version capable to selectively recognize LTR carrying DNA in an in vitro experiments.

Fabrication, Electrical Characterization and Simulation of Thin Film Solar Cells: CdTe and CIGS Materials

CdTe and Cu(In,Ga)Se2 (CIGS) thin film solar cells are fabricated, electrically characterized and modelled in this thesis. We start from the fabrication of CdTe thin ���lm devices where the R.F. magnetron sputtering system is used to deposit the CdS/CdTe based solar cells. The chlorine post-growth treatment is modi���ed in order to uniformly cover the cell surface and reduce the probability of pinholes and shunting pathways creation which, in turn, reduces the series resistance. The deionized water etching is proposed, for the ���rst time, as the simplest solution to optimize the effect of shunt resistance, stability and metal-semiconductor inter-diffusion at the back contact. In continue, oxygen incorporation is proposed while CdTe layer deposition. This technique has been rarely examined through R.F sputtering deposition of such devices. The above experiments are characterized electrically and optically by current-voltage characterization, scanning electron microscopy, x-ray diffraction and optical spectroscopy. Furthermore, for the ���rst time, the degradation rate of CdTe devices over time is numerically simulated through AMPS and SCAPS simulators. It is proposed that the instability of electrical parameters is coupled with the material properties and external stresses (bias, temperature and illumination). Then, CIGS materials are simulated and characterized by several techniques such as surface photovoltage spectroscopy is used (as a novel idea) to extract the band gap of graded band gap CIGS layers, surface or bulk defect states. The surface roughness is scanned by atomic force microscopy on nanometre scale to obtain the surface topography of the ���lm. The modi���ed equivalent circuits are proposed and the band gap graded profiles are simulated by AMPS simulator and several graded pro���les are examined in order to optimize their thickness, grading strength and electrical parameters. Furthermore, the transport mechanisms and Auger generation phenomenon are modelled in CIGS devices.

Morphological instability analysis of a misfit strained core-shell nanowire for the growth of quantum dots

Nell'ambito delle nanostrutture, un ruolo primario �� svolto dai punti quantici. In questo lavoro siamo interessati all'analisi teorica del processo di creazione dei punti quantici: esso pu�� avvenire per eteroepitassia, in particolare secondo il metodo studiato da Stranski-Krastanov. Un film di Germanio viene depositato su un substrato di Silicio in modo coerente, cio�� senza dislocazioni, e, a causa del misfit tra le maglie dei due materiali, c'�� un accumulo di energia elastica nel film. A una certa altezza critica questa energia del film pu�� essere ridotta se il film si organizza in isole (punti quantici), dove la tensione pu�� essere rilassata lateralmente. L'altezza critica dipende dai moduli di Young (E, ��), dal misfit tra le maglie (m) e dalla tensione superficiali (��). Il trasporto di materiale nel film �� portato avanti per diffusione superficiale. Il punto focale nell'analisi delle instabilit�� indotte dal misfit tra le maglie dei materiali �� la ricerca delle caratteristiche che individuano il modo di crescita pi�� rapido dei punti quantici. In questo lavoro siamo interessati ad un caso particolare: la crescita di punti quantici non su una superficie piana ma sulla superficie di un nanofilo quantico a geometria cilindrica. L'analisi delle instabilit�� viene condotta risolvendo le equazioni all'equilibrio: a tal fine sono state calcolate le distribuzioni del tensore delle deformazioni e degli sforzo di un nanofilo core-shell con una superficie perturbata al primo ordine rispetto all'ampiezza della perturbazione. L'analisi �� stata condotta con particolari condizioni al contorno ed ipotesi geometriche, e diverse scelte dello stato di riferimento del campo degli spostamenti. Risolto il problema elastico, �� stata studiata l'equazione dinamica di evoluzione descrivente la diffusione di superficie. Il risultato dell'analisi di instabilit�� �� il tasso di crescita in funzione del numero d'onda q, con diversi valori del raggio del core, spessore dello shell e modo normale n, al fine di trovare il pi�� veloce modo di crescita della perturbazione.

Schriftlicher Text im Film und seine ��bersetzung : die Inserts der BBC-Serie Sherlock (2010-12) ; aus der Perspektive von Untertiteltheorie, Comictheorie und Translationspraxis

Die BBC-Serie SHERLOCK war 2011 eine der meistexportierten Fernsehproduktionen Gro��britanniens und wurde weltweit in viele Sprachen ��bersetzt. Eine der Herausforderungen bei der ��bersetzung stellen die Schrifteinblendungen der Serie (kurz: Inserts) dar. Die Inserts versprachlichen die Gedanken des Protagonisten, bilden schriftliche und digitale Kommunikation ab und zeichnen sich dabei durch ihre visuelle Auff��lligkeit und teilweise als einzige Tr��ger sprachlicher Kommunikation aus, womit sie zum wichtigen ��sthetischen und narrativen Mittel in der Serie werden. Interessanterweise sind in der ��bersetztung alle stilistischen Eigenschaften der Original-Inserts erhalten. In dieser Arbeit wird einerseits untersucht, wie Schrifteinblendungen im Film theoretisch beschrieben werden k��nnen, und andererseits, was sie in der Praxis so ��bersetzt werden k��nnen, wie es in der deutschen Version von Sherlock geschah. Zur theoretischen Beschreibung werden zun��chst die Schrifteinblendungen in Sherlock Untertitelungsnormen anhand relevanter grundlegender semiotischer Dimensionen gegen��bergestellt. Weiterhin wird das Verh��ltnis zwischen Schrifteinblendungen und Filmbild erkundet. Dazu wird gepr��ft, wie gut verschiedene Beschreibungsans��tze zu Text-Bild-Verh��ltnissen aus der Sprachwissenschaft, Comicforschung, ��bersetzungswissenschaft und Typografie die Einblendungen in Sherlock erkl��ren k��nnen. Im praktischen Teil wird die ��bersetzung der Einblendungen beleuchtet. Der ��bersetzungsprozess bei der deutschen Version wird auf Grundlage eines Experteninterviews mit dem Synchronautor der Serie rekonstruiert, der auch f��r die Formulierung der Inserts zust��ndig war. Abschlie��end werden spezifische ��bersetzungsprobleme der Inserts aus der zweiten Staffel von SHERLOCK diskutiert. Es zeigt sich, dass Untertitelungsnormen zur Beschreibung von Inserts nicht geeignet sind, da sie in Dimensionen wie Position, grafische Gestaltung, Animation, Soundeffekte, aber auch Timing stark eingeschr��nkt sind. Dies l��sst sich durch das historisch gepr��gte Verst��ndnis von Untertiteln erkl��ren, die als m��glichst wenig st��rendes Beiwerk zum fertigen Filmbild und -ablauf (notgedrungen) hinzugef��gt werden, wohingegen f��r die Inserts in SHERLOCK teilweise sogar ein zentraler Platz in der Bild- und Szenenkomposition bereits bei den Dreharbeiten vorgesehen wurde. In Bezug auf Text-Bild-Verh��ltnisse zeigen sich die gr����ten Parallelen zu Ans��tzen aus der Comicforschung, da auch dort schriftliche Texte im Bild eingebettet sind anstatt andersherum. Allerdings sind auch diese Ans��tze zur Beschreibung von Bewegung und Ton unzureichend. Die Erkundung der Erkl��rungsreichweite weiterer vielversprechender Konzepte, wie Interface und Usability, bleibt ein Ziel f��r k��nftige Studien. Aus dem Experteninterview l��sst sich schlie��en, dass die ��bersetzung von Inserts ein neues, noch unstandardisiertes Verfahren ist, in dem idiosynkratische praktische L��sungen zur sprach��bergreifenden Kommunikation zwischen verschiedenen Prozessbeteiligten zum Einsatz kommen. Bei hochqualitative Produktionen zeigt ist auch f��r die ersetzende Insert��bersetzung der Einsatz von Grafikern unerl��sslich, zumindest f��r die Erstellung neuer Inserts als ��bersetzungen von gefilmtem Text (Display). Hierbei sind die theoretisch m��glichen Synergien zwischen Sprach- und Bildexperten noch nicht voll ausgesch��pft. Zudem zeigt sich Optimierungspotential mit Blick auf die Bereitstellung von sorgf��ltiger Dokumentation zur ausgangssprachlichen Version. Diese w��re als Referenzmaterial f��r die ��bersetzung insbesondere auch f��r Zwecke der internationalen Qualit��tssicherung relevant. Die ��bersetzten Inserts in der deutschen Version weisen insgesamt eine sehr hohe Qualit��t auf. ��bersetzungsprobleme ergeben sich f��r das genretypische Element der Codes, die wegen ihrer Kompaktheit und multiplen Bez��gen zum Film eine Herausforderung darstellen. Neben weiteren bekannten ��bersetzungsproblemen wie intertextuellen Bez��gen und Realia stellt sich immer wieder die Frage, wieviel der im Original dargestellten Insert- und Displaytexte ��bersetzt werden m��ssen. Aus Gr��nden der visuellen Konsistenz wurden neue Inserts zur ��bersetzung von Displays notwendig. Au��erdem stellt sich die Frage insbesondere bei F��lltexten. Sie dienen der Repr��sentation von Text und der Erweiterung der Grenzen der fiktiv dargestellten Welt, sind allerdings mit hohem ��bersetzungsaufwand bei minimaler Bedeutung f��r die Handlung verbunden.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Biology of FGFRL1, the fifth fibroblast growth factor receptor

FGFRL1 (fibroblast growth factor receptor like 1) is the most recently discovered member of the FGFR family. It contains three extracellular Ig-like domains similar to the classical FGFRs, but it lacks the protein tyrosine kinase domain and instead contains a short intracellular tail with a peculiar histidine-rich motif. The gene for FGFRL1 is found in all metazoans from sea anemone to mammals. FGFRL1 binds to FGF ligands and heparin with high affinity. It exerts a negative effect on cell proliferation, but a positive effect on cell differentiation. Mice with a targeted deletion of the Fgfrl1 gene die perinatally due to alterations in their diaphragm. These mice also show bilateral kidney agenesis, suggesting an essential role for Fgfrl1 in kidney development. A human patient with a frameshift mutation exhibits craniosynostosis, arguing for an additional role of FGFRL1 during bone formation. FGFRL1 contributes to the complexity of the FGF signaling system.

Effects on choroidal neovascularization after anti-VEGF Upload using intravitreal ranibizumab, as determined by spectral domain-optical coherence tomography

It is unclear whether anti-VEGF monotherapy in age-related macular degeneration (AMD) achieves morphologic CNV regression or only stops further CNV growth. In this study, spectral domain-optical coherence tomography (SD-OCT) was used to image CNV structure before and after anti-VEGF treatment.

Extraordinary transmission through a single coaxial aperture in a thin metal film

We investigate experimentally the transmission properties of single sub-wavelength coaxial apertures in thin metal films (t = 110 nm). Enhanced transmission through a single sub-wavelength coaxial aperture illuminated with a strongly focused radially polarized light beam is reported. In our experiments we achieved up to four times enhanced transmission through a single coaxial aperture as compared to a (hollow) circular aperture with the same outer diameter.We attribute this enhancement of transmission to the excitation of a TEM-mode for illumination with radially polarized light inside the single coaxial aperture. A strong polarization contrast is observed between the transmission for radially and azimuthally polarized illumination. Furthermore, the observed transmission through a single coaxial aperture can be strongly reduced if surface plasmons are excited. The experimental results are in good agreement with finite difference time domain (FDTD) simulations.

Femur Specific Polyaffine Model to Regularize the Log-Domain Demons Registration

Osteoarticular allograft transplantation is a popular treatment method in wide surgical resections with large defects. For this reason hospitals are building bone data banks. Performing the optimal allograft selection on bone banks is crucial to the surgical outcome and patient recovery. However, current approaches are very time consuming hindering an efficient selection. We present an automatic method based on registration of femur bones to overcome this limitation. We introduce a new regularization term for the log-domain demons algorithm. This term replaces the standard Gaussian smoothing with a femur specific polyaffine model. The polyaffine femur model is constructed with two affine (femoral head and condyles) and one rigid (shaft) transformation. Our main contribution in this paper is to show that the demons algorithm can be improved in specific cases with an appropriate model. We are not trying to find the most optimal polyaffine model of the femur, but the simplest model with a minimal number of parameters. There is no need to optimize for different number of regions, boundaries and choice of weights, since this fine tuning will be done automatically by a final demons relaxation step with Gaussian smoothing. The newly developed synthesis approach provides a clear anatomically motivated modeling contribution through the specific three component transformation model, and clearly shows a performance improvement (in terms of anatomical meaningful correspondences) on 146 CT images of femurs compared to a standard multiresolution demons. In addition, this simple model improves the robustness of the demons while preserving its accuracy. The ground truth are manual measurements performed by medical experts.

The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor ? through spatial relocalization at helix 7 of its ligand-binding domain

Peroxisome proliferator-activated receptor ? (PPAR?) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR? upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR? is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR? signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR? and enhanced nuclear translocation and ligand-independent transcription of PPAR? target genes. In contrast, Cav1 overexpression sequestered PPAR? in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR?'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR? and to inhibit cell proliferation. Ligand-activated PPAR? also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR? regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR? to ligands, limiting proliferation of gastric epithelial cells.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.