Identification and characterization of CIP2A as a novel oncogenic inhibitor of PP2A

Inhibition of the tumor suppressor protein phosphatase 2A (PP2A) activity has been identified as one of the five key alterations required for human cell transformation. Regardless of this crucial role in human cancer development, the detailed mechanisms by which PP2A inhibition occurs in human cancers remain largely uncharacterized. PP2A regulates a plethora of cellular signaling cascades. One of the targets of PP2A is Myc oncoprotein, which is destabilized and degraded in response to PP2A-mediated dephosphorylation of Myc serine 62. In this study we identify Cancerous Inhibitor of PP2A (CIP2A) as a previously uncharacterized endogenous inhibitor of PP2A in human cancer cells. CIP2A inhibits PP2A activity leading to subsequent stabilization of the Myc protein. CIP2A promotes malignant growth of cancer cells in vitro and xenograft tumor formation in vivo and is overexpressed in cancer. Moreover, we explored the effect of CIP2A on global transcriptional profiles and validated a CIP2A-dependent transcriptional signature. Analysis of the CIP2A signature revealed both Myc-dependent and -independent functions for CIP2A. Importantly, we demonstrate that the CIP2A signature has clinical relevance in human breast cancer subtypes. Finally, we identify the genes potentially mediating the long-term growth suppression in CIP2A depleted cancer cells. Taken together, this work identifies CIP2A as a novel human oncoprotein and describes its function in cancer cells. These results may open novel possibilities for patient stratification and therapeutic intervention of cancer.

Reactive oxygen species in inflammation

Chronic inflammation is the underlying cause of many common disabling conditions such as rheumatoid arthritis (RA), multiple sclerosis, coeliac disease, type I diabetes and coronary artery disease. NOX2 complex derived reactive oxygen species (ROS) are known to regulate joint inflammation in rats and mice, and additionally recent genetic evidence associates phagocyte ROS and the development RA in humans. Ncf1mutated mice have lost the functionality of their NOX2 complex and thus have no phagocyte ROS production. These mice suffer from exacerbated arthritis. The immune suppressive effect of the NOX2 complex derived ROS is mediated by monocytes/macrophages that downregulate the activation of autoreactive T cells. The aim of this thesis was to study how ROS modulate immune responses in different arthritis models and in tumor development. Additionally, genome wide gene expression profiling was carried out to assess the global effects of NOX2 complex derived ROS. Firstly, these results confirmed the potent anti-inflammatory nature of phagocyte ROS in arthritis models that were driven by the adaptive immune system. Secondly, arthritis models with predominantly innate immunity induced pathophysiology were moderately enhanced by phagocyte, more specifically, neutrophil derived ROS. Thirdly, the ROS induced immune suppression mediated by the adaptive immune system allowed development of bigger implanted tumors, while phagocyte ROS production did not affect the development of spontaneously growing tumors. Lastly, genome wide gene expression analysis revealed that both humans and mice with abrogated phagocyte NOX2 complex ROS production had an enhanced type I interferon signature in blood, reflecting their hyperinflammatory immune status.

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25ºC and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M-1, respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DCp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K-1 mol-1 for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

Organisaatioprosessin muutos

Tutkimuksessa tarkasteltiin sähköiseen arkistointiin siirtymisen muutosprosessia Lahden kaupungin organisaatiossa vuosien 2013 – 2015 välillä. Tavoitteena oli sel-vittää kunnallisen organisaation sähköiseen arkistointiin siirtymisen vaatiman muu-tosprosessin toteutuksen ratkaisut sekä muutoksen vaikutukset hallinnollisiin pro-sesseihin. Työ on metodologialtaan kvalitatiivinen tapaustutkimus, jonka tutkimus-aineistona käytettiin haastatteluja sekä organisaation sisäisiä asiakirjoja. Tutkimuk-sessa haastateltiin kohdeorganisaation muutoksen suunnittelun sekä toteutuksen avainhenkilöitä. Tutkimustulokset osoittivat, että muutosprosessi noudatti tieteellisen tutkimuksen kuvaamaa muutoksen vaiheittaista etenemistä, vaikkakin vaiheet me-nivät osittain päällekkäin. Muutosprosessissa tulisi tulosten perusteella kiinnittää huomiota muutoksessa ilmenevien vastakkaisten mielipiteiden vaikutukseen muu-toksen hidastavana tekijänä ja ottaa jo suunnitteluvaiheessa tämä huomioon esi-merkiksi miettimällä toimiva kannustinjärjestelmä tukemaan muutoksen toteutusta. Merkittävimmät havainnot prosessien muuttumiseen liittyen olivat, että arkistointia ei voi ajatella erillisenä prosessina asiakirjan elinkaaren lopussa, vaan sähköisessä toimintaympäristössä arkistointi sulautuu kiinteäksi osaksi organisaation kaikkea toimintaa. Arkistoinnin tapaa muutettaessa on otettava huomioon eri toiminnoille asetetut vaatimukset, kuten tietoturva, allekirjoitus, arkistointi, arkistossa asiointi se-kä hävitys. Sähköiseen arkistointiin siirtyminen muuttaa organisaation eri toimijoi-den rooleja ja jossain kohden tuo säästöjä, mutta toisaalta vaatii organisaatiolta muuttumiskykyä. Osaltaan muutos vauhdittaa myös erilaisten toimintojen keskittä-misen mahdollisuuksia.

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

CLEVER-1 as an immune suppressive molecule

The immune response and immune suppression are equally essential for the immune system to protect the host against an infection and to protect self-molecules in different pathophysiological conditions. Pregnancy is one of the conditions where the maternal immune system remains resistant against microbes and yet attains tolerance to protect the fetus, whose genetic material differs partially from the mother’s. However, if the balance of immune suppression is not precise in the host it can favor conditions which lead to diseases, such as cancer and autoimmune disorders. This study was initiated to investigate the expression and functions of CLEVER-1/Stabilin-1, a multifunctional protein expressed on subsets of endothelial cells and type II macrophages, as an immune suppressive molecule. Firstly, the expression of CLEVER-1/stabilin-1 and its function in human placental macrophages were examined. Secondly, the expression profile and functional significance of stabilin-1 on healthy human monocytes was investigated. The results clarified the expression of CLEVER-1/stabilin-1 on placental macrophages, and verified that CLEVER-1/stabilin-1 functions as an adhesion and scavenging molecule on these cells. The data from normal monocytes revealed that the monocytes with low stabilin-1 expression carried a pro-inflammatory gene signature, and that stabilin-1 can directly or indirectly regulate pro-inflammatory genes in monocytes. Finally, it was shown that monocyte CLEVER-1/stabilin-1 dampens IFN production by T cells. To conclude, CLEVER-1/stabilin-1 is defined as an immune suppressive molecule on monocytes and macrophages. Strikingly, anti-stabilin-1 antibodies may have the potential to promote the Th1 dependent inflammatory response and counteract the tumor induced immune suppression.

Analogisen tunnisteanalyysin käyttö korjauspalveluissa mikropiirien ylijännitteestä aiheutuneiden vaurioiden paikantamisen

Korjauspalveluissa aikaa vieviä tapauksia ovat mikropiirien vaikeasti paikannettavat viat. Tällaista vianetsintää varten yrityksemme oli ostanut Polar Fault Locator 780 –mittalaitteen, jolla voidaan mitata mikropiirien toimintaa käyttämällä analogista tunnisteanalyysiä. Diplomityön tavoitteena oli selvittää, miten mittaustapaa voidaan käyttää korjauspalveluissa. Tutkintaa lähestyttiin joidenkin tyypillisten komponenttien näkökulmasta, mutta pääpaino oli mikropiireissä. Joitain mikropiirejä vaurioitettiin tahallisesti, jolloin mittaustulokset uusittiin ja tutkittiin miten vaurioituminen näkyy mittaustuloksissa. Tutkimusmenetelmänä oli kirjallisuus ja empiirinen kokeellisuus. Diplomityön tuloksena oli, että tätä mittaustapaa käyttämällä mikropiirien kuntoa voidaan tutkia. Ongelmiksi osoittautuivat alkuperäinen oletus mittalaitteen tuloksien tulkinnasta ja taustamateriaalin heikko saatavuus. Täten mittalaite parhaiten soveltuu tilanteisiin, joissa sen antamia tuloksia verrataan suoraan toisen toimivaksi tunnetun yksikön mittaustuloksiin. Vaurioitettaessa komponenteissa oli kuitenkin havaittavissa selvä poikkeavuus.

RECUEIL DES ŒUVRES POÉTIQUES de Jehan Molinet. Ms. in-fol. sur papier de 201 f., demi-rel. v. f, tr. r. (Rel. du XVIIIe siècle.)

Complainte (La) de Grece

Statuts de l'ordre des Hospitaliers de Saint-Jean de Jérusalem, arrêtés sous le grand maître Pierre d'Aubusson. (Fol. 21-106).

Contient : Forme abrégée desdits statuts, précédée (fol. 107 v°) de « la declaracion des rubriques et chapitres des establissemens qui s'ensuivent », et (fol. 108 r°) d'une ordonnance de PIERRE D'AUBUSSON, relative à cette forme de statuts, datée de Rhodes, le 5 août 1493 ; Résumé pour les prieurs et châtelain d'Emposte desdits statuts

[Lettre autographe signée de Giuseppe Verdi adressée à Du Locle, Gênes, 25 décembre 1871]

Comprend aussi une phrase et signature autographe de Teresa Stolz

Psalterium cisterciense.

F. 1 Psautier. F. 134v Cantiques, suivis de « preces » (146). F. 148 Litanies cisterciennes.

Evangelia.

Avec prologues, arguments et « capitula ». « Capitulare Evangeliorum de circulo anni » (1) ; Evang. Matthaei (18v), Marci I,1-XV,39 (62v), Lucae (88v), Johannis (128).

Speculum humanae salvationis.

F. 1-3 Table des chapitres. (Lutz et Perdrizet, Spec. hum. salvat., I, 2-99 et 329).

Expositio in Apocalypsim.

F. 2 « Hoc dicitur quod iste Johannes... Apocalipsis, id est revelatio, ista est commendabilis... — ... ipse venit ad redemptionem humani genen???is. » F. 96 19 vers : « Pax animam nutrit... — ... desperet quisque fidelis. »