Functional analysis of human POT1 and RPA : insights into the role of single stranded DNA binding proteins at telomeres

Abstract Telomeres, the natural ends of chromosomes, need to be protected from chromosome end fusions, aberrant homologous recombination and degradation. In humans, chromosome ends are specified through arrays of tandemly repeated 5'-TTAGGG-3' hexamers, ending in a 3' overhang. A complex formed by the six proteins TRF1, TRF2, hRap1, TIN2, TPP1 and POT1 specifically assocìates with and protects telomeres. Telomeres are maintained by semiconservative DNA replication and by a specialized reverse transcriptase, telomerase, that carries an RNA subunit which templates new telomeric repeat synthesis. The telomeric single stranded (ss) DNA binding protein POT1 protects the telomeric 3' overhang and modulates telomerase-mediated telomere elongation. It is possible that POT1 also influences DNA synthesis during semiconservative DNA replication, which is initiated by the DNA polymerase alpha-primase complex. The heterotrimeric ss DNA-binding protein RPA plays essential roles during DNA replication. RPA binds to ss DNA with high affinity in order to stabilize ss DNA and facilitate nascent strand synthesis at the replication fork. Here we investigate how the two proteins RPA and POT1 contribute to telomere maintenance by regulating semi-conservative DNA replication and telomerase. Using chromatin immunoprecipitation experiments, we show that RPA associates with telomeres during S-phase. Analysis of telomere structure in cells shRNA-depleted for RPA and POT1 reveals that loss of RPA and POT1 causes exposure of single-stranded DNA at telomeres, suggestive of incomplete DNA replication. Biochemical experiments using purified recombinant POT1 and RPA show that saturating telomeric oligonucleotides with POT1 or RPA reduces the primase activity of the DNA polymerase alpha-primase complex and the overall activity of telomerase. POT1 and RPA also increase the primer extension by DNA polymerase alpha-primase complex and the processivity of telomerase under certain conditions, although POT1 increases the activities to a greater extent than RPA. We propose that POT1 is required for proper replication of the lagging strand of telomeres and that some phenotypes observed in POT1-depleted cells may stern from incomplete DNA replication rather than de-protection of the single-stranded overhang. Résumé Les télomères, les extrémités normales des chromosomes linéaires, doivent être protégés des fusions chromosomiques, d'événements de recombinaison homologue aberrants et de phénomènes de dégradation. Chez l'Homme, les extrémités des chromosomes sont constitués d'ADN double brin répétitif de séquence 5'-TTAGGG-3', d'une extension simple brin 3' sortante et d'un complexe protéique formé des six facteurs TRF1, TRF2, hRap1, TIN2, TPP1 et POT1 qui, s'associant à cette séquence, protègent l'ADN télomèrique. Les télomères sont maintenus par la télomérase, une transcriptase inverse capable d'allonger l'extension 3' sortante télomérique. POT1 lie l'ADN simple brin télomérique et module l'élongation des télomères par la télomérase. POT1 pourrait en théorie également influencer la réplication semi-conservative de l'ADN. L'ADN-polymérase Pal alpha-primase amorce et initie la synthèse d'ADN. Pendant la réplication, l'ADN simple brin est stabilisé par RPA, un complexe hétérotrimèrique qui lie l'ADN simple brin. RPA facilite la synthèse du brin naissant à la fourche de réplication. Ici nous avons étudié comment ces deux protéines qui lient l'ADN simple brin, RPA et POT1, régulent la réplication des télomères par la télomérase et la machinerie classique de réplication de l'ADN. Par immunoprécipitation de chromatine (ChIP), nous montrons que RPA est localisé aux télomères lors de la phase S du cycle cellulaire. De plus, l'analyse de la structure des télomeres indique que !a perte de RPA ou de POT1 conduit à l'apparition d'ADN simple brin télomérique, suggérant une réplication incomplète de l'ADN télomérique in vivo. Par une approche complémentaire biochimique utilisant les protéines POT1 et RPA recombinantes purifiées, nous montrons également que la liaison de POT1 ou de RPA à des oligonucléotides télomériques bloque l'activité primase du complexe polymérase alpha/primase et réduit l'activité télomérase sur ces substrats. En revanche, leur liaison augmente l'activité ADN-polymérase du complexe polymérase alpha/primase, ainsi que fa processivité de la télomérase dans certaines conditions, POT1 étant le plus efficace des deux facteurs. Nous proposons que POT1 est nécessaire à la réplication du brin retardé au niveau des télomères, ce qui suggère que certains phénotypes des cellules déplétés en POT1 puissent résulter d'une réplication incomplète de l'ADN télémétrique plutôt que d'une déprotection de l'extrémité sortante des télomères.

Iowa Values Fund and Business Assistance Programs 2007 Annual Report, 2007

Following is the 2007 Annual Report of the Iowa Values Fund (IVF 2005) and Business Assistance Programs covering activity during Fiscal Year 2007 (FY ‘07) and cumulative for the first four years of the Iowa Values Fund Programs. The IVF (2005) is the primary funding source for a menu of financial assistance programs the Iowa Department of Economic Development (IDED) offers as incentives to Iowa companies to expand here, to recruit new companies into Iowa and assist new entrepreneurial ventures. In addition to IDED the law appropriates IVF (2005) funds for economic development activities to the Board of Regents, the Departments of Cultural Affairs and Natural Resources and to Community Colleges for certain workforce training programs. In addition to the IVF (2005), IDED allocates a portion of Community Development Block Grant and Federal Emergency Stimulus funds and several tax credit programs, all of which are included in this report.

Micro-insurance through corporate-NGO partnerships in West Bengal : opportunities and constraints

This Practical Note examines the nascent micro-insurance sector in West Bengal, paying particular attention to the corporate- NGO partnership model for micro-insurance distribution,which has been enabled by India's unique regulatory framework. We challenge the popularconstruction of this model as a 'win - win' for all parties by analysing conflicting understandings of micro-insurance schemes and their purposes by insurance companies, NGOs, and poorvillagers. The article also considers the role of the specific political context of West Bengal inconstricting corporate- NGO micro-insurance

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Iowa Prison Industries Five Year Transition and Business Plan 2011-2015, 2011

IPI is comprised of three divisions. Private Sector funds are handed over to the General Fund. Traditional Industries and Farms funds are managed by IPI. The auditor of the state provides oversight on policies, procedures, and compliance with state law. Each year, the auditor is responsible for providing the Governor, legislature, Director of Corrections, and the public the findings of their comprehensive audits. IPI has received a clean bill of health and has not been cited for any violations in ten (10) years. IPI operates under the guidance of an advisory board, comprised of seven members. The advisory board meets at least four (4) times per year at a location of the board‟s choice, generally at a different prison each quarter. The board reviews the financials, policies, approves any new private sector ventures and offers comprehensive guidance on issues that will impact correctional industries as well as the public and local businesses. Each member serves for two (2) years and may be re-appointed. IPI has found that retaining board members has helped immensely with the continuity of transition and has afforded IPI with superb leadership and guidance. IPI is 100% self-funding. We receive no appropriations from the general fund. We hire our staff, pay their salaries, and pay the stipend of the offenders. We pay for our raw materials, equipment, and construct our buildings all from the proceeds of our sales. We operate with a revolving fund and retain any earnings at year-ends. The retained earnings are used for expansion of our work programs.

Nuclear and cytoplasmic triiodothyronine-binding sites in primary sensory neurons and Schwann cells: radioautographic study during development.

The effects of the thyroid hormones on target cells are mediated through nuclear T3 receptors. In the peripheral nervous system, nuclear T3 receptors were previously detected with the monoclonal antibody 2B3 mAb in all the primary sensory neurons throughout neuronal life and in peripheral glia at the perinatal period only (Eur. J. Neurosci. 5, 319, 1993). To determine whether these nuclear T3 receptors correspond to functional ones able to bind T3, cryostat sections and in vitro cell cultures of dorsal root ganglion (DRG) or sciatic nerve were incubated with 0.1 nM [125I]-labeled T3, either alone to visualize the total T3-binding sites or added with a 10(3) fold excess of unlabeled T3 to estimate the part due to the non-specific T3-binding. After glutaraldehyde fixation, radioautography showed that the specific T3-binding sites were largely prevalent. The T3-binding capacity of peripheral glia in DRG and sciatic nerve was restricted to the perinatal period in vivo and to Schwann cells cultured in vitro. In all the primary sensory neurons, specific T3-binding sites were disclosed in foetal as well as adult rats. The detection of the T3-binding sites in the nucleus indicated that the nuclear T3 receptors are functional. Moreover the concomitant presence of both T3-binding sites and T3 receptors alpha isoforms in the perikaryon of DRG neurons infers that: 1) [125I]-labeled T3 can be retained on the T3-binding 'E' domain of nascent alpha 1 isoform molecules newly-synthesized on the perikaryal ribosomes; 2) the alpha isoforms translocated to the nucleus are modified by posttranslational changes and finally recognized by 2B3 mAb as nuclear T3 receptor. In conclusion, the radioautographic visualization of the T3-binding sites in peripheral neurons and glia confirms that the nuclear T3 receptors are functional and contributes to clarify the discordant intracellular localization provided by the immunocytochemical detection of nuclear T3 receptors and T3 receptor alpha isoforms.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.

Positionnement de l'aire urbaine parisienne dans les réseaux mondiaux des entreprises multinationales

La région-capitale comme principal relais européen vers et depuis l'extérieur Paris est dans le trio de tête des villes globales dans les réseaux mondiaux des entreprises, après Londres et New York, davantage grâce au rayonnement de ses entreprises que par son attractivité aux entreprises étrangères. Sa spécificité tient à son rôle particulier de coordination entre des villes européennes. Très bien placée pour relayer les entrées continentales vers d'autres pays européens, elle occupe un rôle de relais dominant pour la sortie des investissements européens dans le monde en particulier vers le Japon. Par ailleurs, elle est la première ville mondiale en relation avec les villes africaines. Une faiblesse relative de son attractivité La région-capitale ne représente pas la destination première des investissements contrôlés depuis les villes qu'elle investit, notamment en Europe, à l'exception de Bruxelles et Francfort. Elle ne représente la première destination des investissements que pour les deux tiers des villes françaises, et hors d'Europe, elle n'est privilégiée que par les villes africaines. Par ailleurs, elle montre une relative faiblesse dans les activités les plus qualifiées. Vers une métropole francilienne plus high-tech collaborant davantage avec les entreprises étrangères Des mesures incitant les entreprises françaises à davantage s'internationaliser, coopérer avec les entreprises étrangères en créant des joint-ventures à l'exemple de Raytheon et Thalès renforcerait l'attractivité de la métropole francilienne. De même, une meilleure identité du Grand Paris avec la création d'espaces dédiés à l'accueil de secteurs de pointe high tech manquant actuellement de lisibilité et soutenus par de grandes entreprises françaises performantes contribueraient à renforcer son attractivité.

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10 % of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

Strategic alliances. How to make them succeed

Mitjançant aquest treball hem analitzat com en aquest temps en què la internacionalització és un fet necessari i les empreses volen globalitzar, aquestes decideixen unir-se per tal d'aconseguir. Aquí analitzem els diferents tipus d'unió possible centrant-nos més específicament en les aliances estratègiques però veient també les adquisicions. Primer analitzem quan és més convenient aliar-se amb una empresa o adquirir-la. Això ho portarem a terme mitjançant el desenvolupament d'una sèrie d'elements a tenir en compte. Un cop vist això, analitzem els principals motius pels quals les empreses decideixen aliar-se, entre els quals destaquen l'obtenció de coneixement i recursos. A més analitzem les diferents variants d'aliança estratègica com són les llicències, franquícies, contractes de recerca i desenvolupament, Joint Ventures, etc. i quan és més convenient desenvolupar una o una altra. En aquest treball, ja que com veurem les aliances tendeixen a fracassar ja que les empreses no les implementen correctament o no dediquen el temps suficient a analitzar l'entorn i les condicions per veure quina estratègia li convé més a l'empresa, analitzarem els elements necessaris per fer-les funcionar i els factors claus d'èxit d'aquestes aliances com ara l'experiència, la reputació, la confiança, els objectius clars, el Know-how, la cultura ... A més en aquest treball analitzem el cas de l'aliança estratègica sorgida entre Disney i Pixar el 1991. Aquest anàlisi el duem a terme tenint en compte els factors que Dyer et. al. esmentà en el seu estudi. Aquest anàlisi el realitzem amb l'objectiu de conèixer si l'aliança realitzada per ambdues empreses va ser una estratègia encertada o si per contra deurien haver dut a terme una altra estratègia, la qual els hauria aportat més beneficis.

Heavy metal ions are potent inhibitors of protein folding.

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd2+, Hg2+ and Pb2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC(50) in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far.

Business Development Efforts and Performance Improvements in SMEs Case Study of Business Development Projects Implemented in SME

Under the circumstances of the increasing market pressure, enterprises try to improve their competitive position by development efforts, and a business development project is one tool for that. There are not many answers to the question of how the development projects launched to improve the business performance in SMEs have succeeded. Theacademic interest in the business development project success has mainly focused on projects implemented in larger organisations rather than in SMEs. The previous studies on the business success of SMEs have mainly focused on new business ventures rather than on existing SMEs. However, nowadays a large number of business development projects are undertaken in existing SMEs, where they can pose a great challenge. This study focuses on business development success in SMEs thathave already established their business. The objective of the present study is to gain a deep understanding on business development project success in the SME-context and to identify the dimensions and factors affecting the project success. Further, the aim is to clarify how the business development projects implemented in SMEs have affected their performance. The empirical evidence is based on multiple case study. This study builds a framework for a generic theory of business development success in the SME-context, based on literature from the areas ofproject and change management, entrepreneurship and small business management, as well as performance measurement, and on empirical evidence from SMES. The framework consists of five success dimensions: entrepreneurial, project preparation, change management, project management and project success. The framework provides a systematic way for analysing the business development project and its impact on the performance and on the performing company. This case evidence indicates that successful business development projects have a balanced, high performance concerning all the dimensions. Good performance in one dimension is not enoughfor the project success, but it gives a good ground for the other dimensions. The other way round, poor performance in one success dimension affects the others, leading to poor performance of the project. In the SME-context the business development project success seems to be dependent on several interrelated dimensions and factors. Success in one area leads to success in other areas, and so creates an upward success spiral. Failure in one area seems to lead to failure in other areas, creating a downward failure spiral. The study indicates that the internal business development projects have affected the SMEs' performance widely also on areas and functions not initially targeted. The implications cover all thesuccess categories: the project efficiency, the impact on the customer, the business success and the future potentiality. With successful cases, the success tends to spread out to areas and functions not mentioned as the project goals, andwith unsuccessful cases the failure seems to spread out widely to the SMEs' other functions. This study also indicates that the most important key factors for successful business development project implementation are the strength of intention, business ability, knowledge, motivation and participation of the employees, as well as adequate and well-timed training provided to the employees.

Plasmapolttoprosessin kehittäminen ja optimointi

Ydinvoimaloidenprimaarivesikierron puhdistukseen käytetään ioninvaihtohartsia. Käytönjälkeen ioninvaihtohartsi luokitellaan matalaja keskiaktiivisiin jätteisiin. Plasmakäsittelyllä käytetyn ioninvaihtohartsin tilavuutta voidaan pienentää sekä sen orgaaninen luonne poistaa. Plasmakäsittelyn tarkoituksena on hapettaa orgaaninen aines oksideiksi, jotka poistuvat prosessista savukaasuina. Epäorgaaninen aines, joka sisältää radioaktiivisen aineksen, on tarkoitus hapettaa oksideiksi ja sulfideiksi, jotka voidaan kerätä talteen tuhkana. Tässä diplomityössä käsitellään käytetyn ioninvaihtohartsin käsittelyyn suunnitellun plasmapolttoprosessin kehittämistä ja optimointia. Ioninvaihtohartsin plasmakäsittelyssä syntyvien reaktiotuotteiden selvittäminen suoritettiin tarkastelemalla ainetaseita sekä aihetta käsitteleviä tutkimuksia. Näiden perusteella parannettiin jäähdytystä, suunniteltiin jatkuvatoiminen syöttömenetelmä sekä laadittiin toimintaalueen reunaehdot laitteistolle. Koelaitteistossa 6,5 kW:n rfteho syötetään sovitinpiirin ja kuparisen induktiokelan kautta plasmaan. Plasmakaasuna on käytetty hapenja argonin seoskaasua. Plasmapolttoa on seurattu massaspektrometrilla, optisella emissiospektrometrilla, lämpösekä painemittareilla. Laskennan ja kokeiden pohjalta selvitettiin optimaalinen seossuhde plasmakaasulle, paineen ja tehon noston vaikutus hartsin polttonopeuteen sekä jatkuvatoimisen syöttömenetelmän edut panostoimiseen syöttöön. Rfgeneraattorin teho rajoitti jatkuvatoimisen polttonopeuden 130 g/h ja hetkellisen polttonopeuden 175 g/h. Radioaktiivisten aineiden pidätys oli 93,5 % cesiumin osalta. Tulosten perusteella 4 kg/h ioninvaihtohartsia polttavan laitteiston tehon lähteeksi tarvitaan 65 kW rfgeneraattori. Palamattoman hartsin ja tuhkan kulkeutuminen partikkelisuodattimille sekä reaktiotuotteena syntyvien rikinoksidien käsittely vaatii vielä jatkotutkimusta.

The Role and Measurement of Social Capital in New Technology-Based Start-up Context

Tämän Pro gradu-tutkielman tavoitteena olirakentaa esiymmärrys sosiaalisen pääoman roolista ja mittaamisesta uuden teknologian start-up yrityksissä. Pääasiallisena tarkoituksena tässä tutkimuksessa olilöytää sosiaalisen pääoman ja start-up yrityksen tuloksellisuuden välille yhdistävä tekijä. Tutkimuksen empiirinen aineisto kerättiin pääasiallisesti kuuden OKO Venture Capitalin sijoitusportfolioon sisältyvien case-yritysten kvalitatiivisten teemahaastatteluiden sekä kvantitatiivisten kyselylomakkeiden avulla. Kvalitatiivisten haastatteluiden tulosten perusteella sosiaalisen pääoman ja tuloksellisuuden välille löytyi yhdistävä tekijä, jota käytettiin myöhemmin hyväksi kvantitatiivisessa kyselylomakkeessa. Tämän tutkielman tulokset osoittivat, että startegisen päätöksenteon kautta sosiaalinen pääoma vaikuttaa osittain start-up yritysten tuloksellisuuteen. Manageriaalisesti tärkempi löydös tässä tutkimuksessa oli kuitenkin se, että sosiaalista pääomaa voidaan käyttää hyväksi ennustettaessa uuden teknologian start-up yritysten tulevaisuuden kassavirtoja.