Sädekiviliuskeen olemus:liuskekiven käyttö varhaisneoliittisella Simo-Tervola -alueella, tapaustutkimuksena Tainiaro

Tiivistelmä. Tutkielman aiheena on Simon Tainiaron varhaisneoliittisen muinaisjäännöskohteen liuskekivilöydöt vuoden 2018 arkeologisilta kaivauksilta. Alun perin liuskekiviesineiden materiaaliksi oli luokiteltu sädekiviliuske, mutta tutkielmassani pyrin problematisoimaan tämän tulkinnan vanhentuneisiin käsitteisiin perustuvana. Tutkielman tarkoituksena oli selvittää, mitä materiaalia kiviesineet ovat, mistä niiden raaka-aine on peräisin, miksi sitä on käytetty kyseisellä kohteella ja mikä on tämän kivimateriaalin käyttökokemus. Tutkimusaineistona toimi löytöaineiston lisäksi liuskekiviset raaka-ainenäytteet, jotka otettiin kenttätyömatkalla kesällä 2019 Tervolan Tikanmaan muodostuman rekisteröidyillä muinaislouhoksilla ja muilla, mahdollisesti käytettävissä olleilla kalliopaljastumilla, sekä muutama sädekiviliuskeeksi kategorisoitu verrokkikappale Simon Hyötymaan kaivauksilta sekä Kemijärven Lehtolasta. Tutkimusmenetelminä käytin valikoitujen Tainiaron artefaktien ja näytekivien geokemiallista pXRF-analyysiä, kokeellista arkeologiaa sekä artefaktien esteettisten piirteiden luokittelua. Geokemiallisen analyysin tuloksista pyrin muodostamaan klustereita SPSS-tilastolaskuohjelman avulla, ja siten tutkimaan, ryhmittyykö Tainiaron löytöaineisto Tikanmaan muodostuman liuskemateriaalin kanssa samankaltaisena. Valmistamalla näytekivistä esineitä pyrin saamaan tietoa kiven käyttökokemuksesta sekä sen suotuisista ominaisuuksista raaka-aineena. Esteettisellä kategorisoinnilla pyrin havaitsemaan esimerkiksi värin ja hiotun pinnan suhteiden trendejä, mistä saatua dataa pyrin peilaamaan kokeellisten menetelmien tuloksiin. Tutkimuksen perusteella Tainiaron liuskekiviartefaktit vaikuttavat olevan peräisin Tikanmaan muodostumasta, jossa oli saavutettavissa liuskekiveä avokallioina Tainiaron aktiivisen käyttökauden aikana. Kyseisen kivimateriaalin käyttö vaikuttaa perustuneen sen esteettisten ja käytännöllisten ominaisuuksien synteesiin, mihin lukeutuu esimerkiksi vaalean ja tumman kiviaineksen erilainen muokattavuus lohkeamisen ja hioutumisen suhteen, luonnollinen esiintyminen ihmisen muokkaamilta vaikuttavina kappaleina, sekä reagointi vereen erilaisissa käyttöolosuhteissa. Esineet ovat voineet saada useita yliluonnollisina pidettäviä merkityksiä, mikä on voinut vaikuttaa niiden päätymiseen kalmistokontekstiin. Ehdotan tutkielmassani, että virhekäsityksiin perustuvan ”sädekiviliuske” -käsitteen käytöstä luovuttaisiin arkeologisessa tutkimuksessa. Artefaktien täsmällinen kivilaji ei selvinnyt tutkielman puitteissa, joten se vaatii laajempaa geokemiallisilla menetelmillä tehtävää tutkimusta. Lisäksi ehdotan Tainiaron kohteen aikaisempien kaivausten liuskekivilöytöjen systemaattista läpikäyntiä, jotta tutkielmassa esitetyt päätelmät liuskekivimateriaalin käyttötrendeistä voitaisiin varmistaa laajemmalla aineistolla

Parille päällikön pöytään?:olut nuorakeraamisen kivikauden Suomessa

Tiivistelmä. Tutkielmassani pohdin oluenvalmistuksen mahdollisuuksia nuorakeraamisen kivikauden Suomen alueella, sekä vertaan niitä Unto Salon aikaisempaan julkaisutoimintaan aiheesta. Kolme näkökulmaani mahdollisuuksiin ovat kontaktit ja tietotaito, raaka-aineet sekä teknologia. Lopputulokseni oli, että oluenvalmistuksen alkaminen tuolloin olisi nykytiedon varjolla mahdollista, muttei täysin varmistettavissa

Re-evaluation of diagnostic parameters is crucial for obtaining accurate data on idiopathic pulmonary fibrosis

Background: The FinnishIPF registry is a prospective, longitudinal national registry study on the epidemiology of idiopathic pulmonary fibrosis (IPF). It was designed to describe the characteristics, management and prognosis of prevalent and incident IPF patients. The study was initiated in 2012. Methods: We present here results limited to five university hospitals. Patients with IPF were screened from hospital registries using ICD-10 diagnosis codes J84.1 and J84.9. All patients who gave informed consent were included and evaluated using novel diagnostic criteria. Point prevalence on the 31st of December in 2012 was calculated using the reported population in each university hospital city as the denominator. Results: Patients with ICD-10 codes J84.1 and J84.9 yielded a heterogeneous group - on the basis of patient records assessed by pulmonologists only 20-30 % of the cases were IPF. After clinical, radiological and histological re-evaluation 111 of 123 (90 %) of patients fulfilled the clinical criteria of IPF. The estimated prevalence of IPF was 8.6 cases/100 000. 60.4 % were men. Forty four percent of the patients were never-smokers. At diagnosis, the patients' mean age was 73.5 years and mean FVC was 80.4 % and DLCO 57.3 % of predicted. Conclusions: Our results suggest that hospital registries are inaccurate for epidemiological studies unless patients are carefully re-evaluated. IPF is diagnosed in Finland at a stage when lung function is still quite well preserved. Smoking in patients with IPF was less common than in previous reports.Peer reviewe

High expression of tumour-associated trypsin inhibitor correlates with liver metastasis and poor prognosis in colorectal cancer

Increased expression of tumour-associated trypsin inhibitor (TATI) in tumour tissue and/or serum has been associated with poor survival in various cancer forms. Moreover, a proinvasive function of TATI has been shown in colon cancer cell lines. In this study, we have examined the prognostic significance of tumour-specific TATI expression in colorectal cancer, assessed by immunohistochemistry (IHC) on tissue microarrays (TMAs) with tumour specimens from two independent patient cohorts. Kaplan–Meier analysis and Cox proportional hazards modelling were used to estimate time to recurrence, disease-free survival and overall survival. In both cohorts, a high (>50% of tumour cells) TATI expression was an independent predictor of a significantly shorter overall survival. In cohort II, in multivariate analysis including age, gender, disease stage, differentiation grade, vascular invasion and carcinoembryonal antigen (CEA), high TATI expression was associated with a significantly decreased overall survival (HR=1.82; 95% CI=1.19–2.79) and disease-free survival (HR=1.56; 95% CI=1.05–2.32) in curatively treated patients. Moreover, there was an increased risk for liver metastasis in both cohorts that remained significant in multivariate analysis in cohort II (HR=2.85; 95% CI=1.43–5.66). In conclusion, high TATI expression is associated with liver metastasis and is an independent predictor of poor prognosis in patients with colorectal cancer

Relocation of the NIb gene in the tobacco etch potyvirus genome

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.03336-13[EN] Potyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.This research was supported by grant BIO2011-26741 from the Spanish Ministerio de Economia y Competitividad (MINECO) to J.-A.D., grant PROMETEO/2010/019 from Generalitat Valenciana to S. F. E. and J.-A.D., and grants BFU2012-30805 and 22371 from MINECO and the John Templeton Foundation, respectively, to S. F. E. E. M. was supported by a predoctoral fellowship (AP2012-3751) from the Spanish Ministerio de Educacion, Cultura y Deporte. M.P.Z. was supported by a Juan de la Cierva postdoctoral contract (JCI-2011-10379) from MINECO and a Rubicon grant from the Netherlands Organization for Scientific Research (www.nwo.nl).Majer, E.; Salvador, Z.; Zwart, MP.; Willemsen, A.; Elena Fito, SF.; Daros Arnau, JA. (2014). Relocation of the NIb gene in the tobacco etch potyvirus genome. Journal of Virology. (88):4586-4590. https://doi.org/10.1128/JVI.03336-13S4586459088Roossinck, M. J. (2011). The big unknown: plant virus biodiversity. Current Opinion in Virology, 1(1), 63-67. doi:10.1016/j.coviro.2011.05.022Chirico, N., Vianelli, A., & Belshaw, R. (2010). Why genes overlap in viruses. Proceedings of the Royal Society B: Biological Sciences, 277(1701), 3809-3817. doi:10.1098/rspb.2010.1052Belshaw, R., Pybus, O. G., & Rambaut, A. (2007). The evolution of genome compression and genomic novelty in RNA viruses. Genome Research, 17(10), 1496-1504. doi:10.1101/gr.6305707Nguyen, M., & Haenni, A.-L. (2003). Expression strategies of ambisense viruses. Virus Research, 93(2), 141-150. doi:10.1016/s0168-1702(03)00094-7Riechmann, J. L., Lain, S., & Garcia, J. A. (1992). Highlights and prospects of potyvirus molecular biology. Journal of General Virology, 73(1), 1-16. doi:10.1099/0022-1317-73-1-1Urcuqui-Inchima, S., Haenni, A.-L., & Bernardi, F. (2001). Potyvirus proteins: a wealth of functions. Virus Research, 74(1-2), 157-175. doi:10.1016/s0168-1702(01)00220-9Chung, B. Y.-W., Miller, W. A., Atkins, J. F., & Firth, A. E. (2008). An overlapping essential gene in the Potyviridae. Proceedings of the National Academy of Sciences, 105(15), 5897-5902. doi:10.1073/pnas.0800468105Li, X. H., & Carrington, J. C. (1995). Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells. Proceedings of the National Academy of Sciences, 92(2), 457-461. doi:10.1073/pnas.92.2.457Bedoya, L., Martínez, F., Rubio, L., & Daròs, J.-A. (2010). Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector. Journal of Biotechnology, 150(2), 268-275. doi:10.1016/j.jbiotec.2010.08.006Bedoya, L. C., Martínez, F., Orzáez, D., & Daròs, J.-A. (2012). Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis. Plant Physiology, 158(3), 1130-1138. doi:10.1104/pp.111.192922Majer, E., Daròs, J.-A., & Zwart, M. (2013). Stability and Fitness Impact of the Visually Discernible Rosea1 Marker in the Tobacco etch virus Genome. Viruses, 5(9), 2153-2168. doi:10.3390/v5092153Bedoya, L. C., & Daròs, J.-A. (2010). Stability of Tobacco etch virus infectious clones in plasmid vectors. Virus Research, 149(2), 234-240. doi:10.1016/j.virusres.2010.02.004Chen, C.-C., Chen, T.-C., Raja, J. A. J., Chang, C.-A., Chen, L.-W., Lin, S.-S., & Yeh, S.-D. (2007). Effectiveness and stability of heterologous proteins expressed in plants by Turnip mosaic virus vector at five different insertion sites. Virus Research, 130(1-2), 210-227. doi:10.1016/j.virusres.2007.06.014Merits, A., Runeberg-Roos, P., Rajamäki, M.-L., Puustinen, P., Mäkeläinen, K., Saarma, M., … Kekarainen, T. (2002). Proteolytic processing of potyviral proteins and polyprotein processing intermediates in insect and plant cells. Journal of General Virology, 83(5), 1211-1221. doi:10.1099/0022-1317-83-5-1211Parks, T. D., Howard, E. D., Wolpert, T. J., Arp, D. J., & Dougherty, W. G. (1995). Expression and Purification of a Recombinant Tobacco Etch Virus NIa Proteinase: Biochemical Analyses of the Full-Length and a Naturally Occurring Truncated Proteinase Form. Virology, 210(1), 194-201. doi:10.1006/viro.1995.1331KIM, D.-H., PARK, Y. S., KIM, S. S., LEW, J., NAM, H. G., & CHOI, K. Y. (1995). Expression, Purification, and Identification of a Novel Self-Cleavage Site of the NIa C-Terminal 27-kDa Protease of Turnip Mosaic Potyvirus C5. Virology, 213(2), 517-525. doi:10.1006/viro.1995.0024Dolja, V. V., McBride, H. J., & Carrington, J. C. (1992). Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein. Proceedings of the National Academy of Sciences, 89(21), 10208-10212. doi:10.1073/pnas.89.21.10208Fernández-Fernández, M. R., Mouriño, M., Rivera, J., Rodríguez, F., Plana-Durán, J., & García, J. A. (2001). Protection of Rabbits against Rabbit Hemorrhagic Disease Virus by Immunization with the VP60 Protein Expressed in Plants with a Potyvirus-Based Vector. Virology, 280(2), 283-291. doi:10.1006/viro.2000.0762Sánchez, F., Sáez, M., Lunello, P., & Ponz, F. (2013). Plant viral elongated nanoparticles modified for log-increases of foreign peptide immunogenicity and specific antibody detection. Journal of Biotechnology, 168(4), 409-415. doi:10.1016/j.jbiotec.2013.09.002Masuta, C., Yamana, T., Tacahashi, Y., Uyeda, I., Sato, M., Ueda, S., & Matsumura, T. (2000). Development of clover yellow vein virus as an efficient, stable gene-expression system for legume species. The Plant Journal, 23(4), 539-546. doi:10.1046/j.1365-313x.2000.00795.xBeauchemin, C., Bougie, V., & Laliberté, J.-F. (2005). Simultaneous production of two foreign proteins from a potyvirus-based vector. Virus Research, 112(1-2), 1-8. doi:10.1016/j.virusres.2005.03.001Kelloniemi, J., Mäkinen, K., & Valkonen, J. P. T. (2008). Three heterologous proteins simultaneously expressed from a chimeric potyvirus: Infectivity, stability and the correlation of genome and virion lengths. Virus Research, 135(2), 282-291. doi:10.1016/j.virusres.2008.04.006Whitham, S. A., Yamamoto, M. L., & Carrington, J. C. (1999). Selectable viruses and altered susceptibility mutants in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 96(2), 772-777. doi:10.1073/pnas.96.2.772Dolja, V. V., Hong, J., Keller, K. E., Martin, R. R., & Peremyslov, V. V. (1997). Suppression of Potyvirus Infection by Coexpressed Closterovirus Protein. Virology, 234(2), 243-252. doi:10.1006/viro.1997.8660Dietrich, C. (2003). Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants. Journal of General Virology, 84(10), 2871-2876. doi:10.1099/vir.0.19245-0Rajamäki, M.-L., Kelloniemi, J., Alminaite, A., Kekarainen, T., Rabenstein, F., & Valkonen, J. P. T. (2005). A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions. Virology, 342(1), 88-101. doi:10.1016/j.virol.2005.07.01

Rewiring carotenoid biosynthesis in plants using a viral vector

[EN] Plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. This is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. Here we describe the use of a viral vector derived from Tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. The pathway consisted in three enzymes from the soil bacteria Pantoea ananatis. Lycopene is present at undetectable levels in chloroplasts of non-infected leaves. In tissues infected with the viral vector, however, lycopene comprised approximately 10% of the total carotenoid content. Our research further showed that plant viruses that express P. ananatis phytoene synthase (crtB), one of the three enzymes of the heterologous pathway, trigger an accumulation of endogenous carotenoids, which together with a reduction in chlorophylls eventually result in a bright yellow pigmentation of infected tissues in various host-virus combinations. So, besides illustrating the potential of viral vectors for engineering complex metabolic pathways, we also show a yellow carotenoid-based reporter that can be used to visually track infection dynamics of plant viruses either alone or in combination with other visual markers.We thank Veronica Aragones and M. Rosa Rodriguez-Goberna for excellent technical assistance. This research was supported by Spanish Ministerio de Economia y Competitividad (MINECO) grants BIO2014-54269-R to J.-A.D., and BIO2014-59092-P and BIO2015-71703-REDT to M. R.-C. Financial support from the Generalitat Valenciana (PROMETEOII/2014/021), the Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (Ibercarot 112RT0445), and the Generalitat de Catalunya (2014SGR-1434) is also acknowledged. E.M. is the recipient of a pre-doctoral fellowship (AP2012-3751) from the Spanish Ministerio de Educacion, Cultura y Deporte. B.L. is supported by a postdoctoral fellowship (FPDI-2013-018882) from MINECO.Majer, E.; Llorente, B.; Rodríguez-Concepción, M.; Daros Arnau, JA. (2017). Rewiring carotenoid biosynthesis in plants using a viral vector. Scientific Reports. 7. https://doi.org/10.1038/srep41645S7O’Connor, S. E. Engineering of secondary metabolism. Annu. Rev. Genet. 49, 71–94 (2015).Sainsbury, F. & Lomonossoff, G. P. Transient expressions of synthetic biology in plants. Curr. Opin. Plant Biol. 19, 1–7 (2014).Gleba, Y. Y., Tusé, D. & Giritch, A. Plant viral vectors for delivery by Agrobacterium. Curr. Top. Microbiol. Immunol. 375, 155–192 (2014).Chen, Q., He, J., Phoolcharoen, W. & Mason, H. S. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum. Vaccin. 7, 331–338 (2011).Pogue, G. P., Lindbo, J. A., Garger, S. J. & Fitzmaurice, W. P. Making an ally from an enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40, 45–74 (2002).Peyret, H. & Lomonossoff, G. P. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol. J. 13, 1121–1135 (2015).Bedoya, L. C., Martínez, F., Orzáez, D. & Daròs, J. A. Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. Plant Physiol. 158, 1130–1138 (2012).Majer, E., Daròs, J. A. & Zwart, M. P. Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome. Viruses 5, 2153–2168 (2013).Bedoya, L., Martínez, F., Rubio, L. & Daròs, J. A. Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector. J. Biotechnol. 150, 268–275 (2010).Kelloniemi, J., Mäkinen, K. & Valkonen, J. P. Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths. Virus Res. 135, 282–291 (2008).Carrington, J. C., Haldeman, R., Dolja, V. V. & Restrepo-Hartwig, M. A. Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo . J. Virol. 67, 6995–7000 (1993).Li, X. H. & Carrington, J. C. Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells. Proc. Natl. Acad. Sci. USA 92, 457–461 (1995).Fraser, P. D. & Bramley, P. M. The biosynthesis and nutritional uses of carotenoids. Prog. Lipid Res. 43, 228–265 (2004).Meléndez-Martínez, A. J., Mapelli-Brahm, P., Benítez-González, A. & Stinco, C. M. A comprehensive review on the colorless carotenoids phytoene and phytofluene. Arch. Biochem. Biophys. 572, 188–200 (2015).Rodríguez-Concepción, M. & Boronat, A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol. 130, 1079–1089 (2002).Giuliano, G. Plant carotenoids: genomics meets multi-gene engineering. Curr. Opin. Plant Biol. 19, 111–117 (2014).Cazzonelli, C. I. & Pogson, B. J. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci. 15, 266–274 (2010).Ruiz-Sola, M. A. & Rodríguez-Concepción, M. Carotenoid biosynthesis in Arabidopsis: a colorful pathway. Arabidopsis Book 10, e0158 (2012).Nisar, N., Li, L., Lu, S., Khin, N. C. & Pogson, B. J. Carotenoid metabolism in plants. Mol. Plant 8, 68–82 (2015).Misawa, N. et al. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli . J. Bacteriol. 172, 6704–6712 (1990).Hasunuma, T. et al. Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. Plant J. 55, 857–868 (2008).Lu, Y., Rijzaani, H., Karcher, D., Ruf, S. & Bock, R. Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons. Proc. Natl. Acad. Sci. USA 110, E623–632 (2013).Mann, V., Harker, M., Pecker, I. & Hirschberg, J. Metabolic engineering of astaxanthin production in tobacco flowers. Nat. Biotechnol. 18, 888–892 (2000).Wurbs, D., Ruf, S. & Bock, R. Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J. 49, 276–288 (2007).Cordero, M. T. et al. Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana . Mol. Plant-Microbe Interact. doi: 10.1094/MPMI-11-16-0239-R (2016).Ye, X. et al. Engineering the provitamin A (b-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305 (2000).Ravanello, M. P., Ke, D., Alvarez, J., Huang, B. & Shewmaker, C. K. Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production. Metab. Eng. 5, 255–263 (2003).Fujisawa, M. et al. Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation. J. Exp. Bot. 60, 1319–1332 (2009).Ohara, K., Ujihara, T., Endo, T., Sato, F. & Yazaki, K. Limonene production in tobacco with Perilla limonene synthase cDNA. J. Exp. Bot. 54, 2635–2642 (2003).Gutensohn, M. et al. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits. Plant J. 75, 351–363 (2013).Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H. & Misawa, N. Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 58, 1112–1114 (1994).Bahieldin, A. et al. Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter. Plasmid 72, 18–28 (2014).Xie, W., Lv, X., Ye, L., Zhou, P. & Yu, H. Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metab. Eng. 30, 69–78 (2015).Li, Y., Cui, H., Cui, X. & Wang, A. The altered photosynthetic machinery during compatible virus infection. Curr. Opin. Virol. 17, 19–24 (2016).Tilsner, J. & Oparka, K. J. Tracking the green invaders: advances in imaging virus infection in plants. Biochem. J. 430, 21–37 (2010).Kumagai, M. H. et al. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc. Natl. Acad. Sci. USA 92, 1679–1683 (1995).Kumagai, M. H., Keller, Y., Bouvier, F., Clary, D. & Camara, B. Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana . Plant J. 14, 305–315 (1998).Zhai, S., Xia, X. & He, Z. Carotenoids in staple cereals: metabolism, regulation, and genetic manipulation. Front. Plant Sci. 7, 1197 (2016).Zhang, H. et al. A Narcissus mosaic viral vector system for protein expression and flavonoid production. Plant Methods 9, 28 (2013).Nielsen, A. Z. et al. Redirecting photosynthetic reducing power toward bioactive natural product synthesis. ACS Synth. Biol. 2, 308–315 (2013).Sainsbury, F., Saxena, P., Geisler, K., Osbourn, A. & Lomonossoff, G. P. Using a virus-derived system to manipulate plant natural product biosynthetic pathways. Methods Enzymol. 517, 185–202 (2012).Geisler, K. et al. Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants. Proc. Natl. Acad. Sci. USA 110, E3360–3367 (2013).Kanagarajan, S., Muthusamy, S., Gliszczynska, A., Lundgren, A. & Brodelius, P. E. Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana . Plant Cell Rep. 31, 1309–1319 (2012).Mozes-Koch, R. et al. Expression of an entire bacterial operon in plants. Plant Physiol. 158, 1883–1892 (2012).Thole, V., Worland, B., Snape, J. W. & Vain, P. The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation. Plant Physiol. 145, 1211–1219 (2007).Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).Cunningham, F. X. Jr., Chamovitz, D., Misawa, N., Gantt, E. & Hirschberg, J. Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of b-carotene. FEBS Lett. 328, 130–138 (1993).Shivprasad, S. et al. Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255, 312–323 (1999).Schürer, H., Lang, K., Schuster, J. & Mörl, M. A universal method to produce in vitro transcripts with homogeneous 3′ ends. Nucleic Acids Res. 30, e56 (2002).Lu, R. et al. High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J. 22, 5690–5699 (2003).Dickmeis, C., Fischer, R. & Commandeur, U. Potato virus X-based expression vectors are stabilized for long-term production of proteins and larger inserts. Biotechnol. J. 9, 1369–1379 (2014).Nakagawa, T. et al. Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci. Biotechnol. Biochem. 71, 2095–2100 (2007).Bedoya, L. C. & Daròs, J. A. Stability of Tobacco etch virus infectious clones in plasmid vectors. Virus Res. 149, 234–240 (2010).Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025 (2006).Llorente, B. et al. Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism. Plant J. 85, 107–119 (2016)

Novel factors regulating cardiac remodeling in experimental models of cardiac hypertrophy and failure

Abstract Cardiac loading induces left ventricular hypertrophy and cardiac remodeling which when prolonged, leads to heart failure, a complex syndrome affecting approximately 1-2% of the adult population of the Western world with a prevalence increasing with age. Pathological remodeling involves functional and structural changes that are associated with fetal gene expression, sarcomeric re-organization, hypertrophy of cardiomyocytes, fibrosis, inflammation, oxidative stress and impairment of metabolism. The aim of this study was to investigate the role of three novel factors during the cardiac remodeling process with different experimental models of cardiac overload. Phosphatase and actin regulator 1 (Phacr1) expression was rapidly downregulated due to myocardial infarction (MI). Adenovirus-mediated Phactr1 overexpression changed the skeletal α-actin to cardiac α-actin ratio in both healthy and infarcted rat hearts and cultured cardiomyocytes. Phactr1 could regulate the actin isoform switch via the serum response factor (SRF). The expression of transforming growth factor (TGF)- β-stimulated clone 22 (TSC-22) was rapidly induced by multiple hypertrophic stimuli and was also evident post-MI. In addition, TSC-22 could regulate collagen 3a1 expression in the heart. The expression of retinal degeneration 3-like (Rd3l) was downregulated in response to pressure overload and also downregulated post-MI. Rd3l knockout mice expressed increased myocyte hypertrophy and cardiac dysfunction in response to a transverse aortic constriction (TAC) induced pressure overload. This thesis provides novel information about Phactr1, TSC-22 and Rd3l in load-induced cardiac hypertrophy and remodeling. Collectively these studies increase our understanding of the regulatory mechanisms underlying the progression of heart failure.Tiivistelmä Sydämen kuormitus saa aikaan vasemman kammion liikakasvun eli hypertrofian ja sydämen uudelleenmuovautumisen, mikä pitkittyessään johtaa sydämen vajaatoimintaan. Sydämen vajaatoiminta on monimutkainen oireyhtymä, josta länsimaissa kärsii noin 1-2 % aikuisväestöstä, ja esiintyvyys nousee iän myötä. Patologisessa uudelleenmuovautumisessa tapahtuu toiminnallisia ja rakenteellisia muutoksia, joihin liittyy muutoksia geenien ilmentymisessä, sarkomeerin uudelleen järjestäytymistä, sydänlihassolujen koon kasvua, fibroosia, tulehdusta, oksidatiivista stressiä ja aineenvaihdunnan huonontumista. Tämän työn tarkoituksena oli tutkia kolmen uuden tekijän roolia sydämen uudelleenmuovautumisessa erilaisissa kokeellisissa sydämen kuormituksen malleissa. Fosfataasin ja aktiinin säätelijä 1:n (Phactr1) ilmentyminen väheni nopeasti infarktin seurauksena. Adenovirusvälitteinen Phactr1:n ylituotanto muutti luusto- ja sydänlihasaktiinien isomuotojen suhdetta sekä terveessä että infarktisydämessä, samoin viljellyissä sydänlihassoluissa. Phactr1 saattaa säädellä isomuotojen suhdetta seerumiresponsiivisen tekijän (SRF) avulla. Transformoituvan kasvutekijä β1:n stimuloima proteiini 22:n (TSC-22) ilmentyminen nousi nopeasti usean hypertrofisen stimuluksen seurauksena sekä infarktin jälkeen. Lisäksi TSC-22 voisi säädellä kollageeni 3a1:n ilmentymistä sydämessä. Retinan degeneroituvan proteiinin 3 kaltaisen tekijän (Rd3l) ilmentyminen väheni sekä painekuormituksen että infarktin seurauksena. Rd3l-poistogeenisillä hiirillä aortan ahtauman aiheuttama painekuormitus sai aikaan lisääntynyttä sydänlihassolujen hypertrofiaa ja sydämen toimintahäiriöitä. Tämä väitöskirjatutkimus tuo uutta tietoa Phactr1-, TSC-22- ja Rd3l-geeneistä kuormituksen aiheuttamassa sydämen hypertrofiassa ja uudelleenmuovautumisessa. Nämä tulokset auttavat osaltaan ymmärtämään monimutkaisia molekyylitason mekanismeja, jotka johtavat sydämen vaajatoiminnan kehittymiseen