Susceptibilitat genètica al càncer gàstric: caracterització de les interaccions de segon ordre entre gens i variants identificades en l’estudi EPIC‐EURGAST

Curs 2012-2013En aquest Treball de Final de Grau s’exposen els resultats de l’anàlisi de les dades genètiques del projecte EurGast2 "Genetic susceptibility, environmental exposure and gastric cancer risk in an European population”, estudi cas‐control niat a la cohort europea EPIC “European Prospective lnvestigation into Cancer and Nutrition”, que té per objectiu l’estudi dels factors genètics i ambientals associats amb el risc de desenvolupar càncer gàstric (CG). A partir de les dades resultants de l’estudi EurGast2, en el què es van analitzar 1.294 SNPs en 365 casos de càncer gàstric i 1.284 controls en l’anàlisi Single SNP previ, la hipòtesi de partida del present Treball de Final de Grau és que algunes variants amb un efecte marginal molt feble, però que conjuntament amb altres variants estarien associades al risc de CG, podrien no haver‐se detectat. Així doncs, l’objectiu principal del projecte és la identificació d’interaccions de segon ordre entre variants genètiques de gens candidats implicades en la carcinogènesi de càncer gàstric. L’anàlisi de les interaccions s’ha dut a terme aplicant el mètode estadístic Model‐based Multifactor Dimensionality Reduction Method (MB‐MDR), desenvolupat per Calle et al. l’any 2008 i s’han aplicat dues metodologies de filtratge per seleccionar les interaccions que s’exploraran: 1) filtratge d’interaccions amb un SNP significatiu en el Single SNP analysis i 2) filtratge d’interaccions segons la mesura Sinèrgia. Els resultats del projecte han identificat 5 interaccions de segon ordre entre SNPs associades significativament amb un major risc de desenvolupar càncer gàstric, amb p‐valor inferior a 10‐4. Les interaccions identificades corresponen a interaccions entre els gens MPO i CDH1, XRCC1 i GAS6, ADH1B i NR5A2 i IL4R i IL1RN (que s’ha validat en les dues metodologies de filtratge). Excepte CDH1, cap altre d’aquests gens s’havia associat significativament amb el CG o prioritzat en les anàlisis prèvies, el que confirma l’interès d’analitzar les interaccions genètiques de segon ordre. Aquestes poden ser un punt de partida per altres anàlisis destinades a confirmar gens putatius i a estudiar a nivell biològic i molecular els mecanismes de carcinogènesi, i orientades a la recerca de noves dianes terapèutiques i mètodes de diagnosi i pronòstic més eficients.In this Final Degree Project the results of the genetic analysis of the nested‐case/control EurGast2 project "Genetic susceptibility, environmental exposure and gastric cancer risk in an European population” of the European EPIC cohort “European Prospective lnvestigation into Cancer and Nutrition” are exposed. Its aim is to study the genetic and environmental factors associated to the risk of developing gastric cancer (GC). In base of the EurGast2 study which analyzed 1.294 SNPs in 365 gastric cancer cases and 1.284 controls in a previous Single SNP analysis, the present hypothesis of this Final Degree Project defends that some variants with a weak marginal effect which may be associated to GC together with other variants could have not been detected. The aim of this project is to identify second order interactions between genetic variants from candidate genes implicated in gastric cancer carcinogenesis. The interaction’s analysis has been carried out applying the statistical Model‐based Multifactor Dimensionality Reduction Method MB‐MDR developed by Calle et al. in 2008 and two different methodologies have been applied in order to select the best second order interactions to explore: 1) interactions’ filtering with one significant SNP in the Single SNP analysis and 2) interactions’ filtering using the Synergy measure. The results of the project have identified that 5 second order interactions between different SNPs are statistically associated with a major risk of developing gastric cancer at a p‐value<10‐4. These interactions identified correspond to interactions between SNPs from MPO and CDH1 genes, XRCC1 and GAS6 genes, ADH1B and NR5A2 genes and IL4R and IL1RN genes (which has been validated in both independent filter methods). Except CDH1, any other of these genes have been associated statistically to gastric cancer or has been prioritized in the previous univariant analysis. This fact confirms the interest of analyzing genetic second order interactions which can represent a start point for other analysis to confirm putative genes and study at a biological and molecular level the mechanisms underpinning gastric cancer carcinogenesis which can represent a step closer to the research of new therapeutic targets and the definition of more efficient diagnosis methods

Transfection of Capsaspora owczarzaki, a close unicellular relative of animals

How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki. We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity.Fil: Parra Acero, Helena. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Ros Rocher, Núria. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Perez Posada, Alberto. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Kożyczkowska, Aleksandra. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Sánchez Pons, Núria. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Nakata, Azusa. Prefectural University of Hiroshima; JapónFil: Suga, Hiroshi. Prefectural University of Hiroshima; JapónFil: Najle, Sebastián Rodrigo. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ruiz Trillo, Iñaki. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España. Institució Catalana de Recerca i Estudis Avancats; España. Universidad de Barcelona; Españ

What is it like to be a choanoflagellate? Sensation, processing and behavior in the closest unicellular relatives of animals

International audienceAll animals evolved from a single lineage of unicellular precursors more than 600 million years ago. Thus, the biological and genetic foundations for animal sensation, cognition and behavior must necessarily have arisen by modifications of pre-existing features in their unicellular ancestors. Given that the single-celled ancestors of the animal kingdom are extinct, the only way to reconstruct how these features evolved is by comparing the biology and genomic content of extant animals to their closest living relatives. Here, we reconstruct the Umwelt (the subjective, perceptive world) inhabited by choanoflagellates, a group of unicellular (or facultatively multicellular) aquatic microeukaryotes that are the closest living relatives of animals. Although behavioral research on choanoflagellates remains patchy, existing evidence shows that they are capable of chemosensation, photosensation and mechanosensation. These processes often involve specialized sensorimotor cellular appendages (cilia, microvilli, and/or filopodia) that resemble those that underlie perception in most animal sensory cells. Furthermore, comparative genomics predicts an extensive “sensory molecular toolkit” in choanoflagellates, which both provides a potential basis for known behaviors and suggests the existence of a largely undescribed behavioral complexity that presents exciting avenues for future research. Finally, we discuss how facultative multicellularity in choanoflagellates might help us understand how evolution displaced the locus of decision-making from a single cell to a collective, and how a new space of behavioral complexity might have become accessible in the process

The Origin of multicellularity in animals : a functional approach from a unicellular perspective : Developing Capsaspora owczarzaki as an emerging model

The origin of animal multicellularity is a major evolutionary question. Recent genome data from the closest extant unicellular relatives of animals revealed that they actually possess and express a complex repertoire of genes related to animal development and multicellularity. Thus, assessing the functions of those genes in those unicellular relatives is key to gaining insight into to how they were co-opted at the onset of animals. However, such analyses have been hampered by the lack of genetic tools. Overall, this thesis advances our understanding of how co-option worked by using two different approaches. First, I provide a reliable transfection method for the filasterean Capsaspora owczarzaki, the close unicellular relative of animals with the richest repertoire of genes related to transcriptional regulation. This accomplishment converts Capsaspora into a unique experimentally tractable organism to investigate the origin and evolution of animal multicellularity. Then, I provide evidence of a remarkable degree of conservation between several transcription factor (TFs) networks in Capsaspora, suggesting that complex regulatory networks of TFs existed in the unicellular ancestor of animals.L’origen de la multicel·lularitat dels animals és un gran enigma evolutiu. Dades genòmiques recents dels parents unicel·lulars més propers als animals han revelat que codifiquen i expressen un repertori complex de gens relacionats amb el desenvolupament i amb la multicel·lularitat animal. Per aquest motiu, investigar les funcions d’aquests gens en aquests parents unicel·lulars és clau per entendre com es van co-optar a l’origen dels animals. No obstant, aquestes anàlisis han estat obstaculitzades per la falta d’eines genètiques. En conjunt, aquesta tesi avança el nostre coneixement de com la co-opció va funcionar de dues maneres. En primer lloc, aporto un protocol fiable de transfecció per al filasteri Capsaspora owczarzaki, el parent unicel·lular més proper als animals amb el repertori genètic més ric en gens relacionats amb la regulació transcripcional. Això converteix Capsaspora en un organisme tractable experimentalment únic per investigar l’origen i l’evolució dels animals. Després, aporto evidència d’un grau considerable de conservació entre diverses xarxes de factors de transcripció a Capsaspora, suggerint que xarxes de factors de transcripció complexes van existir en l’ancestre unicel·lular dels animals

Chemical factors induce aggregative multicellularity in a close unicellular relative of animals

Regulated cellular aggregation is an essential process for development and healing in many animal tissues. In some animals and a few distantly related unicellular species, cellular aggregation is regulated by diffusible chemical cues. However, it is unclear whether regulated cellular aggregation was part of the life cycles of the first multicellular animals and/or their unicellular ancestors. To fill this gap, we investigated the triggers of cellular aggregation in one of animals’ closest unicellular living relatives—the filasterean Capsaspora owczarzaki. We discovered that Capsaspora aggregation is induced by chemical cues, as observed in some of the earliest branching animals and other unicellular species. Specifically, we found that calcium ions and lipids present in lipoproteins function together to induce aggregation of viable Capsaspora cells. We also found that this multicellular stage is reversible as depletion of the cues triggers disaggregation, which can be overcome upon reinduction. Our finding demonstrates that chemically regulated aggregation is important across diverse members of the holozoan clade. Therefore, this phenotype was plausibly integral to the life cycles of the unicellular ancestors of animals.We thank the Light Microscopy Center at the Indiana University for support in image acquisition and analysis (funding provided by the NIH grant NIH1S10OD024988-01) and the Advanced Light Microscopy Unit of the Center for Genomic Regulation for support on image acquisition. We also thank the Indiana University Nanoscale Characterization Facility, Electron Microscopy Center, and Laboratory for Biological Mass Spectrometry for use of their instruments. We want to special thank Claudio Scazzocchio for fruit-ful discussions and Sebastián R. Najle for feedback and support in early work on Capsaspora aggregation. We acknowledge Omaya Dudin for feedback and support in image acquisition and analysis, Michelle M. Leger for feedback on the manuscript, and Koryu Kin for support in immunocytochemistry experi-ments, image acquisition and analysis, and stimulating discussions. We also thank many people from the lab for insights and support that helped advance this project. This work was supported by a NIH grant (R35GM138376) to J.P.G. and grants BFU2017-90114-P from Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI), and Fondo Europeo de Desarrollo Regional and PID2020-120609GB-I00 by Ministerio de Ciencia e Innovación/AEI/10.13039/501100011033 and “European Regional Development Fund: A way of making Europe” to I.R.-T. N.R.-R. was supported by a “Formación del Profesorado Universitario (FPU13/01840)” predoctoral fellowship from Ministerio de Educación, Cultura y Deporte and R.Q.K. was supported by a NIH training grant (T32GM131994