Memoria para optar al Título Profesional de Ingeniera Forestal / Cambios en composición y diversidad del sotobosque podrían indicar variaciones del ambiente, por ello es considerado un ente regulador en el ecosistema. También, una determinada asociación vegetal podría reflejar una condición ambiental específica. Los rasgos funcionales de las especies (morfológico, fisiológico o fenológico) podrían además ser indicadores claves de cómo las comunidades de plantas responden a cambios en el ambiente. El objetivo de este estudio es analizar la composición de especies y la diversidad del sotobosque en un bosque de Nothofagus pumilio establecido después del retroceso de los glaciares O'Higgins y Chico en Campo de Hielo Sur. A lo largo de un transecto, que abarca parte de las cuencas de ambos glaciares, se realizó un levantamiento florístico y ambiental dentro del bosque de Nothofagus pumilio. Se estableció un total de 20 parcelas (4 x 4 m). La cobertura de especies fue estimada mediante la escala de Londo. Se realizaron análisis de clasificación y ordenación para determinar grupos o asociaciones de especies del sotobosque y para analizar la influencia de las variables ambientales en la composición florística. A través del análisis de la cuarta esquina se exploró la influencia de los rasgos funcionales de las especies sobre las asociaciones y las interacciones con variables ambientales.

Evolutionary transitions between the two major predator avoidance strategies aposematism and crypsis are expected to be associated with changes in many important traits of animals. However, empirical studies on populations experiencing ongoing or recent transitions between these strategies are rare. This thesis investigates the co-evolution of traits among populations of the Strawberry poison frog D.pumilio in Bocas del Toro, Panama. I found that all investigated populations were genetically distinct but that colour and pattern did not correlate with genetic or geographic distance, which suggests that selection needs to be invoked to explain the observed variation. Based on the chromatic contrast between frog dorsal colour and the natural habitat substrates used by the frogs, the populations were defined as bright or dull coloured. I found that frogs from bright coloured populations were larger. This is expected if aposematism is enhanced by large signals while crypsis is enhanced by small size. Further, individuals from bright coloured populations had a coarser black dorsal pattern, which is expected if crypsis is impaired by a bold pattern. The importance of pattern coarseness was confirmed by an avian detection experiment showing that coarse patterned dark green prey were more easily detected than dark green prey without pattern or with fine pattern. I put forward the hypothesis that enhanced protection, gained by aposematism, may affect behaviours that influence dispersal and pairing patterns. Indeed, males from bright coloured populations displayed at more exposed sites and showed a tendency to be more explorative and aggressive. In summary, my results show that the bright and dull coloured populations most likely represent an aposematic and a cryptic strategy, respectively. Furthermore, I show that evolutionary changes between aposematism and crypsis can be associated with coevolution of both morphology and behaviour. I argue that this coevolution may increase the likelihood of both pre- and post-zygotic reproductive isolation. This is because greater phenotypic differences between populations increase the likelihood of selection against badly adapted migrants and hybrids with intermediate traits.

Les propriétés centrales des cellules souches sont la pluripotence et la capacité d'auto-renouvellement. Les cellules souches hématopoïétiques (CSHs) sont dotées de ces caractéristiques qui leur permettent de générer toutes les cellules du compartiment hématopoïétique, tout en maintenant en parallèle leur compartiment. Nous menons des approches visant à amplifier ex vivo les CSHs en les activant par HOXB4 exogène (CSHs humaines) ou via la signalisation Notch/DLL-4 (CSHs murines). Or deux analyses transcriptomiques indépendantes de ces deux modes d'activation ont de manière étonnante convergé sur une augmentation de l'expression de deux gènes jamais identifiés auparavant comme étant impliqués dans le maintien des CSHs : Pumilio1 (Pum1) et Pumilio2 (Pum2). Pum1 et Pum2 sont des régulateurs post-transcriptionnels appartenant à la famille Pumilio-FBF (PUF) des protéines liant l'ARN. Bien qu'il ait été établi que le rôle princeps de ces protéines PUF est de soutenir la prolifération des cellules souches chez les Invertébrés, jusqu'à présent on ne sait rien du rôle de Pum1 et Pum2 dans les CSH humaines et murines.Pour toutes ces raisons, nous avons étudié le rôle et les mécanismes d'action de Pum1 et Pum2 dans les CSH murines et humaines en utilisant l'interférence ARN (ARNi). L'invalidation de Pum1 ou de Pum2 dans les CSHs murines conduit à une réduction de l'expansion et du potentiel clonogénique ex vivo, associée à une apoptose accrue et l'arrêt du cycle cellulaire en phase G0/G1. L'invalidation concomitante de Pum1 et Pum2 majore ces effets ce qui suggère un effet coopératif entre les deux protéines. L'expansion et le potentiel clonogénique des CSH invalidées pour Pum1 sont restaurés suite à l'expression forcée de Pum1 (insensible au shRNA utilisé), validant ainsi la spécificité de nos shRNAs. Par contre la surexpression de Pum1 dans les CSHs invalidées pour Pum2 ne restaure pas leurs fonctions, soulignant le rôle non redondant de chaque protéine. En outre, lorsque les CSHs invalidées pour Pum1 ou Pum2 sont inoculées à des souris irradiées létalement de suivre le potentiel hématopoïétique à long terme, seules quelques rares cellules de la moelle osseuse issues des CSH KD pour Pum1 ou Pum2 sont mises en évidence après 4 mois de reconstitution, contrairement aux CSH contrôles. Des résultats identiques ont été obtenus en invalidant Pum1 ou Pum2 dans les CSH humaines.En conclusion, nos résultats démontrent l'implication des facteurs Pumilio dans le maintien du potentiel souche, l'expansion et la survie des CSHs murines et humaines. L'identification des facteurs Pumilio et de leurs cibles comme nouveaux régulateurs des CSHs permettra d'envisager de nouveaux outils en vue de perspectives thérapeutiques. / The central properties of stem cells are the pluripotency and the capacity of self-renewal. Hematopoietic stem cells (HSCs) posses such common features that allows them to generate all the cells of the hematopoietic compartments, maintaining in the same time the HSC pool. We develop approaches focused on ex vivo HSC expansion through activation by exogenous HOXB4 (human HSCs) or Notch/Dll-4 ligand (murine HSCs). Two independent transcriptomic analyses surprisingly converged toward an increased expression of two genes never identified sofar as crucial for HSC functions: Pumilio1 (Pum1) and Pumilio2 (Pum2). Pum1 and Pum2 are posttranscriptional regulators belonging to the Pumilio-FBF (PUF) family of RNA-binding proteins. Although it was established that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells in Invertebrates, so far nothing is known about the role of Pum1 and Pum2 in human and murine HSCs.For these reasons, we have investigated the roles and mechanisms of action of Pum1 and Pum2 in murine and human HSCs through shRNA strategy. Pum1 and Pum2 knockdown (KD) in murine HSCs led to a decreased HSC expansion and clonogenic potential ex vivo, associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase. KD of both Pum1 and Pum2 enhanced these effects, suggesting a cooperative effect. Expansion and clonogenic potential of KD Pum1 HSCs were rescued by enforced expression of Pum1 (insensitive to our shRNA), thus validating the specificity of our shRNA. Enforced expression of Pum1 could not rescue the functions of Pum2 KD HSCs, highlighting the non-redundant role of these proteins. Furthermore, when Pum1 or Pum2 KD HSCs were inoculated into lethally irradiated mice to follow the long-term hematopoietic potential, only rare bone marrow cells derived from Pum1 and Pum2 KD HSCs were evidenced after 4 months, contrary to control HSCs. Identical results were obtained with human Pum1 or Pum2 KD HSCs.In conclusion, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of murine and human HSCs. Identification of Pumilio factors and their targets as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.

Les protéines de liaison à l’ARN PUMILIO 1 et 2 (PUM1/2) exercent un rôle central dans le maintien des cellules souches chez les Invertébrés en se fixant, en association avec des partenaires protéiques, sur la région 3’ UTR de certains ARNm, régulant ainsi leur devenir. A ce jour, le rôle de PUM1/2 dans les cellules souches/progénitrices hématopoïétiques (CSPHs) a été peu étudié. La perte de la coordination entre auto-renouvellement et différenciation des CSPHs peut aboutir à des hémopathies chez l'Homme, d’où la nécessité de comprendre les mécanismes sous-jacents. Notre équipe a mis en évidence, par une approche de shARN, que l’invalidation des protéines PUM1/2 dans les CSHs humaines et murines conduit à une réduction de leur expansion, associée à une apoptose accrue et un arrêt du cycle cellulaire en phase G0/G1, et aussi à une perte du potentiel clonogénique in vitro et du potentiel de reconstitution in vivo. L’objectif de notre travail a consisté à : a/ évaluer les effets de la surexpression de PUM1/2 dans les CSPHs, b/ déterminer l’implication de PUM1/2 dans les processus leucémiques, c/ étudier les mécanismes moléculaires responsables de l’activité de PUM1/2 en identifiant les cibles et les partenaires protéiques par une approche de protéomique globale. Nos résultats suggèrent qu’une surexpression modérée de PUM1 (2/3 fois) dans les cellules CD34+ limite la perte du potentiel clonogénique alors qu’une expression plus élevée (5/10 fois et plus) est toxique. L’analyse de l’expression de PUM1/2 par RT-qPCR dans les échantillons de Leucémies Aigue Myeloïdes (LAM) (GOELAMSthèque) montre une augmentation significative dans les échantillons les plus immatures (LAM0-2) comparés aux contrôles sains. La perte de PUM1/2 par shARN dans les cellules primaires de leucémies ainsi que dans des lignées issues de différents processus leucémiques réduit fortement leur survie. La recherche des partenaires associés à PUM par spectrométrie de masse a permis de découvrir Argonaute2 et MOV10 (tous les 2 impliqués dans la machinerie des miRNA), ainsi que des protéines de liaison aux ARNs, ELAV1 déjà connue pour son implication dans le maintien des CSH murines et IMP3, impliqué dans de nombreux cancers et dans la régulation du cycle cellulaire. L’invalidation de IMP3 ou ELAV1 dans les CSPHs conduisent, in vitro, aux mêmes effets observés avec la perte du PUM 1/2, une diminution de l’expansion avec une augmentation de l’apoptose, et la perte du potentiel clonogénique. Enfin, nous avons identifié FoxP1 (Forkhead box P1) comme nouvelle cible directe de PUM1/2, dont le rôle est encore très peu décrit dans l’hématopoïèse. L’étude fonctionnelle de FoxP1 sur les CSPHs par shARN mime les effets observés avec les facteurs PUM1/2. De plus, la surexpression de FoxP1 restaure partiellement les activités antiprolifératives et pro-apoptotiques générées par les shPUM1/2. Enfin, le profil d’expression de FoxP1 dans les LAM corrèle avec le profil d’expression de PUM1/2. Nos résultats confirment le rôle majeur joué par les protéines PUM1/2 en partie via la régulation positive de FoxP1 qui contribue au maintien les CSPHs normales et pathologiques. / Pumilio 1 and 2 (PUM1/2) RNA-binding proteins exert a central role in stem cell maintenance among Invertebrates by binding the 3'UTR of mRNA targets in association with protein partners, thus regulating mRNA stability/translation. Nothing is known regarding normal and pathologic hematopoietic stem and progenitor cells (HSPCs). Loss of coordination between self-renewal and differentiation of HSPCs can lead to leukemia in humans, hence the need to understand the mechanisms. Our team has highlighted the fundamental role played by the post-transcriptional regulators Pumilio (PUM) 1/2 on normal HSPC properties. By a shRNA approach, PUM 1/2 knockdown in human and murine HSPCs leads to: a/ a reduced expansion associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase, b/ the loss of their clonogenic capacity and their in vivo reconstitution potential. The objective of our work is to: a/ evaluate the effects of PUM 1/2 overexpression in HSPC, b/ determine PUM1/2 involvement in leukemic processes; c/ investigate the molecular mechanisms responsible of PUM activity in HSPC by identifying protein targets and partners. Our results showed that a moderate overexpression of PUM1 (2 to 3 fold) in normal CD34+ HSPCs limits the loss of their clonogenic potential, while a higher expression (5 to 10 fold or more) is toxic. The expression analysis of PUM1/2 transcripts in Acute Myeloid Leukemia (AML) (GOELAMSthèque) showed a significant increase in the most immature samples (AML0-2) as compared to healthy controls. PUM1/2 knockdown by shRNA in AML cells significantly reduced their survival. The same effect was observed in cell lines from several leukemic processes. We identified various PUM-associated partners by mass spectrometry, Argonaute2 and MOV10 (involved in the miRNA machinery), and the RNA-binding proteins IMP3 (involved in several cancer and in cell cycle regulation) and HuR/ELAV1 (already known to be involved in murine HSPCs maintenance). IMP3 or ELAV1 knockdown in HSPCs in vitro lead to the same effect of a PUM1/2 invalidation, a decreased expansion with an increased apoptosis and the loss of clonogenic potential. Finally, we identify the forkhead box P1 (FOXP1) transcription factor as a new direct target up-regulated by PUM1 and PUM2. Functional study of FoxP1 knockdown by shRNA in HSPCs mimic PUM1/2 activities. Moreover, FOXP1 overexpression partially rescued shPUM antiproliferative and pro-apoptotic effects. Also, the PUM1/2 and FOXP1 expression levels in leukemic primary cells were measured by RT-qPCR and revealed a positive correlation. Our results reveal that PUM1/2 are direct positive regulators of FOXP1 which contributes to the maintenance of normal and leukemic HSPCs.

Flamboyant colors are widespread throughout the animal kingdom. While many of these traits arise through sexual selection, bright coloration can also evolve through natural selection. Many aposematic species, for example, use conspicuous warning coloration to communicate their noxiousness to predators. Recent research suggests these signals can also function in the context of mate choice. Studies of warning color evolution can therefore provide new insights into how the interplay of natural and sexual selection impact the trajectory of conspicuous signal evolution. For my dissertation, I investigated the potential for male-male competition to impact the warning color evolution of a species of poison frog. I focused my work on an exceptionally bright and toxic population of the strawberry poison frog (Oophaga pumilio) where males are brighter than females, a classic signature of sexual selection. In Chapter 1, I used theoretical models of predator and frog visual systems to determine which can see the variation in bright warning coloration within this population. I found that birds, the presumed major predator, likely cannot see this variation, indicating that sexual selection can work under the radar of predators in this species. In Chapter 2, I tested the aggressive responses of males using a two-way choice paradigm that manipulated the perceived brightness of stimulus males. I found that males directed more of their behaviors to bright stimulus frogs, and brighter focal frogs more readily approached stimuli and directed more of their attention to the brighter rival. In Chapter 3, I tested the outcomes of dyadic interactions between males of varying brightness and observed male reactions to simulated intruders in their territories. I found that brighter males initiated aggressive interactions with rivals more readily, and brightness asymmetries between males settled interactions in a way that is consistent with classic hypotheses about male sexual signals. In Chapter 4 I sought to describe physiological correlates of male warning color brightness. While male brightness did not co-vary with classic measures of body condition (circulating testosterone and skin carotenoids), it did correlate with toxins sequestered from the diet and thus appears to be a reliable signal of toxicity in this population. / text

Plant viruses have developed a number of strategies that enable them to become obligate intracellular parasites of many agricultural crops. Potato virus X (PVX) belongs to a group of positive-sense, single-stranded plant RNA viruses that replicate on host membranes and form elaborate structures known as viral replication complexes (VRCs) that contain viral RNA (vRNA), proteins and host cellular components. VRCs are the principal sites of viral genome replication, virion assembly and packaging of vRNA for export into neighbouring cells. For many animal viruses, host membrane association is crucial for RNA export. For plant viruses, it is not yet known how vRNA is transported to and through plant plasmodesmata. PVX encodes genetic information required for its movement between cells; three viral triple gene block (TGB) movement proteins and a viral coat protein are essential for viral trafficking. This research project studies the relationship between PVX and its host plants, Nicotiana benthamina and Nicotiana tabacum. A particular focus of this project is exploration of the structural and functional significance of the PVX VRC and how the virus recruits cell host components for its replication and movement between cells. The role of specific viral proteins in establishing the VRC, and the ways in which these interact with host organelles, was investigated. A combination of different approaches was used, including RNA-binding dyes and a Pumilio-based bimolecular fluorescence complementation assay for detection of the vRNA, fluorescent reporters for virusencoded proteins, fluorescent reporters for host organelles involved in viral replication, and also transgenic tobacco plants expressing reporters for specific plant components (endoplasmic reticulum, Golgi, actin, microtubules and plasmodesmata). In addition, mutagenesis was used to study the functions of individual viral proteins in replication and movement. All of these approaches were combined to achieve live-cell imaging of the PVX infection process. The PVX VRC was shown to be a highly compartmentalised structure; (+)-stranded vRNA was concentrated around the viral TGB1 protein, which was localised in discrete circular compartments within the VRC while coat protein was localised to the external edges of the VRC. The vRNA was closely associated with host components (endoplasmic reticulum and actin) shown to be involved in the formation of the VRC. The TGB2/TGB3 viral proteins were shown to colocalise with the host endomembranes (ER) and to exit these compartments in the form of motile granules. vRNA, TGB1, TGB2 and CP localised to plasmodesmata of the infected cells. TGB1 was shown to move cell-to-cell and recruit ER, Golgi and actin in the absence of viral infection. In the presence of virus, TGB1 targeted the VRCs in several neighbouring cells. A model of PVX replication and movement is proposed in which TGB1 functions as a key component for recruitment of host components into the VRC to enable viral replication and spread.

Memoria para optar al Título Profesional de Ingeniero Forestal / La lenga (Nothofagus pumilio) es una especie nativa de madera de gran calidad, que presenta una amplia distribución y constituye un importante recurso forestal especialmente para la XI y XII Región. El manejo silvícola de esta especie se realiza mediante el sistema de corta de protección, donde sólo en la primera intervención (corta de regeneración) se extrae alrededor del 50% de las existencias del bosque en área basal, generando condiciones favorables para el establecimiento y desarrollo de la regeneración, una vez que la regeneración se ha establecido satisfactoriamente en forma homogénea sobre la superficie, y ha alcanzado una altura de entre 50 cm y 1 m, los árboles remanentes son extraídos en la corta final. La aplicación de cortas de protección en forma extensiva en los bosques de lenga de la XII Región datan de 1992, y a la fecha existen aproximadamente 25.000 ha intervenidas bajo esta modalidad (Schmidt et al., 2001). De acuerdo a lo expuesto por Schmidt (1993) la realización de cortas de protección en bosques de lenga permite extraer un volumen maderable de alrededor de 10 a 20% de las existencias originales del bosque, y además mejora la producción a futuro. Es este sentido Schmidt y Urzúa (1982) indican que en condiciones naturales el crecimiento diametral de los árboles en promedio es de 1,7 mm/año; y en bosques intervenidos este crecimiento puede elevarse en promedio a valores superiores a 4 mm/año en diámetro. La apertura del dosel como consecuencia de la intervención permite un mayor ingreso de luz al piso del bosque que favorece tanto el desarrollo de la regeneración establecida, como el establecimiento de nuevas plantas de la que surgirá el bosque futuro.

In Drosophila, early embryonic development is controlled by maternally loaded RNAs and proteins. For proper development to occur it is vital these maternal transcripts are post-transcriptionally regulated. SMAUG, a major post-transcriptional regulator, has been found to be responsible for the destabilization of two thirds of the unstable maternal transcripts upon egg activation (Tadros et al., 2007). smg mRNA is translationally repressed in stage 14 oocytes, but its translation is activated upon egg activation in a PAN GU kinase dependent manner. Here I show that redundant translational repression elements reside in the smg 3’UTR, and PUMILIO mediates repression through one of these elements. I also show that these elements are sufficient to cause translational repression in stage 14 oocytes. smg mRNA appears to be regulated post-initiation in stage 14 oocytes in a large repression complex which is similar to smg mRNA repression in a png mutant.

In Drosophila, early embryonic development is controlled by maternally loaded RNAs and proteins. For proper development to occur it is vital these maternal transcripts are post-transcriptionally regulated. SMAUG, a major post-transcriptional regulator, has been found to be responsible for the destabilization of two thirds of the unstable maternal transcripts upon egg activation (Tadros et al., 2007). smg mRNA is translationally repressed in stage 14 oocytes, but its translation is activated upon egg activation in a PAN GU kinase dependent manner. Here I show that redundant translational repression elements reside in the smg 3’UTR, and PUMILIO mediates repression through one of these elements. I also show that these elements are sufficient to cause translational repression in stage 14 oocytes. smg mRNA appears to be regulated post-initiation in stage 14 oocytes in a large repression complex which is similar to smg mRNA repression in a png mutant.

<p>Post-transcriptional regulation plays an important role in governing various processes in all organisms. The development of the early embryo of <italic>Drosophila melanogaster</italic> is governed solely by post-transcriptional mechanisms; therefore, further insights into post-transcriptional regulation can be gained by studying the <italic>Drosophila </italic> embryo. This thesis addresses the actions of the translational repressor, Pumilio, in regulating two mRNAs during early embryogenesis. First, we examined the ability of Pumilio to regulate the mRNA stability of <italic>bicoid</italic>, a gene required for <italic>Drosophila </italic> head development. <italic>bicoid</italic> mRNA contains the canonical Pumilio recognition site, termed the Nanos response element (NRE), within the 3'UTR. Interestingly, we show that Pumilio binds to the NRE both in vitro and in vivo; however, no physiological significance is associated with this interaction. Furthermore, in <italic> pumilio</italic> mutant embryos <italic>bicoid</italic> mRNA stability and translation are unaltered, demonstrating that Pumilio does not regulate <italic>bicoid</italic> mRNA. Second, Pumilio has been shown to negatively regulate <italic>Cyclin B</italic>, the cyclin necessary for mitotic entry, in the somatic cytoplasm of the embryo and this repression is alleviated by the PNG Kinase complex through currently unidentified mechanisms. We further investigated the actions of Pumilio in regulating <italic>Cyclin B</italic> and discovered that the canonical partner of Pumilio, Nanos, is not involved in repressing somatic <italic>Cyclin B</italic>. Furthermore, we show that the 3'UTR of <italic>Cyclin B</italic> is not required for the regulation by Pumilio and the PNG Kinase complex. Lastly, through genetic analyses, we conclude that Pumilio may actually act upstream of the PNG Kinase complex to regulate <italic>Cyclin B</italic>.</p> / Dissertation