Dissertations / Theses on the topic 'Organism in biology'

In this dissertation I criticize and reconfigure the ontological framework within which discussions of the organization, ontogeny, and evolution of organic form have often been conducted. Explanations of organismal form are frequently given in terms of a force or essence that exists prior to the organism’s life in the world. Traits of organisms are products of the selective environment and the unbroken linear inheritance of genetically coded developmental programs. Homological traits share unbroken vertical inheritance from a single common ancestor. Species are the product of exclusive gene flow between conspecifics and vertical genetic inheritance. And likewise, race is ascribed on the basis of pre-existing essential features. In place of this underlying preformationism which locates the source of form either in the informational program of inherited genes or within a selecting environment, I suggest form is the product of an organism’s self-construction using diverse resources. This can be understood as a modification of Kant’s view of organisms as self-organizing, set out in his Critique of Judgment (1790). Recast from this perspective the meaning and reference of “trait,” “homology,” “species,” and “race” change. Firstly, a trait may be the product of the organism’s self-construction utilizing multiple ancestral resources. Given this, homologous traits may correspond in some but not all of their features or may share some but not all of their ancestral sources. Homology may be partial. Species may acquire epigenetic, cellular, behavioural, and ecological resources both vertically and horizontally. As such, they are best conceived of as recurrent successions of self-constructed and reconstructed life cycles of organisms sharing similar resources, a similar habitus, similar capacities for sustaining themselves, and repeated generative processes. Lastly, race identity is not preformed but within the control of human organisms as agents who self-construct, interpret, and ascribe their own race identities utilizing diverse sets of dynamic relationships, lived experiences, and histories.

Rapid prototyping tools exist in many fields of science and engineering, but are rare in biology especially not general tools that can handle the diversity and complexity of the many spatial and temporal scales in nature. In this thesis a general use, cell-based, middle-out biology emulation programming framework (outlining a programming paradigm) is presented, that enables biologists to emulate and use virtual biological systems of previously unimaginable complexity and potentially get results accurate enough to be used in research and ultimately, in clinical practice, such as diagnosis or operations. With this technology, virtual organisms can be created that are viable, fit and can be optimised for any task that arises. The tool, realised with a programming framework created for the C++ language is detailed and demonstrated through several examples of increasing complexity, namely several example organisms and a cancer emulation, showing both viable virtual organisms and usable experimental results.

In this thesis I present a critical examination of the role played by mechanistic ideas in shaping our understanding of living systems. I draw on a combination of historical, philosophical, and scientific resources to uncover a number of problems which I take to result from the adoption of mechanistic thinking in biology. I provide an analysis of the historical development of the conflict between mechanistic and vitalistic conceptions of life since the seventeenth century, and I argue that the basic terms of this conflict remain central to current disputes over the nature of the organism as well as the question of how far the theories, concepts, and methods of physics, chemistry, and engineering can ultimately take us in the explanation of life. I offer a detailed critique of the machine conception of the organism, which constitutes the central unifying idea of mechanistic biology. I argue that this notion, despite its undeniable heuristic value, is fundamentally inadequate as a theory of the organism due to a number of basic differences between organisms and machines. Ultimately, I suggest that the neglected vitalistic tradition in biology actually possesses the best conceptual tools for coming to terms with the nature of living systems. I also undertake a philosophical analysis of the concept of mechanism in biology. I argue that the term ‘mechanism’ is actually an umbrella term for three distinct notions, which are unfortunately conflated in philosophical discussions. I explore the relation between mechanistic biology and the new philosophical interest in the concept of mechanism and I show that these two research programs have little to do with one another because each of them understands the concept of mechanism in a different way. Finally, I draw on the historical and philosophical foundations of cell theory to propose an epistemological perspective which enables the reductionistic explanation of the organism without having to give up the distinctive features of life in the process. In this way, I show this perspective to have significant advantages over the classic physicochemical reductionism of mechanistic biology.

Around 375 million years ago, the first tetrapods appeared, marking one of the most important events in vertebrate evolutionary history. The fin to limb transition saw the appearance of fingers and a weight bearing pelvic girdle. While very little research has been done on the evolution of the tetrapod pelvic girdle, a fair amount has been done on the origins of fingers but some aspects remained controversial. A combination of palaeontology, developmental biology and comparative morphology was therefore used in this thesis to better understand the fin to limb transition. The pectoral fin of Panderichthys, a sarcopterygian fish closely related to tetrapods was CT-scanned and modeled in three dimensions and its pelvic girdle and fin were examined with traditional techniques. This information from the fossil record was integrated with comparisons of the development of the Australian lungfish, Neoceratodus forsteri, our closest living fish relative and the axolotl (Ambystoma mexicanum), a salamander representing well the condition of early tetrapods. Development of bone and cartilage was studied through clearing and staining and development of skeletal muscles through immunostaining. In situ hybridizations were performed on the lungfish to study the expression of Hoxd13, associated with the formation of digits in tetrapods. This work shows that the late expression phase of Hoxd13 is present in Neoceratodus and is associated with the formation of radials. Redescription of the pectoral fin of Panderichthys reveals that distal radials are present, which, in addition to other information, lead us to conclude that digits are not novelties in tetrapods but rather have evolved from the distal radials present in the fins of all sarcopterygian fish. The earliest tetrapods lack a full set of wrist + carpals/ankle + tarsal bones. Here, we propose that this region of the limbs evolved after fingers and toes through an expansion of the region between the proximal limb bones and the digits. As for the pelvic girdle, it is very primitive in Panderichthys but comparison of its development in Neoceratodus and Ambystoma suggest that the ischium evolved through the posterior expansion of the pubis and the ilium, through an elongation of the iliac process already present in sarcopterygian fishes. The results of this thesis help to better understand the fin to limb transition and show that it is more gradual than previously believed.

Na teoria da experiência da terceira Crítica, Kant estabelece a articulação das três faculdades de conhecimento. A possibilidade das coisas em geral, segundo o modelo da analítica do entendimento, está ainda sendo pensada pelas categorias, contudo, em conformidade com as leis do entendimento, estas mesmas coisas dotadas de formas particulares estão sendo pensadas pela legalidade da faculdade de julgar, e ao cabo a razão pensa a possibilidade da totalidade dessas formas arranjadas em ordem em vista de um sistema, segundo a dialética da razão. A partir dessa articulação entre as faculdades mentais em função da experiência, buscou-se determinar os conceitos que possibilitam pensar o organismo mostrando que o organismo é finalidade interna produzida por epigênese e pré-formação. Para pensar a possibilidade de articulação dessas duas teorias embriológicas da história da filosofia, Kant mobilizou conceitos do entendimento, da faculdade de julgar e da razão. A terceira Crítica operou uma série de deslocamentos e articulações conceituais para pensar o organismo. A contingência que era pensada pela razão é deslocada para a faculdade de julgar em sua legalidade do contingente. A terceira Crítica ainda deslocou a produção do organismo da intenção divina para a espontaneidade natural tendo o princípio da finalidade interna como fundamento mental. Na medida do possível buscamos introduzir a lógica transcendental na representação do organismo, e por esse caminho revelamos que entre os seus conceitos estão a liberdade, a técnica, o sistema e a comunidade, sob os quais Kant articulou as opostas teorias da pré-formação e da epigênese.
In the theory of experience of the third Critique, Kant establishes the articulation of the three faculties of knowledge. The possibility of things in general, according to the model of the analytic of the understanding, is still being thought by the categories, but in conformity with the laws of the understanding these same things endowed with particular forms are being thought by the lawfulness of the faculty of judgement, and finally reason thinks the possibility of the totality of these forms arranged in the order of a system, according to the dialectic of reason. From this articulation between the mental faculties for the experience, we tried to determine the concepts that make it possible to think the organism by showing that the organism is an internal purposiveness produced by epigenesis and pre-formation. In order to think about the possibility of articulating these two embryological theories of the history of philosophy, Kant mobilized concepts of the understanding, the faculty of judgement and reason. The third Critique operated a series of dislocations and conceptual articulations to think the organism. The contingency that was thought by reason is shifted to the faculty of judgement in its lawfulness of the contingent. The third Critique still shifted the organisms production from divine intention to natural spontaneity by having the principle of internal purposiveness as a mental foundation. As far as possible we seek to introduce transcendental logic into the representation of the organism, and through this path we reveal that among its concepts are freedom, technique, system and community, under which Kant articulated the opposing theories of pre-formation and of epigenesis.

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Na descrição hierárquica do conhecimento biológico, o ser vivo é considerado como ponto central nas relações engendradas pelos seguintes níveis: ambiente externo (ecológico/evolutivo), organismo e ambiente interno (genético/ molecular). O organismo compreendido como nível focal da discussão biológica pode ressaltar a autonomia da Biologia em relação às outras áreas do conhecimento científico. No contexto do ensino, assume-se que as discussões epistemológicas do conhecimento biológico podem promover uma compreensão mais integrada dos fenômenos biológicos. Assim, organizou-se um grupo de pesquisa com graduandos de um curso de Ciências Biológicas para discutir conceitos centrais do conhecimento biológico, entre eles, o conceito de organismo. Esta pesquisa teve como objetivos: 1) Elaborar uma caracterização do conceito de organismo, partindo de uma abordagem hierárquica, integrando as discussões advindas da Filosofia da Biologia contemporânea referentes aos conceitos de auto-organização, autonomia agencial, propriedades emergentes e níveis hierárquicos; 2) Analisar como o conceito de organismo se impõe frente às explicações de vida presentes na literatura contemporânea da Filosofia da Biologia; 3) Utilizar a discussão teórica relativa ao conceito de organismo como fundamentação de um grupo de “Pesquisas em Epistemologia da Biologia”, verificando as contribuições desse aporte teórico para a formação de alunos de Licenciatura em Ciências Biológicas na área de Epistemologia da Biologia e Ensino de Ciências; 4) Analisar as discussões e produções escritas ocorridas no desenvolvimento do grupo de “Pesquisas em Epistemologia da Biologia” que abordaram o conceito de organismo, com a finalidade de verificar se uma abordagem hierárquica tendo o organismo como nível focal contribui para uma visão integrada do conhecimento biológico pelos...
In the hierarchical of biological knowledge, the living being could be considered as central point in the relations produced by three levels: external environment (ecological/evolution), organism and environment intern (genetic/molecular). The comprehension of the organism as a focal level in the biological debate can underline the autonomy of Biology among the other areas of the scientific knowledge. In the education context it is assumed that the epistemological discussions of the biological knowledge can promote an integrated understanding of the biological phenomena. Thus, a research group consisting of Biological Sciences undergraduates was organized to debate central concepts of the biological knowledge in which the discussions of the organism concept are included. This research aimed to: 1) develop a characterization of the concept of organism from a hierarchical approach by integrating the resulting discussions from contemporary philosophy of biology that are related to the concepts of self-organization, autonomy agents, emergent properties and hierarchical levels; 2) to analyze how the concept of organism is placed in front of the explications of life in the contemporary literature of philosophy of biology; 3) to use the theoretical discussion on the concept of organism as fundamentation for a group of Studies in Epistemology of Biology, noting the help of this theoretical contribution to the Biological Sciences students training in the Biology and Epistemology in Science Teaching; 4) to analyze discussions and written productions that occurred in the development of the ‘Research in Epistemology of Biology’s group which addressed the concept the organism in order to verify if a hierarchical approach in which the organism is the focal level contributes to an integrated view of biological knowledge for biology students. On the basis of the theoretical referential... (Complete abstract click electronic access below)

Palmitoylation is a reversible post-translational modification of proteins which involves the addition of the C16 saturated fatty acid palmitic acid to sulfhydryl groups on cysteine residues, forming a thioester linkage. The addition of palmitate allows proteins to associate with different cellular membranes and membrane subdomains. Palmitoylation is catalysed by the DHHC family of palmitoyl-acyl transferases (PATs), named for their characteristic DHHC motif in a cysteine-rich domain (CRD). Reversibility is conferred by palmitoyl-protein thioesterases (PPTs), which cleave the thioester linkage. The study of palmitoylation has recently gathered pace with the development of methods which allow proteome-scale identification of candidate palmitoyl-proteins. Despite the importance of model organisms in several key studies in the field, palmitoylation has barely been studied in the simple eukaryote Caenorhabditis elegans, the nematode worm. This study commenced with the use of the C. elegans genome data to identify its PATs and PPTs, using the DHHC-CRD and homology respectively. The 15 DHHC PATs were officially named using a dhhc-x system and the two previously known PPTs were confirmed as the only ones with homology to other known PPTs. The current knowledge on palmitoylation enzymes and substrates was collated and analysed to predict possible phenotypes resulting from mutation or knockdown of the enzymes and potential substrates. All available C. elegans strains containing a mutation in an individual PAT or PPT were obtained, covering about half of the PATs and both PPTs, and assayed for various gross phenotypes. In addition a complete library of bacteria able to express double-stranded RNA against PAT or PPT genes was sourced and used to perform similar assays using feeding RNA interference (RNAi). A number of small but significant differences were seen both with mutant and RNAi-treated strains, especially in lifespan assays. To test for possible redundancy and compensation amongst the enzymes, double RNAi was performed against selected closely related PATs and both PPTs. This resulted in the largest phenotype seen: a reduction in lifespan after simultaneous knockdown of both ppt-1 and ath-1. As there are no known palmitoyl-proteins in C. elegans, the proteomic approaches acyl-biotin exchange (ABE) and acyl-resin-assisted capture (acyl-RAC) were employed to provide a list of candidates. These were first optimised using rat brain material and the results compared with previous proteomic studies to find that two-thirds of the hits had been previously found. With this validation, both methods were applied to wild-type C. elegans lysates to give 91 hits as putative palmitoyl-proteins. Mutants for the PPT ath-1 were also profiled by ABE, showing 33 hits which were not present in the wild-type profile. These are potential ATH-1 substrates whose lack of depalmitoylation in the mutant leads to their enrichment relative to wild-type. However, further repeats of these analyses are required for rigorous statistical testing. Taken together, this study shows the first characterisation of palmitoylation in C. elegans, encompassing all of the DHHC PAT and PPT enzymes, putative palmitoyl-proteins and potential substrates of the PPT enzyme ATH-1.

The objective of this study was to explore Macrostomum lignano, a free-living organism, as a model organism for parasitic trematodes, such as Fasciola and Schistosoma, in order to better understand the role of their cysteine proteases (cathepsins). Using a bioinformatics approach, two novel cysteine proteases genes (mlcl1 and mlcb2) were identified and phylogenetically characterized. These genes were synthesized, cloned into the yeast secretory system Pichia pastoris (Invitrogen), and functionally-active recombinant proteins were expressed and purified. These recombinant peptidases were then characterized biochemically in terms of activity and stability in various conditions including temperature, salinity and pH. Antibodies specific for the recombinant proteins were generated through a peptide or whole-protein immunization, and tested against both the recombinant proteins and the worm extract proving that the proteins exist in the worm. These studies lay the foundation for further investigations on the biological function of the cysteine peptidases using RNAi and confocal microscopy. In conclusion, M. lignano is a tractable model organism for its parasitic counterparts.
L'objectif de cette recherche était d'explorer l'organisme, vivant en liberté dans la nature, Macrostomum lignano en tant qu'organisme modèle pour ses cousins parasites, Fasciola et Schistosoma, et de mieux comprendre le rôle de leurs protéases cystéines (cathepsins). En utilisant une approche bioinformatique, deux nouveaux gènes de protéases cystéines (mlcl1 et mlcb2) ont été découverts et caractérisés phylogénétiquement. Ces gènes ont été synthétisés, clonés dans un système de sécrétion employant la levure Pichia pastoris (Invitrogen) et exprimés en tant que protéases recombinantes et actives. Ces protéases recombinantes ont alors été caractérisées biochimiquement en termes d'activité et de stabilité dans diverses conditions telles que la température, la salinité et le pH. Des anticorps spécifiques aux protéines recombinantes ont été générés en immunisant des mammifères avec des séquences de peptides ou la protéine recombinante entière, et ont été testés avec l'extrait du vers prouvant ainsi que les protéines sont bel et bien exprimées. Ces études jettent la base pour l'investigation sur la fonction biologique des protéases cystéines par l'emploi du RNAi et de la microscopie confocale. En conclusion, M. lignano est un organisme modèle tractable pour ses cousins parasites.

The frequency of oscillatory behaviors, like shivering, depends on the animal size and the properties of the muscles driving them. Titin and other muscular proteins play an important role in determining muscle properties, such as stiffness. Because the frequency of oscillatory behaviors depends on muscle properties, we predict that changes in titin's structure would affect these behaviors. The muscular dystrophy with myositis ( mdm) mouse model is characterized by a deletion in the N2A region of titin. Homozygous mdm mutants are substantially smaller (body mass is ½ to ⅓), have a stiffer gait, and have reduced lifespans compared to their wildtype and heterozygous siblings. In addition, we observed that mutants were heterothermic while wildtypes and heterozygotes were homeothermic when exposed to ambient temperatures ranging from 20-37 °C. We measured the relationship between metabolic rate and the differential between body and ambient temperatures for all three genotypes. As the temperature differential increased, metabolic rates increased more rapidly in the mutants than in wildtype or heterozygous mice, indicating that the mutants have a much higher conductance than their age-matched siblings. We measured shivering frequency in the mdm mice. The frequency of tremor during shivering is expected to be directly proportional to (k/ m)^0.5 where k is stiffness and m is body mass. Using an allometric relationship between body mass and shivering frequency, we calculated expected values for all three genotypes based on body mass alone. These predicted values allowed us to take into account the much lower body masses of the mdm mutants. The difference between expected and observed values was significantly larger for mutant mice than wildtypes or heterozygotes. Together, the heterothermy in mutants, the very high conductance, and the decreased tremor frequency demonstrate the thermoregulatory challenges faced by mice with the mdm mutation. Previous work at the whole-muscle level showed that despite the higher passive stiffness observed in mdm mutant muscles, these muscles are more compliant when activated compared to muscles from wildtype mice. The lower tremor frequencies in mutants are consistent with a reduced active muscle stiffness in vivo. These observations suggest that titin affects the tuning of shivering frequency by playing a role in setting active muscle stiffness.

Estuaries are dynamic physical environments. The stability of the sediment-water interface is influenced by sources and rates of sediment delivery and physical reworking of sediments by currents, tides, waves and biology, but effects of disruption of this interface on benthic biology are poorly resolved. For this study, I investigated effects of prevalent gradients in seabed disturbance processes and associated seabed characteristics on estuarine benthic community structure and function in the mesohaline York River, a tributary of Chesapeake Bay, USA. I used a variety of approaches to characterize the seabed, including sediment grain size, sediment water content, maximum depth of 7Be, depth of the oxidized sediment layer, profiles of sediment Eh, physical structure of the sediment, sediment chlorophyll a, and sediment organic content. Differences in magnitude of deposition and subsequent reworking of sediments by physical processes were documented among the five benthic subenvironments sampled (south shoal, secondary channel, main channel flank, main channel, and north shoal). Temporal and spatial variations in spring recruitment were observed among subenvironments sampled weekly for recruits: the south shoal, secondary channel and main channel flank. Total recruitment was greatest in the main channel flank, which experienced the highest sediment deposition, and was limited in the secondary channel, which had the strongest tidal currents. The five benthic subenvironments sampled for patterns of community structure and estimates of secondary production were dominated by estuarine opportunist species. Total abundance was greatest in the north shoal, which experienced minimal deposition and physical reworking of sediment. Biomass and secondary production estimates were driven by presence of deep-dwelling bivalves, and were greatest in subenvironments that experienced deposition. These results suggest that variations in seabed characteristics across relatively small spatial scales can influence estuarine benthic community structure and function. Laboratory experiments were conducted to further elucidate the effect of sediment deposition on estuarine organism survival. Species representing both infaunal and epifaunal taxa ranged from highly susceptible to highly tolerant of burial by sediment. Survival was a function of organism motility, residence depth and perhaps physiological adaptations. Small, shallow-dwelling juveniles of some common estuarine species were highly tolerant of burial.

Biology
Ph.D.
Wnt signaling plays important role in many aspects of embryogenesis such as cell proliferation, cell fate specification, cell polarity and organogenesis(Clevers 2006, van Amerongen and Nusse 2009). Wnt ligands have been shown to activate several intra-cellular signaling cascades, including the canonical or Wnt/-catenin dependent pathway and the non-canonical or -catenin independent pathway. Dishevelled (Dvl) occupies a key position at crossroads of all branches of Wnt signaling cascade. To understand, how Dishevelled (Dvl) may channel signaling into the downstream branches, we sought to identify novel effectors for Dishevelled (Dvl) using a yeast-two hybrid screen. In this study, we used the PDZ domain of Dishevelled (Dvl) as a bait and from this screen, we identified a new binding protein of Dishevelled (Dvl)-termed as Custos. To characterize the functional role of Custos in Wnt signaling pathway, we used mammalian cell culture and zebrafish as a model vertebrate organism. We confirmed the interaction between Custos and Dvl using co-immunoprecipation and GST pull-down. Custos also interacted with -catenin in vivo and this interaction was positively regulated by Wnt stimulation. Immunofluorescence experiments in mammalian cells showed that Custos co-localizes with the nuclear envelope marker, lamin and inhibits translocation of -catenin to the nucleus. In zebrafish embryos, Custos is a maternal gene and expressed throughout development. Spatial in situ hybridization studies showed that Custos was expressed in the dorsal region of the embryo at early stages and in the nervous system in zebrafish at 24hpf. To delineate the biological role of Custos during embryogenesis, we conducted a gain of function and loss of function studies. Overexpression of exogenous Custos and morpholino knockdown of Custos revealed that Custos is critical for embryonic patterning. Knockout of Custos in zebrafish revealed that Custos delays embryonic development and exhibits defects in pigmentation suggesting a plausible role in neural crest development. Taken together, our studies demonstrate that Custos is a novel component of canonical Wnt signaling and required for -catenin translocation into the nucleus and important for embryonic patterning.
Temple University--Theses

Includes bibliographical references.
The effects of the exploitation of prawns (Callianassa & Upogebia spp.) in Langebaan Lagoon, primarily for bait collecting, were assessed using field experiments and observations. Langebaan Lagoon has been a proclaimed National Park for 20 years and a proclaimed Marine Protected Area since 2000. The area is zoned for different human uses, about one third being reserved as a sanctuary, one third for non-extractive visitation and one third for extractive purposes including bait-collecting and fishing. Four species of prawns were identified in Langebaan Lagoon - C. kraussi, C. rotundicaudata, U. africana and U. capensis.

Paramecium tetraurelia is a ciliated single cell used as a model organism for the study of ciliopathies. Ciliopathies are mammalian diseases involving the dysfunction of cilia, including cilia maintenance, construction, and signaling. P. tetraurelia and its cilia provides an excellent non-canonical system for the investigation and elucidation of proteins important for the structure, maintenance and function of cilia and ciliary beating. We utilize features of this cell such as its 1000's of cilia and highly organized and patterned cell surface to observe changes in swimming behavior or disruptions in the ordered cell surface which are not feasible in mammalian cells. Here, we present research on three proteins in Paramecium, two of which are homologs to human ciliopathy genes. Using combinations of epitope-tagging, RNA interference (RNAi), immunofluorescence, immunoprecipitations, LC-MS/MS analysis and electrophysiology, we have attempted to elucidate the location, function, and potential interacting partners of these three proteins. The first protein, meckelin (MKS3), is a contributing factor in Meckel-Gruber syndrome, among other ciliopathies. Using epitope tagging, we identified the location of the Mks3 protein above each basal body. Depletion of MKS3 using RNAi leads to global loss of cilia, a severe disruption in the surface organization and a mislocalization of basal bodies out of the anterior-posterior axis of the cell. We show that depletion of Mks3 leads to abnormal backward swimming in ionic stimuli and depleted secretion of trichocysts. Based on our data, we propose two functions for Mks3 in P. tetraurelia. The first function is a transition zone component important for proper regulation of ciliary protein content, consistent with MKS3 function in other organisms. The depletion of MKS3 led to global ciliary loss, but also an imbalance in the ciliary ion channels that was different from the loss of cilia due to interference with intraflagellar transport as observed in cells depleted of IFT88. The second novel role for MKS3 is as a transient connection to the kinetodesmal fiber which is important for basal body guidance when daughter basal bodies migrate away from the mother basal body before cell division. We also examine the contribution of the non-selective cation channel Polycystin-2 (Pkd2) in Paramecium to Mg2+ permeability and Mg2+-induced behavior. When mutated in humans, Pkd2 leads to 15% of the cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). When PKD2 is depleted using RNAi in Paramecium, cells show short backward swimming in Mg2+ solutions, a resistance to heavy metal paralysis, and depleted membrane permeability to Mg2+. The channel-like protein XntA which is unique to Paramecium and Tetrahymena, is also important for these phenotypes. Therefore, we utilized the Paramecium XntA1 mutant in our studies, which lacks Mg2+-induced behavior. We demonstrate that both Pkd2 and XntA are present in the cell membrane and in the cilia. Co-IP assays show that the IP of XntA-myc co-IPs the Pkd2-FLAG channel, but not vice versa, possibly because of an occluded FLAG epitope due to protein interactions. To tease apart the contributions of Pkd2 in the cilia and the cell membrane, electrophysiology was used to measure membrane potential of ciliated and deciliated cells. Depletion of BBS8 eliminates Pkd2 in the cilia, allowing us to examine Pkd2 activity restricted to the cell membrane of ciliated cells. Depletion of Pkd2 or XntA decreases membrane permeability to Mg2+. When Pkd2 was restricted to the cell membrane via BBS8 depletion, the membrane permeability to Mg2+ increased, much like over-expressing the Pkd2 protein. Depletion of Pkd2, especially in the deciliated XntA1 mutant, leads to a dramatic decrease in Mg2+ membrane permeability. Based on these data, we propose that Pkd2 is the Mg2+ channel in Paramecium and XntA is not a channel, but is perhaps important for stabilizing Pkd2 in membrane microdomains. We have uncovered novel function roles for the proteins mentioned here, leading to a broader understanding of their function. These studies also highlight to usefulness and importance of the model organism Paramecium tetraurelia to the study of human ciliopathy genes.

Cette thèse porte sur deux aspects complémentaires, le développement et l’implémentation de nouvelles approches de cristallisation et de cristallographie sérielle ainsi que leur mise en œuvre dans l’étude structurale de complexes enzymes : ARNt. La cristallographie est la méthode la plus employée en biologie structurale, mais elle présente encore des points délicats. Plusieurs méthodes avancées ont été déployées dans ce travail pour y pallier qui ont conduit à la résolution de la structure de l’ARNt nucléotidyltransférase du psychrophile Planococcus halocryophilus et à l’étude de son adaptation structurale au froid ; des puces microfluidiques de cristallisation qui ont servi à la résolution de plusieurs structures à température ambiante par cristallographie sérielle ; enfin le Xtal Controller utilisé pour l’étude d’évènements de nucléation et de croissance cristalline dans un but de préparation d’échantillons pour analyse sous rayonnement XFEL. Entre autres systèmes biologiques, cette thèse présente la caractérisation de deux familles d’inhibiteurs visant les aspartyl-ARNt synthétases, notamment du pathogène Pseudomonas aeruginosa
This thesis focuses on two complementary aspects, the development and implementation of new approaches of crystallization and of serial crystallography as well as their use in the structural study of enzymes/tRNA complexes. Crystallography is the most used method in structural biology, but it presents delicate points. Different methods were implemented in this work to overcome these points, which led to the resolution of the structure of the CCA-adding enzyme of the psychrophilic organism Planococcus halocryophilus and to the study of its structural adaptation to the cold; novel microfluidic crystallization chips that have been used for the resolution of several structures by serial crystallography at room-temperature; finally the Xtal Controller used for the study of nucleation and crystal growth events with the purpose of preparing samples for analysis under XFEL radiation. Among other biological systems, this thesis presents the study and characterization of two families of inhibitors targeting aspartyl-tRNA synthetases, including the one of the pathogenic organism Pseudomonas aeruginosa

Gnathostomes, or jawed vertebrates, make up the overwhelming majority of modern vertebrate diversity. Among living vertebrates, they comprise the chondrichthyans (“cartilaginous fishes” such as sharks, skates, rays, chimaeras) and the osteichthyans (“bony fishes” or bony vertebrates, inclusive of tetrapods). Gnathostomes appear to have originated in the early Palaeozoic Era, but their early fossil record is fairly scant. The best fossils appear first in the Late Silurian and Devonian periods. Much of gnathostome diversity owes to unique adaptations in the internal skeleton of their head (the endocranium). The endocranium is composed of the braincase, jaws, hyoid arch, and branchial arches, which sometimes fossilise when they are composed of bone or calcified cartilage. The purpose of this thesis is to describe and compare the fossilised cranial endoskeletons of a variety of different Palaeozoic gnathostomes. The objective is to test current conceptions of gnathostome interrelationships (i.e. phylogeny) and infer aspects of key morphological transformations that took place during the evolution of Palaeozoic members of this group. Two key areas are examined: the morphology and interrelationships of Palaeozoic gnathostomes and the morphology of the visceral arches in sarcopterygian fishes. New data on the visceral arches are described from the stem tetrapods Panderichthys and rhizodontids. These provide insight into the sequence of character acquisition leading to the tetrapod middle ear. Panderichthys shows key features of the tetrapod middle ear chamber were established prior to the origin fo digited limbs. New morphological data are described from the “acanthodian” fish Ptomacanthus. Ptomacanthus provides only the second example of a well-preserved braincase from any member of this group. It shows dramatic differences from that of its counterpart, Acanthodes, providing new evidence for acanthodian paraphyly. New interpretations of basal gnathostome and osteichthyan phylogeny are presented, challenging or enriching existing views of these problems.

Grey Leaf Spot (GLS) is a fungal disease of Zea mays (maize) that is caused by Cercospora zeina. It thrives in sub-tropical climates and causes devastating crop losses of up to 60% in southern Africa where maize is grown as a staple food source. Phytoalexins are low molecular weight anti-microbial bio-chemicals that are synthesised in planta in response to biotic stress. Related studies have characterised many phytoalexins produced in various plants against several diseases. In maize, phytoalexins fall into to two terpenoid groups: kauralexins and zealexins. To date no studies have been carried out that examine the accumulation in maize of phytoalexins in response to C. zeina. This research project found that in maize samples inoculated with C. zeina, kauralexin accumulation significantly increased with disease development stages (T0 - 0 days post inoculation, T1 - 17 dpi, T2 - 18 dpi and T3 - 24 dpi) while zealexins did not change. Gene expression of the phytoalexin biosynthesis genes TPS6 and TPS11 (both encoding the protein terpene synthase 6/11, specific for zealexins) and CPPS2 (encoding ent-copalyl diphosphate synthase 2, specific for kauralexins) increased significantly at each time point, reaching a maximum level at T2. Infiltration of maize leaves with a chitosan elicitor to mimic fungal pathogen associated molecular pattern (PAMP), and a subsequent callose assay showed positive induction of a callose defence response. However, gene expression and phytoalexin accumulation did not change following chitosan treatment, although zealexin accumulation was higher than kauralexins. Previous studies have shown that phytoalexins accumulate transiently in seedlings. Six diverse Southern African maize lines were compared for phytoalexin accumulation at seedling stage. Zealexin accumulation was generally higher than kauralexins and there were significant differences in both zealexin and kauralexin accumulation in different lines. Gene expression analysis using Genevestigator looked at microarray files and found that expression of TPS6/11 (zealexin biosynthesis) and CPPS2 (kauralexin biosynthesis) genes to be largely co-regulated and highly expressed in response to fungal pathogens, nematodes, insect pests and abiotic stresses; Ustilago maydis, Phytophthora cinnamomi, Fusarium moniliforme, Colletotrichum graminicola, Sporisorium reilianum, Meloidogyne incognita, Ostrinia nubilalis, waterlogging and drought stress. Finally promoter region analysis showed similar cis-acting regulatory elements in the 1kb region upstream of the promoter of both genes and defence specific elements. Thus kauralexin phytoalexins are produced in response to C. zeina inoculation, chitin is not likely to be the key PAMP leading to phytoalexin accumulation, phytoalexin accumulation in seedlings is genotype-dependent and phytoalexin biosynthesis genes are expressed under different conditions suggesting a wider range of action beyond repelling fungal pathogens.

One of the most unforgiving processes in nature is that of Muller's ratchet, a seemingly irreversible accumulation of deleterious mutations that all organisms have to deal with or face extinction. The most obvious way to avoid fitness collapse is recombination, though asexual populations usually do not have the luxury of recombining freely.  With the aid of computational and mathematical models, we have studied other situations where this threat is averted and the organism can survive the ratchet. The results show that a ratchet where all mutations have the same deleterious fitness effect is very effectively stalled for large effects. However, if mutations are allowed to have a broad range of effects, the fitness-loss rate can be substantial even with the same mean effect as the one-type ratchet, but we have  identified parameter regions where even the broad-range effects are effectively stopped. The fitness-loss from a ratchet is very sensitive to the mutation rate and a mutation that increases the mutation rate (mutator) can easily start an otherwise stalled ratchet. Large effect mutators are heavily counter-selected, but smaller mutators can spread in the population. They can be stopped by reversals (antimutators), but even if the mutation rate is equilibrated in this way, there will be large fluctuations in mutation rate and even larger in the fitness-loss rate due to the feedback amplification in their coupling.    Another way of preventing the ratchet is by reversal of the deleterious mutations themselves through back-mutations or compensatory mutations. The rate required to stop the ratchet using only back-mutations before the fitness collapses is very large. A detailed comparison between the deleterious mutations in the ratchet and in a sexual population was made and the difference was found to be greatest for large populations with large genomes. There are obviously many ways to survive the ratchet, but even more ways to drive a species to extinction by enhancing and speeding up the ratchet. By modelling and testing the ratchet for numerous different situations, we show the effects of some of these threats and benefits.

'Cavity disease', defined as the rapid degradation of the sporocarps of white button mushrooms, is caused by Burkholderia gladioli pv. agaricicola, a member of Pseudomonaceae. The specific strain analysed in this thesis, BG 164, was isolated from New Zealand, but the disease has also been sporadically reported in Europe. Nine mutants that did not cause cavity symptoms ('no-cavity') were isolated by transposon mutagenesis of the BG 164 derivative, BG 164R. Eight mutants had altered flagella number, did not secrete protease, had a highly reduced capacity to secrete chitinase and did not degrade mushroom mycelia. All these eight mutations were clustered in the GSP (General Secretory Pathway) operon. A cosmid, carrying 23.4kb genomic DNA, pCosGSP, complemented all mutations, which along with mapping of the cosmid, confirms that the cosmid has most or all of the functional gsp operon. The ninth mutant, BGII-107, secreted protease, although it did not form cavity on mushrooms. The DNA sequence analysis suggests that the mutation is in a polyketide synthetase gene. Interestingly, the mutant can also be complemented by the gsp operon-containing cosmid, suggesting the presence of a polyketide synthetase gene adjacent to the GSP genes. Cavity disease initiation, in contrast to most of the other diseases, requires a very high inoculum of the pathogen, suggesting a probable reason for the sporadic appearance of the disease. Studies on the indigenous non-pathogenic bacterial population of mushrooms led to the identification of a natural antagonist of BGI64R, Ewingella americana (PRC120), in the sporocarp tissue. Population studies indicated the involvement of polymicrobial interactions and a quorum sensitivity-dependent induction of Cavity disease expression.

reinhardtii, a unicellular green alga, is a model organism to study the circadian clock. Cryptochromes are the blue light photoreceptors that entrain the clock in some organisms. The CPH1 protein of C. reinhardtii resembles the cryptochromes of the plant model Arabidopsis, but whether CPH1 entrains the circadian clock in C. reinhardtii is not yet known. Recent reports have suggested the existence of one more cryptochrome in C. reinhardtii, which resembles the cryptochromes of animals. However, the amino acid sequence of this protein shows even higher sequence similarity with the 6-4 DNA photolyase of Arabidopsis. DNA photolyases are involved in the repair of UV light-induced DNA damage using the energy of blue light. In order to determine, if the “animal cryptochrome” gene of C. reinhardtii actually encodes a 6-4 DNA photolyase rather than a photoreceptor, an experimental design was developed to test whether the protein product is able to rescue an E. coli mutant defective in its DNA photolyase gene. The design is as follows: In a first step, the coding region of the “animal cryptochrome” cDNA is cloned. In a second step, the cDNA is inserted in-frame into an E. coli expression vector. In a third step, the construct is transformed into an E. coli photolyase mutant, its expression induced, and the strain tested for better survival after UV light exposure. To accomplish the first step, the cloning of “animal cryptochrome” cDNA, total RNA was successfully extracted from C. reinhardtii 4 hrs into the light phase of a 12 h light/12 h dark cycle and reverse transcribed into cDNA using oligo(dT) primers. After initially unsuccessful attempts at amplifying animal cryptochrome from cDNA or genomic template with a variety of primers and conditions, a short fragment with the expected size of 186 bp was amplifiable with both templates. However, even this fragment was not reliably obtained in every PCR assay. Because of this difficulty, real-time PCR was finally performed in the presence of DMSO (Dimethylsulfoxide) and Betaine. These two adjuvants were reported to improve amplifications particularly for GC-rich templates. C. reinhardtii DNA is especially GC-rich with an average of 64% Gs and Cs. The improved conditions allowed the reliable amplification of the 186 bp fragment from genomic template. It also enabled the amplification of a larger fragment of 528 bp from the same template. The results suggest that a combination of 5% DMSO and 1M Betaine is optimal for the amplification of C. reinhardtii DNA and thus can serve as the basis for successful amplification of the entire 1788 bp coding region of the animal cryptochrome cDNA.

In the course of experiments designed to identify and characterize structural proteins of the nuclear matrix, one antibody was generated which recognized an extraction-resistant cytoplasmic protein. This antibody was used as the starting point in the cloning and molecular characterization of a novel protein of the inter-membrane space of the mitochondrion which has been named mitofilin. Mitofilin is expressed in all human cell types, and murine homologues also exist. Mitofilin associates only with mitochondria and not with other membrane-bounded organelles such as Golgi or endoplasmic reticulum. This observation has been confirmed both by biochemical fractionation and multi-label fluorescence microscopy. Recombinant mitofilin, purified to homogeneity by affinity chromatography and preparative electrophoresis, was used to raise second-generation antibodies. Results of Western blot and immunofluorescence microscopy experiments, identical to those obtained using the original monoclonal antibody, verify the cloning and biochemical characterization. The mitofilin polypeptide contains several regions which are predicted to interact by forming coiled coils; a mitochondrial targeting signal; and a hydrophobic, membrane-spanning domain. During the course of this work, a sequence match was found with a cDNA reported by Icho, et al (1994) for a mRNA preferentially expressed in heart muscle, which they have called HMP. Evidence is presented which contradicts those authors' contention that HMP is a kinesin-like motor protein. In the course of these investigations, methods were developed to detect and quantitate the expression of solubilization-resistant proteins of the nuclear matrix and the nuclear matrix-intermediate filament scaffold. This was accomplished by combining SDS-PAGE, high sensitivity chemiluminescent Western blots, and scanning densitometry. Sensitivity in the picogram range was obtained, and reproducibility was assessed. For semi-quantitative measurements of protein expression in tissue samples, cell number was normalized by measurement of lamin B, the major protein of the nuclear envelope. Results of screening several cell and tissue types for the expression of mitofilin and for the nuclear matrix proteins NuMA, the nucleoporin tpr, and lamin B are presented. These preliminary data suggest a potential connection of over-expression of NuMA, tpr, and mitofilin with ovarian carcinoma. In addition, quantitative analysis of mitofilin expression in a variety of human cell types was done using purified recombinant protein antigen as the standard. The presence of coiled coil domains in these and other proteins associated with cellular sub-structures gave rise to the third area of investigation described here. Experimental observations of the nuclear matrix-intermediate filament scaffold (NMIF), a tissue-wide structure greatly enriched in coiled coil proteins, led to the following hypothesis: that the differentiated cell and tissue architecture which characterizes Metazoa has evolved through the propagation and selective expression of genes encoding a wide variety of coiled coil proteins, and the integration of the gene products into a tissue-wide matrix based on coiled coil interactions. This hypothesis was explored by computer searches of sequence data files. The GenBank phylogenetic sequence files were examined with a heptad repeat analysis program to assess the occurrence of coiled coil proteins. how heptad repeat domains are organized within these proteins, and what structural/functional categories they comprised. Of 102,007 proteins analyzed, 5.95% (6074) contained coiled coil domains: 1.26% (1289) contained "extended" (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were 4-fold more frequent in the animal kingdom, and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two categories: 1) myosins and motors, or 2) components of the NMIF. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1-2% of the cell mass while accurately retaining morphological features of living epithelium. The NMIF incorporates many proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in Metazoa compared to plants or protists supports the hypothesis that a tissue-wide matrix of coiled coil interactions underlies metazoan differentiated cell and tissue structure.

Resumo: Na descrição hierárquica do conhecimento biológico, o ser vivo é considerado como ponto central nas relações engendradas pelos seguintes níveis: ambiente externo (ecológico/evolutivo), organismo e ambiente interno (genético/ molecular). O organismo compreendido como nível focal da discussão biológica pode ressaltar a autonomia da Biologia em relação às outras áreas do conhecimento científico. No contexto do ensino, assume-se que as discussões epistemológicas do conhecimento biológico podem promover uma compreensão mais integrada dos fenômenos biológicos. Assim, organizou-se um grupo de pesquisa com graduandos de um curso de Ciências Biológicas para discutir conceitos centrais do conhecimento biológico, entre eles, o conceito de organismo. Esta pesquisa teve como objetivos: 1) Elaborar uma caracterização do conceito de organismo, partindo de uma abordagem hierárquica, integrando as discussões advindas da Filosofia da Biologia contemporânea referentes aos conceitos de auto-organização, autonomia agencial, propriedades emergentes e níveis hierárquicos; 2) Analisar como o conceito de organismo se impõe frente às explicações de vida presentes na literatura contemporânea da Filosofia da Biologia; 3) Utilizar a discussão teórica relativa ao conceito de organismo como fundamentação de um grupo de "Pesquisas em Epistemologia da Biologia", verificando as contribuições desse aporte teórico para a formação de alunos de Licenciatura em Ciências Biológicas na área de Epistemologia da Biologia e Ensino de Ciências; 4) Analisar as discussões e produções escritas ocorridas no desenvolvimento do grupo de "Pesquisas em Epistemologia da Biologia" que abordaram o conceito de organismo, com a finalidade de verificar se uma abordagem hierárquica tendo o organismo como nível focal contribui para uma visão integrada do conhecimento biológico pelos... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In the hierarchical of biological knowledge, the living being could be considered as central point in the relations produced by three levels: external environment (ecological/evolution), organism and environment intern (genetic/molecular). The comprehension of the organism as a focal level in the biological debate can underline the autonomy of Biology among the other areas of the scientific knowledge. In the education context it is assumed that the epistemological discussions of the biological knowledge can promote an integrated understanding of the biological phenomena. Thus, a research group consisting of Biological Sciences undergraduates was organized to debate central concepts of the biological knowledge in which the discussions of the organism concept are included. This research aimed to: 1) develop a characterization of the concept of organism from a hierarchical approach by integrating the resulting discussions from contemporary philosophy of biology that are related to the concepts of self-organization, autonomy agents, emergent properties and hierarchical levels; 2) to analyze how the concept of organism is placed in front of the explications of life in the contemporary literature of philosophy of biology; 3) to use the theoretical discussion on the concept of organism as fundamentation for a group of "Studies in Epistemology of Biology," noting the help of this theoretical contribution to the Biological Sciences students training in the Biology and Epistemology in Science Teaching; 4) to analyze discussions and written productions that occurred in the development of the 'Research in Epistemology of Biology's group which addressed the concept the organism in order to verify if a hierarchical approach in which the organism is the focal level contributes to an integrated view of biological knowledge for biology students. On the basis of the theoretical referential... (Complete abstract click electronic access below)
Orientador: Ana Maria de Andrade Caldeira
Coorientador: Charbel Niño El-Hani
Banca: Fernando Bastos
Banca: Maurício de Carvalho Ramos
Banca: Luzia Marta Bellini
Banca: Marcelo Carbone Carneiro
Doutor

Despite the two-fold advantage to asexual reproduction and its prevalence in a variety of organisms, sexual reproduction is prevalent across all taxa. The maintenance of two sexes is required to ensure genetic diversity and to prevent “evolutionary dead ends,” especially in clonal organisms. Many mechanisms have been proposed for the maintenance of two sexes, ranging from environmental variation and stochasticity, parasites and predators, and mutation rates. Spatial configuration, the size and location of populations with respect to other populations, can allow two competitors to coexist when one would normally be lost. This is especially important when the two competitors are the two sexes. In the clonal organism Marchantia inflexa, I determined that spatial configuration of populations can directly influence the maintenance of both sexes in a population and in an aggregate of populations (a metapopulation) using a combination of theoretical models and field studies. Based on field studies, population size has a significant influence on whether a subpopulation will contain both sexes, with populations smaller than 1m2 being more likely to contain only one sex while populations greater than 1m2 are more likely to contain both sexes. Based on mathematical models, the spatial arrangement of subpopulations within a metapopulation can greatly influence whether a metapopulation maintains both sexes as well as whether the metapopulation persists once one sex has been lost. Field data suggest that distance to nearest neighbor, a measurement of spatial arrangement, influences the maintenance of the sexes within subpopulations, but could affect maintenance differently depending on the metapopulation identity. In some metapopulations, both sexes are maintained when the nearest neighbor is close, while in other streams, one sex is lost when the nearest neighbor is close. When mathematical models are used to explicitly simulate natural metapopulations, the mathematical model predicts the observed sex ratios in one metapopulation, predicts the observed bias in another metapopulation, and fails to predicted observed values in two other metapopulations. Understanding spatial configuration helps us understand the maintenance and loss of sex, but other factors, such as environmental differences, may be required to accurately predict which sex will be lost.

I use morphological, vocal, molecular, behavioural, ecological and distributional data to re-evaluate the systematics of three passerine bird groups, the Mirafraassamica complex (bush-larks), the genus Seicercus ("spectacled-warblers"; with emphasis on the the S. burkii complex) and the genus Motacilla (wagtails). Two new species are described: Seicercus soror and Motacilla samveasnae. I propose that the polytypic species M. assamica should be treated as four separate species: M. assamica, M. affinis, M. microptera and M. marionae (it is also remarked that the proper name of the latter is M. erythrocephala). That is primarily supported by vocalisations and mitochondrial DNA. The latter data set also suggests that M. assamica sensu lato is paraphyletic, since M. erythroptera, which is always treated as a separate species, is nested within the M. assamica complex. I propose that the polytypic species S. burkii comprises six sibling species. Some of these are found to breed sympatrically, although mainly or entirely segregated altitudinally. Mitochondrial DNA suggests that the S. burkii complex is non-monophyletic, and also that the divergence of the different taxa is much older than indicated by morphological and vocal data. According to the molecular phylogeny, both the genera Seicercus and its assumed sister genus Phylloscopus are paraphyletic. That is corroborated by independent data. The phylogenetic study of the genus Motacilla reveals incongruence between mitochondrial DNA, nuclear DNA and non-molecular data. I conclude that the nuclear gene tree reflects the organismal phylogeny more faithfully than the mitochondrial gene tree. The latter is likely to have been affected by introgressive hybridisation, possibly also stochastic lineage sorting. The most remarkable result that is strongly supported by both mitochondrial and nuclear DNA is that M. flava is non-monophyletic.

This thesis deals with evolutionary relationships within the asterids, a group of plants comprising about one-third of all flowering plants.

Two new families are recognised: Pennantiaceae and Stemonuraceae. The woody Pennantia from New Zealand and Australia is the sole genus of Pennantiaceae. Stemonuraceae consist of a dozen woody genera with a pantropical distribution and a centre of diversity in South East Asia and the Malesian islands. They are characterised by long hairs on their stamens and/or fleshy appendages on their fruits. Both families were formerly included in Icacinaceae. While Pennantiaceae are unrelated to any of the former Icacinaceae and placed in the order Apiales, other former Icacinaceae genera are related to Cardiopteris, a twining herb from South East Asia and Malesia. The monogeneric family Cardiopteridaceae is enlarged as to include also these. Cardiopteridaceae and Stemonuraceae are sister groups and placed in Aquifoliales. The three other families of Aquifoliales are monogeneric and closely related. The Asian Helwingiaceae and the Central/South American Phyllonomaceae are suggested to be merged into Aquifoliaceae (hollies). The genera of Icacinaceae in the traditional sense not placed in any of the above families (all euasterids II) are members of early diverging lineages of the euasterids I and possibly included in the order Garryales.

The three woody Australasian families Alseuosmiaceae, Argophyllaceae, and Phellinaceae are confirmed as members of Asterales, despite traditional placements not close to that order. They are, moreover, supported as each other’s closest relatives.

The results are based mainly on parsimony analysis of DNA sequence data, but morphological studies have revealed characters in support for the molecularly based conclusions. The gene that has provided most new information is the chloroplast ndhF gene. The results are, however, drawn from combined analyses of sequences from one or several additional genes (atpB, matK, rbcL, 18S rDNA). The data have also been explored with Bayesian analysis, a statistical, model-based method that most recently has been developed for phylogeny reconstruction.

Echiochilon, Ogastemma and Sericostoma are revised resulting in the recognition of 15 species of Echiochilon and one Ogastemma species. Several species are placed in synonymy and three new species are described, E. baricum, E. callianthum and E. cyananthum. The single species of Sericostoma is shown to be nested within Echiochilon. The plastid atpB gene was sequenced for Echiochilon and Ogastemma from the Old World and Antiphytum from the New World, plus for a selection of 33 other Boraginaceae taxa. They were analysed together with selected outgroup taxa to give a framework of the tribes of Boraginoideae. The analysis gave support for establishing the new tribe Echiochileae for Antiphytum, Echiochilon and Ogastemma, and for merging the traditionally accepted tribe Eritrichieae with Cynoglosseae. The ITS region was sequenced for all but one species of Echiochilon and for representatives of Antiphytum and Ogastemma. Phylogenetic analysis of Echiochilon revealed that the strongly zygomorphic-flowered species form a paraphyletic group. The morphological data gave results fairly congruent with the ITS phylogeny. Biogeographic interpretations of the ITS and atpB phylogenies indicated a trans-Atlantic dispersal of Antiphytum as the most plausible explanation to the Old/New World disjunction. Analyses using DIVA (Dispersal Vicariance Analysis) of the distributions of the Echiochilon species indicated an ancestor to Echiochilon with a wide distribution over northern Africa and Arabia to India.

Invasive species can have a variety of effects on the behavior and ecology of native species. Currently in New Orleans, Louisiana, both A. sagrei and A. carolinensis lizards are relatively abundant, but the A. sagrei population is expanding rapidly. I used a combination of laboratory and field studies to investigate factors that might be influencing local dominance of invasive A. sagrei over native A. carolinensis populations, including habitat use, display behavior, interspecific aggressive interactions, and plasticity. When comparing display behavior and habitat use in anole populations across three field sites in southern Louisiana, I found differences in male display behavior of both species, and also that A. carolinensis perched higher when A. sagrei was present. In staged interspecific interactions, I discovered that A. sagrei females achieved consistently higher aggressive scores than A. carolinensis females, suggesting that female interspecific behavior is probably more important than male behavior in driving changes in habitat use. Lastly, I studied plasticity in several morphological and whole-organism performance variables by rearing males and females of each species on two different perch diameters. I found that sprinting performance in A. sagrei was significantly different between treatment groups, although the morphological differences between perch treatments were subtler than those reported in previous studies. I also found that A. carolinensis females exhibited significant differences in both sprinting and clinging performance, despite no significant differences in male or female morphology between perch size treatments, highlighting the potential for both species-specific and sex-specific plasticity.

In order to study the potential effects of an oil spill on coral reef organisms, the marine sponge, Cinachyrella spp. was investigated. In this study, Cinachyrella spp. was placed in a closed aquaculture system and exposed to sub-lethal water-accommodated fractions (WAFs) of Macondo crude oil and chemically-enhanced water accommodated fractions (CE-WAFs) of the dispersant, Corexit 9500, over a 24-hour time course, in order to model the BP Deepwater Horizon oil spill and oil spill sponge response. Illumina RNA sequencing and gene expression analysis utilizing hierarchical clustering, principal component analysis, and KEGG bioinformatic database generated 34,147 unique transcripts with differential expression of 483 transcripts across all samples related to metabolism, genetic, environmental, and cellular processes, and associations with pathways involved in human disease development and progression. These pathways highlight the induction of Rac1, a GTPase in the Ras superfamily responsible for cell proliferation, differentiation, and senescence and SOS, a set of specialized Ras-GTP activators. These Ras-regulated signaling proteins are thought to play a significant role in the development of human malignancies, specifically Rac1. The data reported here helps support the possible role of Cinachyrella spp. as an ecotoxicological model for oil and dispersant pollution as well as the identification of potential biomarkers of stress and environmental perturbation. These results have important implications in identifying stress response in coral reef associated communities, and will ultimately be useful in coral reef conservation, management, and oil spill mitigation activities.

Wing polyphenism is a type of phenotypic plasticity present in several insect species whereby a genotype have the ability to develop alternative wing morphs when exposed to different environmental cues. One organism demonstrating a clear case of wing polyphenism is the water strider species, Gerris buenoi, which develop long- or short wings depending on exposure to different photoperiods (the time the organism is exposed to light during a 24 h period). The molecular mechanism behind wing polyphenism in insects in general, and in water striders in particular, is largely unknown. From a study on wing polyphenism in the Brown planthopper (Nilaparvata lugens), some candidate genes have been identified and include two insulin receptor genes and the Forkhead transcription factor (FOXO). Since these genes have been demonstrated to affect wing polyphenism in Brown planthopper (BPH) and since G. buenoi contains an additional insulin receptor homolog, the potential role of these genes in regulating wing polyphenism in G. buenoi have in this project been investigated. The functional genetic technique RNA interference (RNAi) was used to evaluate the function of the genes. This method knock down gene expression in the genes mentioned above, one at a time, to investigate if they have a function in wing polyphenism in G. buenoi. DsRNA with specific homology to each target gene was successfully produced. However, when attempting to inject the dsRNA through micro injection all injected liquid leaked out from the body cavity, and the RNAi was therefore not successful. Further optimisation of the injection protocol has to be done to be able to perform RNAi properly in the future. Thereafter, RT-qPCR was used to evaluate whether the insulin receptor genes and FOXO are differentially expressed between the two photoperiods giving rise to the different wing morphs. The differential gene expression experiment showed differences between the mRNA levels of all target genes between G. buenoi being reared in the two different photoperiods. More specific upregulation of the genes FOXO and insulin receptor 2 in short winged G. buenoi were demonstrated. Further, insulin receptor 1-like, was also demonstrated to be upregulated in the short winged morph. Results presented in this project are in line with the previously identified regulation pattern in BPH, still the results need further evaluation. Since gene expression differences were present for all candidate genes between G. buenoi reared in the different photoperiods, theses genes could still be seen as potential candidate genes in wing polyphenism in water striders.

Performance is key to survival. From day-to-day foraging events, to reproductive activities, to life-or-death crises, how well an organism performs these tasks can determine success or failure. Selection, therefore, both natural and sexual, act upon performance, and performance demands on individuals shape a population’s morphological and physiological trait distributions. While studies of morphological adaptations to ecological pressures implicitly center on the idea that responses to selection improve performance via changes in morphology, the relationships between morphology, performance, and fitness are not always well understood. In this dissertation, I investigate these relationships explicitly, as well as determine the effects that different ecological and genetic contexts have on selection and how populations respond to performance pressures. Using a model of lizard locomotor performance, I address three issues that may impact selection on performance that are often overlooked in performance studies. First, performance is not a static trait. Rather, individuals possess a range of performance abilities or intensities that can be expressed as needed. Using a novel, individual-based, quantitative genetic simulation model, I demonstrate the effects of variable performance expression and genetic constraints on how a population experiences and responds to selection on sprint and endurance performance. Second, sex differences in performance are expected in sexually dimorphic species, but empirical evidence for this is lacking. To this end, I measured and analyzed multivariate morphology and performance in Anolis carolinensis to identify sex-specific patterns in functional morphology and functional trade-offs within a broad suite of performance traits. Third, intralocus sexual conflict should constrain the evolution of the multivariate performance phenotype in both sexes. By extending the simulation model to include correlated trait inheritance between sexes and sex-specific selection on certain performance traits, I demonstrate the extent to which this sexual conflict constrains performance evolution. In combining studies of natural populations with simulation studies of selection, this dissertation embraces the complexity of performance to address the multiple contributing factors and constraints on performance evolution, and demonstrates the importance of accounting for such complexity when studying animal performance.

Small GTP binding proteins function as molecular switches which cycles between GTP-bound ON and GDP-bound OFF states, and regulate a wide variety of cellular processes as biological timers. The first characterized member of the small GTPase family, the mutated oncogene p21 src, later known as Harvey-Ras, was identified in the early 1980s (Shih, T. Y. et al. 1980). In the following years small Ras-lik GTPases were found in several organisms and it was soon discovered that they took part in processes, such as signal transduction, gene expression, cytoskeleton reorganisation, microtubule organisation, and vesicular and nuclear transport. The first Rho (Ras homology) gene was cloned in 1985 from the sea slug Aplysia (Madaule, P. et al. 1985) and because of their homology to Ras it was first suspected that they could act as oncogenes. Later studies have shown that even though they participate in processes such as cell migration and motility they are not mutated in cancers.

The first indications that Rho was a signaling protein regulating the actin cytoskeleton, came from experiments where activated forms of human RhoA was microinjected into 3T3 cells (Paterson, H. F. et al. 1990). Another Rho-like GTPase Rac1 (named after Ras-related C3 botulinum toxin substrate) was later shown to regulate actin cytoskeletal dynamics as well, suggesting that Rho-family members cooperate in controlling these processes (Ridley, A. J. et al. 1992). The Rac GTPase was also implicated in regulating the phagocytic NADPH oxidase, which produce superoxide for killing phagocytized microorganisms (Abo, A. et al. 1991). Thus, it soon became clear that Rac/Rho and the related GTPase Cdc42 (cell division cycle 42) had central functions in many important cellular processes.

There are at least three types of regulators for Rho-like proteins. The GDP/GTP exchange factors (GEFs) which stimulates conversion from the GDPbound form to the GTP-bound form. GDP dissociation inhibitors (GDIs) decrease the nucleotide dissociation from the GTPase and retrieve them from membranes to the cytosol. GTPase activating proteins (GAPs) stimulates the intrinsic GTPase activity and GTP hydrolysis. In addition there are probably regulators that dissociate GDI from the GTPase leaving it open for activation by the RhoGEFs.

Ras and Rho-family proteins participate in a coordinated regulation of cellular processes such as cell motility, cell growth and division. The Ral GTPase is closely related to Ras and recent studies have shown that this GTPase is involved in crosstalk between both Ras and Rho proteins (Feig, L. A. et al. 1996; Oshiro, T. et al. 2002). Ral proteins are not found in plants and they appear to be restricted to animalia and probably yeast. During a screen for small GTPases in Drosophila melanogaster I discovered in 1993 several new members of the Ras-family, such as Drosophila Ral (DRal), Ric1 and Rap2. The functions of Ral GTPases in Drosophila have until recently been poorly known, but in paper 2 we present some of the new findings.

Rho-like GTPases have been identified in several eukaryotic organisms such as, yeast (Bender, A. et al. 1989), Dictyostelium discoideum (Bush, J. et al. 1993), plants (Yang, Z. et al. 1993), Entamoeba histolytica (Lohia, A. et al. 1993) and Trypanosoma cruzi (Nepomuceno-Silva, J. L. et al. 2001). In our first publication, (Winge, P. et al. 1997), we describe the cloning of cDNAs from RAC-like GTPases in Arabidopsis thaliana and show mRNA expressions pattern for five of the genes. The five genes analyzed were expressed in most plant tissues with the exception of AtRAC2 (named Arac2 in the paper), which has an expression restricted to vascular tissues. We also discuss the evolution and development of RAC genes in plants. The third publication, (Winge, P. et al. 2000), describe the genetic structure and the genomic sequence of 11 RAC genes from Arabidopsis thaliana. As most genomic sequences of the AtRACs we analyzed came from the Landsberg erecta ecotype and the Arabidopsis thaliana genome was sequenced from the Columbia ecotype, it was possible to compare the sequences and identify new polymorphisms. The genomic location of the AtRAC genes plus the revelation of large genomic duplications provided additional information regarding the evolution of the gene family in plants. A summary and discussion of these new findings are presented together with a general study of small Ras-like GTPases and their evolution in cellular organisms. This study suggests that the small GTPases in eukaryots evolved from two bacterial ancestors, a Rab-like and a MglA/Arp-like (Arf-like) protein. The MglA proteins (after the mgl locus in Myxococcus xanthus) are required for gliding motility, which is a type of movement that take place without help of flagella.

The second publication describes experiments done with the Drosophila melanogaster DRal gene and its effects on cell shape and development. Ectopic expression of dominant negative forms of DRal reveals developmental defects in eye facets and hairs, while constitutive activated forms affects dorsal closure, leaving embryos with an open dorsal phenotype. Results presented in this publication suggest that DRal act through the Jun N-terminal kinase (JNK) pathway to regulate dorsal closure, but recent findings may point to additional explanations as well. The results also indicate a close association between processes regulated by Rac/Rho and Ral proteins in Drosophila.

Pattern formation is found widely throughout nature serving important roles in many different biological contexts. Multicellular organisms form patterns to enable new functions and pattern formation is also a crucial step for biological adaptation. The work presented here lays the foundations for the creation and control of multicellular growth patterns with a normally single-celled organism. The natural capacity for strains of Saccharomyces cerevisiae yeast to perform multicellular growth is here genetically and externally-controlled using synthetic biology tools. This allows S. cerevisiae grow in multicellular filamenting patterns, following a unique phenotype called pseudohyphal growth. Fine-control of the dynamic behaviour and gene expression of a colony of cells growing with this phenotype should enable fractal-like growth formations to be genetically-encoded. In this work, synthetic gene regulatory networks were first constructed in order to control genetic targets than induce or repress the pseudohyphal growth phenotype. This generated haploid and diploid yeast strains that can quickly switch to growing as filaments when simple chemical stimuli are externally provided or removed. In order to enable control over pattern formation with these strains, further genetic targets such as the BUD genes were investigated and the pseudohyphal growth phenotype was linked to previously described timer gene regulatory networks that dynamically change the expression of target genes over hours and days. These timer networks allowed cells to be programmed to transition from pseudohyphal growth back to normal yeast growth as the cells are grown in a colony over days. Finally, in an attempt to create controlled growth from pseudohyphal yeast so that fractal-like patterns could be made, new hybrid promoters with multiple modes of regulation were generated. These externally-inducible promoters were constructed to only express in mother cells after they have budded a daughter cell, and were designed in order to control how often filaments form new branches when S. cerevisiae grows in pseudohyphal form. While a full integration of the whole system to generate controllable fractal-like patterns was not achieved in the end, this study delivers several valuable new tools for yeast research and yeast synthetic biology.

El creixent nombre de genomes seqüenciats està posant de manifest que la pèrdua gènica és un fenomen recurrent que pot haver generat diversitat al llarg de la història evolutiva dels diferents grups d’organismes. En aquest sentit, un dels grans reptes en el camp de la EvoDevo és entendre l’impacte de la pèrdua gènica en l’evolució dels mecanismes del desenvolupament animal considerant diferents escenaris evolutius. En escenaris de selecció positiva, la pèrdua hauria comportat canvis morfològics o fisiològics que haurien permès l’adaptació de les espècies a noves condicions ambientals. En escenaris de selecció neutre (o quasi neutre), la pèrdua hauria afectat a gens que haurien esdevingut prescindibles per l’existència de robustesa mutacional o perquè les condicions ambientals haurien canviat. Saber si una pèrdua gènica ha estat adaptativa o neutre és generalment molt complex ja que molts cops es difícil establir relacions directes de causalitat entre la pèrdua d’un gen i l’aparició d’una nova característica evolutivament avantatjosa. Malgrat aquesta dificultat, en aquesta tesi doctoral vàrem voler estudiar l’impacte de la pèrdua gènica en l’EvoDevo a partir d’analitzar l’evolució de les vies de senyalització de l’àcid retinoic (RA) i de Wnt -vies essencials pel desenvolupament de tots els cordats- a l’urocordat Oikopleura. dioica com a sistema model de referència. Per establir O. dioica com a model animal, però, a més de les característiques biològiques que la fan atractiva per aquests tipus estudis –desenvolupament embrionari i cicle de vida extremadament ràpid, simplicitat i transparència corporal, genoma reduït i totalment seqüenciat, possibilitat de manipulació gènica per estudis funcionals– ha estat fonamental desenvolupar unes instal·lacions per cultivar-la en el laboratori, així com protocols de manteniment assequibles (low-cost) per qualsevol grup d’investigació. Gràcies a aquest sistema de cultiu, em pogut analitzar els components de la xarxa gènica del metabolisme de l’RA (RA-MGN), així com els lligands de la via de senyalització per Wnt. En referència a la RA-MGN, el nostre treball ens permet concloure que les pèrdues gèniques que han afectat a aquesta via durant l’evolució de O. dioica han tingut lloc en un sistema genètic no robust, on el desballestament de la via no han tingut un impacte dramàtic en el pla corporal típic de cordats que preserva aquest organisme. Per altre banda, l’anàlisi de la família Wnt a cordats, ens ha permès concloure que a més de la duplicació gènica o de la redundància funcional per processos d’evolució convergent, els fenòmens de reassignació de funcions (function shuffling) augmenten també la robustesa mutacional i faciliten les pèrdues gèniques en les famílies de gens. En resum, els resultats d’aquesta tesi doctoral posen de manifest que O. dioica és un model animal atractiu per estudiar tant aspectes bàsics de l’impacte de les pèrdues gèniques en l’evolució dels mecanismes del desenvolupament, com en aspectes aplicats en que certes pèrdues confereixen a O. dioica la condició de knockout evolutiu que pot ser interessant per l’estudi de mecanismes moleculars concrets com la toxicitat de les PUAs en el desenvolupament embrionari d’organismes marins.
The bloom of genomics is revealing gene loss as a pervasive evolutionary force generating genetic diversity that shapes the evolution of species. In this sense, one of the great challenges in the field of EvoDevo is to understand the impact of gene loss on the evolution of animal developmental mechanisms. In this doctoral thesis we wanted to study the impact of gene loss in EvoDevo, using the analysis of the evolution of retinoic acid (RA) and Wnt signaling pathways - essential pathways in the development of all the chordates- in the urochordate Oikopleura. dioica as a model reference system. To establish O. dioica as an animal model, in addition to the biological characteristics that make it attractive for his type of studies-embryonic development and extremely fast life cycle, simplicity and body transparency, reduced and fully sequenced genome, possibility of gene manipulation for functional studies - It has been essential to develop a facility to grow O. dioica in the laboratory, as well as low-cost maintenance protocols affordable for any research group. Thanks to this culture system, we were able to analyze the components of the RA metabolic gene network (RA-MGN), as well as the ligands of the Wnt signaling pathway. In reference to the RA-MGN, our work allows us to conclude that the gene losses that have affected this pathway during the evolution of O. dioica have taken place in a non-robust genetic system, where the dismantling of the network has not had a dramatic impact on the typical body plan of chordates that preserves this organism. On the other hand, the analysis of the Wnt family in chordates, has allowed us to conclude that in addition to gene duplication or functional redundancy due to processes of convergent evolution, function shuffling also increase the mutational robustness and facilitate gene losses in gene families. In summary, the results of this doctoral thesis show that O. dioica is an attractive animal model for studying both basic aspects of the impact of gene losses in the evolution of development mechanisms, and in applied aspects. For instance, certain losses give to O. dioica an evolutionary knockout condition that may be interesting for the study of concrete molecular mechanisms such as the toxicity of PUAs in the embryonic development of marine organisms.

Tyrimo objektas: Maisto papildų vartojimo paplitimas tinklininkų ir rankininkų, bei jų fizinis pajėgumas. Tyrimo problema: Maisto papildų vartojimo paplitimas tinklininkų ir rankininkų, bei jų fizinis pajėgumas yra mažai nagrinėti teoriniu ir empiriniu lygmeniu. Tikslas: Nustatyti maisto papildų vartojimo paplitimą tinklininkų ir rankininkų, bei jų fizinį pajėgumą. Uždaviniai: 1) Nustatyti ir palyginti įvairių maisto papildų vartojimo ypatumus tinklininkų ir rankininkų grupėse. 2) Nustatyti ir palyginti maisto papildų daromą įtaką fiziniam pajėgumui tinklininkų ir rankininkų grupėse. Hipotezė: Maisto papildų daroma įtaka sportininkų organizmui yra susijusi su geresniu fiziniu pajėgumu. Išvados: 1. Tyrimo dalyviai daugiausia vartojo baltymų ir angliavandenių mišinius, o mažiausiai tarp tiriamųjų buvo vartojami HMB maisto papildai. Tinklininkai labiausiai buvo linkę vartoti baltymų ir angliavandenių mišinius, rankininkai L-karnitiną, amino rūgštis bei baltymų ir angliavandenių mišinius. Rankininkų ir tinklininkų maisto papildų vartojamų preparatų pasirinkimas gana skirtingas. 2. Tinklininkų grupėje, praėjus 6 mėn. nuo tyrimo pradžios, geresniems staigiosios jėgos rezultatams, taikant šuolio į tolį pratimą, reikšmės turėjo amino rūgščių maisto papildų vartojimas, o rankininkų grupėje – kreatino maisto papildų vartojimas. Taikant šuolio aukštyn atsispiriant pratimą, geresniems tinklininkų rezultatams, praėjus 6 mėn. nuo tyrimo pradžios, reikšmės turėjo kreatino, o... [toliau žr. visą tekstą]
Object of this study: Prevalence of food supplements usage for volleyball and handball players’ and their physical fitness. Problem of this study: Prevalence of food supplements usage and their physical fitness (particularly for volleyball and handball players) is a low-examined theoretically and empirically. Aim: Identify the prevalence of food supplements usage for volleyball and handball players‘ and their physical fitness. Tasks: 1) Identify and compare the food supplements usage features for volleyball and handball players. 2) Identify and compare the influence of food supplements for volleyball and handball players’ physical fitness. Hypothesis: The influence of food supplements for athletes’ organism is related with better physical fitness. Findings: 1. Participants of this study mainly used protein and carbohydrates’ mixtures and the least among the subjects were used the HMB supplements. Volleyball players was the most likely to use mixtures’ of protein and carbohydrates, handball players – L-carnitine, amino acids, protein and carbohydrate mixtures. As can be seen, handball and volleyball players select quite different food supplements to use. 2. The emergency power, when the long jump exercise was used, after 6 months from the beginning of this study was better for those volleyball players‘ who used amino acids and for handball players’ – who used supplements of creatine. When the jump up exercise was used, better results (after 6 months from the... [to full text]

BACKGROUND:Systems Biology research tools, such as Cytoscape, have greatly extended the reach of genomic research. By providing platforms to integrate data with molecular interaction networks, researchers can more rapidly begin interpretation of large data sets collected for a system of interest. BioNetBuilder is an open-source client-server Cytoscape plugin that automatically integrates molecular interactions from all major public interaction databases and serves them directly to the user's Cytoscape environment. Until recently however, chicken and other eukaryotic model systems had little interaction data available.RESULTS:Version 2.0 of BioNetBuilder includes a redesigned synonyms resolution engine that enables transfer and integration of interactions across species
this engine translates between alternate gene names as well as between orthologs in multiple species. Additionally, BioNetBuilder is now implemented to be part of the Gaggle, thereby allowing seamless communication of interaction data to any software implementing the widely used Gaggle software. Using BioNetBuilder, we constructed a chicken interactome possessing 72,000 interactions among 8,140 genes directly in the Cytoscape environment. In this paper, we present a tutorial on how to do so and analysis of a specific use case.CONCLUSION:BioNetBuilder 2.0 provides numerous user-friendly systems biology tools that were otherwise inaccessible to researchers in chicken genomics, as well as other model systems. We provide a detailed tutorial spanning all required steps in the analysis. BioNetBuilder 2.0, the tools for maintaining its data bases, standard operating procedures for creating local copies of its back-end data bases, as well as all of the Gaggle and Cytoscape codes required, are open-source and freely available at http://err.bio.nyu.edu/cytoscape/bionetbuilder/ webcite.

Haematophagous parasites and disease vectors such as leeches, ticks, mites, lice, bed bugs, mosquitoes, and myiasis-causing fly larvae are common health problems in Lao Peoples Democratic Republic (Lao PDR). A main aim of my field work in Lao PDR in 2006-2010 was to document traditional knowledge among different ethnic groups about plants that people use to repel or to kill blood-feeding invertebrates. We carried out structured interviews in 66 villages comprising 17 ethnic groups, covering a range of ethnic group, throughout Lao PDR and recorded a total of 92 plant species - in 123 different plant-ectoparasite combinations - that are used as traditional repellents and/or as “pesticides” to kill "pest" invertebrates. Traditional use was confirmed in the scientific literature for 74 of these plant species, and for an additional 13 species based on literature on closely related species. We concluded that repellents and pesticides from many plant species are commonly used in the Lao countryside. We also investigated traditionally used Lao plants for their activity to repel or to kill certain disease vectors and parasites. Target organisms were mosquitoes (Diptera, Culicidae), fly larvae (Diptera, Cyclorrhapha) in fermented fish production, and terrestrial blood-sucking leeches (Hirudinea, Haemadipsidae). The potential mosquito repellent activities of essential oils of Croton roxburghii (Euphorbiaceae), Hyptis suaveolens (Lamiaceae), and Litsea cubeba (Lauraceae) were evaluated in the field near Vientiane. Oils at concentrations of 1.7-6.7 µg/cm2 were significantly repellent to Aedes, Armigeres and Culex attracted to human baits. The activities against fly larvae, infesting fermenting fish, of three plant species, Tadehagi triquetrum (Fabaceae), Uraria crinita (Fabaceae) and Bambusa multiplex (Poaceae) were investigated: When fresh material of the plants was added on top of fermenting fish infested with fly larvae significant proportions of the larvae were repelled or killed. The total protective effect, i.e., repellent and killing effect combined, of T. triquetrum, U. crinita, and B. multiplex was 60-83 %, 77-90 %, and 60-93 %, respectively. Field evaluation of the potential leech repellent activities of water extracts of Sapindus rarak (Sapindaceae), Catunaregam spathulifolia (Rubiaceae) and Vernonia elaeagnifolia, (Asteraceae) impregnated on stockings and worn by persons in two leech-infested biotopes revealed leech repellent activities of 82.6%, 62.6% and 63.0%, respectively. The corresponding repellencies of deltamethrin and diethyl-3-methyl-benzamide (DEET) were 73.1% and 88.4%, respectively. Identification of the active components in certain of the plants with the ultimate aim to develop more optimal, less costly repellents, insecticides, acaricides, and anti-leech compounds as alternatives to synthetic repellents and pesticides against blood-feeding insects, ticks, mites, and leeches is in progress.

The origin of animal multicellularity is a major evolutionary transition and a poorly understood one. In recent years, the sequencing of the genomes of several earlybranching metazoans, such as sponges and cnidarians, has allowed to define a common and exclusive molecular toolkit of animal multicellularity. Most of these genes are involved in cell adhesion, cell-cell communication and control of cell proliferation and differentiation, all of them essential functions for a multicellular organism. The access to genomic data of close unicellular relatives of animals, such as the choanoflagellates, has revolutionized the comparative genomic studies to understand the origin of animals and the molecular toolkit of the Urmetazoa (the common ancestor of all animals), showing that some genes that were considered exclusive to animals are, in fact, present in their unicellular relatives and were later co-opted to function in a multicellular context. The sequencing of the genome of the unicellular holozoan Capsaspora owczarzaki offers a new opportunity for further studying this topic. Thus, the main objectives of this thesis have been: first, to perform comparative genomic studies to reconstruct the evolutionary history of animal molecular toolkit; second, to study the functional conservation of some of the genes identified in Capsaspora owczarzaki by performing heterologous expression in model systems such as Xenopus laevis and Drosophila melanogaster; and finally, to study the life cycle and cell biology of Capsaspora owczarzaki to further understand the unicellular functional context in which this genetic toolkit of the multicellularity can act and may have evolved first. One of the main results of our comparative genomic studies is the presence of a complete integrin adhesome in Capsaspora owczarzaki and an almost full adhesome (including integrins) in the more distantly related Thecamonas trahens. This results allowed us to reconstruct the step-wise evolutionary assembly of this key machinery of animal multicellularity. Another important result is the finding of several transcription factors (essential tools for regulating cell differentiation and cell proliferation in animals) in Capsaspora owczarzaki previously considered exclusive to animals, for example NFkappaB, Myc/Max, Mef2, T-box, Runx,... Finally, we found in Capsaspora owczarzaki homologs of the components of the Hippo signalling pathway, an essential mechanism controlling cell proliferation and organ size in animals. Through heterologous expression studies we could study in detail the functional conservation of two of these machineries discovered in Capsaspora owczarzaki by comparative genomic studies. First, we studied the Capsaspora owczarzaki homolog of Brachyury (CoBra) using Xenopus laevis, the classical model system for studying Brachyury function. Brachyury is a T-box transcription factor involved in gastrulation and mesoderm specification in metazoans. We demonstrated that CoBra is functionally conserved and that it has similar DNA binding sites to those of animal T-box genes. Second, we used Drosophila melanogaster to demonstrate the functional conservation of three components of Capsaspora owczarzaki's Hippo pathway. One the most striking morphological features of Capsaspora owczarzaki is the presence of long filopodia. Using this organism and the choanoflagellate Salpingoeca rosetta we studied the origin and evolution of metazoan filopodia and microvilli and the associated molecular toolkit. We found that most components are innovations of animals and these two close unicellular lineages and that the microvilli originated in the common ancestor of animals and choanoflagellates. Finally, we studied the life cycle of Capsaspora owczarzaki using microscopy, flow cytometry and comparative transcriptomics of different life stages. We demonstrated the existence of an aggregative pluricellular stage in Capsaspora owczarzaki and that the transition between this and other stages is tightly regulated at the level of gene expression and alternative splicing. We also found evidence that the integrin adhesome is expressed in the aggregative stage, we an extracellular matrix is produced between the cells. The study of Capsaspora owczarzaki has provided valuable insights into the origin of animal multicellularity and emphasizes the fact that gene co-option was a major mechanism to generate to multicellular molecular toolkit of animals.
El origen de la multicelularidad animal es una de las mayores transiciones evolutivas de la historia de la vida. La secuenciación, en los últimos años, de genomas de animales basales como esponjas y cnidarios ha permitido establecer la maquinaria genética común a todos los animales. La mayoría de estos genes son aquellos involucrados en la adhesión celular, la comunicación celular y el control de la proliferación y la diferenciación. El acceso a datos genómicos de organismos unicelulares muy cercanos a los animales, como Capsaspora owczarzaki, es esencial para entender mejor esta transición. Los objetivos principales de esta tesis ha sido estudiar la presencia de genes de multicelularidad y su conservación funcional en Capsaspora owczarzaki, así como su ciclo vital. Analizando su genoma, descubrimos una completa maquinaria de adhesión por integrinas en C.owczarzaki. Hemos podido reconstruir en detalle la historia evolutiva de este mecanismo crucial de adhesión y comunicación celular. También hemos encontrado un amplio repertorio de factores de transcripción, elementos esenciales para regular la diferenciación y la proliferación en los animales, que se creían exclusivos de animales en el genoma de C.owczarzaki; por ejemplo, genes NFkappaB, T-box o p53. Por último, hemos descrito que una importante vía de señalización en animales, la llamada vía Hippo, también está presente en C.owczarzaki. Este mecanismo es esencial para controlar la proliferación y el tamaño de los órganos en los animales. Mediante estudios de función heteróloga en Xenopus y Drosophila demostramos la conservación funcional de los homólogos del gen Brachyury (un factor de transcripción de la clase T-box) y de la vía Hippo de C.owczarzaki. C.owczarzaki y el coanoflagelado Salpingoeca rosetta nos sirvieron para estudiar la evolución y el origen de los filopodios y microvilli animales y su maquinaria molecular. Por último, el estudio de la biología y el ciclo de C.owczarzaki, mediante el uso de técnicas microscopias, citometría y transcriptómica comparada de los distintos estadios vitales del organismo. Demostramos la existencia de un estadio de pluricelularidad agregativa y que la transición entre éste y otros estadios está finamente regulada a nivel de expresión génica y de splicing alternativo. El estudio de C.owczarzaki nos ha dado valiosos ejemplos de cómo, más allá de la innovación génica, la co-opción de maquinaria pre-existente en un contexto unicelular fue un mecanismo esencial para el origen de la multicelularidad animal.

Lipids are a group of biologically important compounds that may be used as indicators of relationships between an organism and its environment. Lipid class composition identifies types of storage lipid which can suggest a pelagic or benthic existence and may be used to measure the condition of an organism. Fatty acid composition may reflect trophic ecology. Lipid profiling has been used to elucidate elements of the ecology of organisms from Mid-Atlantic and Juan de Fuca Ridge hydrothermal vents and shrimp from below a permanent oxygen minimum zone. Lipid profiles of these organisms from extreme deep-sea environments are compared. Lipid profiles of adult Mid-Atlantic Ridge hydrothermal vent shrimp, Rimicaris exoculata. support a benthic existence and primarily chemoautotrophic bacterial nutrition. Postlarval alvinocarid shrimp lipid profiles suggest a pelagic life cycle stage with a reliance on phototrophically derived organic matter. This result supports previous molecular evidence that Rimicaris exoculata travels between hydrothermal vents on the Mid-Atlantic Ridge. The branchial area of Rimicaris exoculata has been reported to be colonised with bacteria and elevated levels of bacterial fatty acids are present in these tissues. Lipid analyses of the reproductive organs of Rimicaris exoculata reveal increases in triglycerides, sterols, phosphatidyl choline and the proportion of n-3 fatty acids with advancing reproductive maturity. Preliminary studies of the lipid profiles of the Mid-Atlantic vent shrimp Alvinocaris markensis support the hypothesis that it is a scavenger and contains a lower proportion of bacterially derived fatty acids. The lipid profiles of the deep-sea shrimp, Nematocarcinus gracilis, from the Indian Ocean, were consistent with a benthic lifestyle, opportunistic feeding and a reliance on phototrophically derived organic matter. Despite the presence of a permanent oxygen minimum zone above the habitat of the shrimp, Nematocarcinus gracilis contain low levels of highly unsaturated fatty acids, reflecting a reduction in the availability of labile organic matter with depth. The hydrothermal vent tube worm Ridgeia piscesae contains only low levels of storage lipid, reflecting an extreme reliance on endosymbiotic bacteria. Wax esters detected are thought to be stored in oocytes. The fatty acid profile of Ridgeia piscesae reveal mainly bacterial biomarker fatty acids, but some phototrophically derived fatty acids are also present. Adult Ridgeia piscesae have no gut, so the mechanism by which these fatty acids are assimilated is uncertain. Lipid profiles of the hydrothermal vent palm worm, Paralvinella palmiformis vary with the conditions of the different microenvironments in which specimens were found. Fatty acids reflect a mixed diet based mainly on chemoautotrophic bacteria, but with significant inputs of phototrophically derived organic matter, such as diatom debris. Phototrophically derived fatty acids were also detected in the hydrothermal vent clam Calyptogena pacifica. suggesting that the reduced filter-feeding ability of this species is used to supplement nutrition from endosymbiotic bacteria. Endosymbiont-bearing gill tissues contain the highest proportion of bacterial fatty acids and also high concentrations of triglycerides. The presence of triglycerides in gill tissues suggests that energy may be transferred from symbionts by hydrolysis of bacterial symbiont membrane lipids and their conversion to triglyceride. The lipid profiles presented in this work increase understanding of the life history strategies and ecology of the species studied. This thesis shows that even the most highly adapted hydrothermal vent organisms do not rely solely on a chemoautotrophic source of nutrition, but also appear to require phototrophically derived elements in their diet.