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Elektroporation: Eine Methode zur Transfektion von Wirbeltierembryonen / Electroporation: A transfection method for vertebrate embryosVukovich, Wolfgang 24 April 2002 (has links)
No description available.
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Rôle de l'ADN dans l'activation du TLR9 lors de l'infection par Leishmania major : propriétés des séquences génomiques et implication des facteurs protéiques / TLR9 activation by Leishmania major DNA : role of genomic sequences and implication of DNA cofactorErin Khan, Melissa 21 March 2014 (has links)
La plus grande sensibilité des souris TLR9-/- a révélé le rôle de ce récepteur dans l'infection par Leishmania major. Les cellules dendritiques (DCs) sont activées de manière TLR9-dépendante par l'ADN du L. major et d'autres Trypanosomatidae et non par l'ADN de vertébré. La nature de l'ADN capable d'activer le TLR9 reste controversée quant à la séquence/charpente de l'ADN et l'implication de cofacteurs se liant avec le TLR9 ou l'ADN. Nous avons démontré l'importance de la séquence d'ADN. Contrairement aux génomes de parasites, l'ADN de vertébré présente une contre-sélection des motifs activateurs du TLR9 au profit des motifs inhibiteurs. De plus, l'activation du TLR9 par l'ADN du parasite est augmentée en présence de la protéine HMGB1, qui se fixe mieux sur l'ADN de parasite que de vertébré. La maturation du TLR9 requiert un clivage protéolytique par des protéases endosomales, dont les cathepsines (Cat) B, S, L et l'asparagine endopeptidase (AEP) qui interviennent différemment dans les macrophages et les DCs. Après infection par L. major, nous avons montré que les souris AEP-/-, CatS-/- et CatL-/- ont une pathologie identique aux souris WT, ce qui peut être dû à la redondance de leur fonction. Etonnamment, les souris CatB-/- sont plus résistantes. Leurs lésions et la charge parasitaire dans les ganglions se résolvent plus rapidement, reflétant une réponse immune plus précoce et un contrôle plus rapide de la réaction inflammatoire.En conclusion, ces résultats contribuent à une meilleure compréhension des mécanismes permettant au TLR9 de discriminer entre l'ADN de pathogène et de vertébré et soulèvent le rôle non protecteur de la cathepsine B dans l'infection par L. major. / As TLR9-deficient mice are more sensitive to Leishmania major infection, we have shown previously that TLR9 receptor mediates this parasite infection. Dendritic cells (DCs) are activated by L. major and other Trypanosomatidae DNA and not by vertebrate DNA. There is an ongoing controversy concerning the properties of DNA required for TLR9 activation, regarding the DNA sequence or backbone or the implication of a cofactor interacting with TLR9 or DNA. We have established the importance of DNA sequences. In contrast to parasite genome, vertebrate genome have counter-selected stimulatory sequences and over-represented inhibitory motifs for TLR9. In addition, host proteins contribute to TLR9-dependent DC activation. HMGB1 enhances TLR9 activation only in the presence of L. major DNA and, surprisingly, HMGB1 binds more abundantly L. major than vertebrate DNA. TLR9 activation requires a proteolytic cleavage by endosomal proteases, as cathepsins (Cat) B, S and L and asparagine endopeptidase (AEP) that have a differential activity in macrophages and DCs. After L. major infection, we have showed that AEP-/-, CatS-/- and CatL-/- mice have a similar pathology than WT mice, likely due to their functionnally redundant activites. In contrast, CatB-/- mice are more resistant to the infection. Their lesion sizes and the parasite burdens in lymph nodes are significantly decreased, reflecting an earlier immune response and a more rapid control of the inflammatory response. In conclusion, our results bring further insights into how TLR9 discriminates between Trypanosomatidae and vertebrate DNA and reveal a non protective role of cathepsin B in L. major infection.
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Revisão sistemática do gênero Mourasuchus (Alligatoroidea, Caimaninae), com comentários sobre filogenia, biogeografia e paleoecologia de Caimaninae / Systematic revision of the Mourasuchus genus (Alligatoroidea, Caimaninae), with comments on the phylogeny, biogeography and paleoecology of CaimaninaeCidade, Giovanne Mendes 09 June 2015 (has links)
Mourasuchus (Alligatoroidea, Caimaninae) é um táxon extinto de crocodilianos restrito ao Cenozoico da América do Sul. Representa um dos grupos de crocodilianos mais peculiares de todos os tempos, devido ao formato longo, largo e achatado de seu rostro (lembrando o bico de um pato) entre outras características. Apesar dessas peculiaridades, relativamente poucos trabalhos foram feitos sobre esse grupo. A maioria das descrições morfológicas dos fósseis do gênero são sucintas e breves, incluindo as dos holótipos de duas das quatro espécies a ele assinaladas: M. amazonensis e M. arendsi. Do mesmo modo, as diagnoses das quatro espécies também se mostram sucintas. Poucas também são as análises filogenéticas realizadas com Mourasuchus a maioria das quais, porém, recupera Orthogenysuchus olseni, do Eoceno dos Estados Unidos, como táxon-irmão de Mourasuchus, gerando um impasse biogeográfico. Além disso, a maneira exata pela qual Mourasuchus se alimentava, fazendo uso de seu peculiar rostro bico de pato, bem como seus itens alimentares, ainda não foram plenamente esclarecidos, ainda que algumas teorias tenham sido propostas na literatura. Assim, este trabalho se propôs a: oferecer uma re-descrição dos holótipos de M. amazonensis e M. arendsi; revisar as diagnoses das espécies e do próprio gênero; realizar uma análise filogenética investigando as relações das espécies entre si e do gênero, como um todo, em Caimaninae; investigar a relação de Orthogenysuchus olseni como táxon-irmão de Mourasuchus; elucidar o modo de forrageio e os itens alimentares consumidos pelo grupo. As re-análises das diagnoses das espécies revelaram que duas delas constituem, na verdade, espécies não-válidas, enquanto a análise de um novo material craniano descrito neste trabalho (MCNC-PAL-110-72V) revelou a existência de uma nova espécie de Mourasuchus, fazendo com que este trabalho reconheça três espécies válidas para o gênero. A análise filogenética revelou M. atopus como o táxon mais basal, enquanto M. amazonensis e a nova espécie proposta formam um clado mais derivado. Orthogenysuchus olseni não foi recuperado como táxon-irmão de Mourasuchus em nenhuma das análises, mas seu posicionamento ainda dentro de Caimaninae faz com que impasses biogeográficos permaneçam. Este trabalho defende que Mourasuchus coletava presas em grande quantidade usando a musculatura da parte de baixo de seu rostro uma alimentação coletora enquanto ainda não há evidências de que esses animais fariam uma filtração da massa alimentar coletada. É possível, também, que tal hábito tenha evoluído a partir do hábito alimentar durófago exibido por Caimaninae basais, especialmente o gênero Gnatosuchus. / Mourasuchus (Alligatoroidea, Caimaninae) is an extinct crocodilian taxon restricted to the Cenozoic of South America. It represents one of the most peculiar crocodilian groups of all time, due to the long, wide, flattened shape of its rostrum (resembling the beak of a duck), among other features. Regardless these peculiarities, relatively few works have been done about this group. Most of morphological descriptions of the fossils belonging to this genus are shot and brief, including those of two from the four species assigned to it: M. amazonensis and M. arendsi. Similarly, the diagnoses of all the four species are also very brief. The phylogenetic analyses involving Mourasuchus are also very few most of them, however, recover Orthogenysuchus olseni, from the Eocene of the United States, as Mourasuchus sister-taxon, creating a biogeographically problematic scenario. Furthermore, the exact way by which Mourasuchus feed itself, using its peculiar beak of duck rostrum, as well as its prey items, are yet to be determined, even though some proposals have already been made in the literature. As such, this work had the following objectives: offer a redescription of the holotypes of M. amazonensis and M. arendsi; re-evaluate the diagnoses of Mourasuchus species and the genus itself; perform a phylogenetic analysis to evaluate the relationships between Mourasuchus species and of this group, as a whole, within Caimaninae; evaluate the position of Orthogenysuchus olseni as a sister-taxon of Mourasuchus; elucidate the foraging tactics and the prey items consumed by this group. The re-evaluation of the diagnoses of the species revealed that two are in reality non-valid species, while the analysis of a new cranial material described in this work (MCNC-PAL-110-72V) revealed the existence of a new species of Mourasuchus, taking this work to recognize the existence of three valid species for the genus. The phylogenetic analysis recovered M. atopus as the basalmost taxon, while M. amazonensis and the new species proposed in this work form a more derived clade. Orthogenysuchus olseni was not recovered as a Mourasuchus sister-taxon in any of the analyses made, but its position still within Caimaninae maintains biogeographically problematic scenarios in this clade. This work defends that Mourasuchus collected a high number of prey items using the musculature between the lower jaws a collecting foraging tactic while there is still no evidence that these animals could perform a straining of the entire concentration of food it collected. It is possible, as well, that such habit may have evolved from the durophagous feeding habit of some basal Caimaninae, especially Gnatosuchus.
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A Multimedia Atlas of Dissection for Comparative Anatomy of the VertebratesCurran, Anthony A. 08 1900 (has links)
Traditional methods of teaching the laboratory course for Comparative Anatomy of the Vertebrates could be improved by applying current computer technology to construct an interactive, multimedial atlas of dissection. Five specimens used in comparative anatomy courses at most institutions were chosen as representative members of the Phylum Chordata: amphioxus, lamprey, dogfish shark, mud puppy, and cat. Specimens were dissected according to the modified method of Wischnitzer, 1993, and each stage was photographed with a Kodak DC120 digital zoom camera. These images were processed on a Power Macintosh 7600 computer with Adobe Photoshop v. 5.0. The atlas was constructed from these images using Macromedia Authorware v. 4.0.3. Each image contains a series of interactive objects that display a highlight and descriptive text as the cursor passes over each object.
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From pup to predator : ontogeny of foraging behaviour in grey seal (Halichoerus grypus) pupsCarter, Matt January 2018 (has links)
For young animals, surviving the first year of nutritional independence requires rapid development of effective foraging behaviour before the onset of terminal starvation. Grey seal (Halichoerus grypus) pups are abandoned on the natal colony after a brief (15-21 days) suckling period and must learn to dive and forage without parental instruction. Regional and sex-specific differences in diet and foraging behaviour have been described for adults and juveniles, but the early-life behaviour of pups during the critical first months at sea remains poorly understood. This thesis investigates sources of intrinsic and extrinsic variation in the development of foraging behaviour and resource selection in grey seal pups. The studies presented here feature tracking and dive data collected from 52 recently-weaned pups, tagged at six different breeding colonies in two geographically-distinct regions of the United Kingdom (UK). Original aspects of this thesis include: (Chapter I) a comprehensive review of analytical methods for inferring foraging behaviour from tracking and dive data in pinnipeds; (Chapter II) description and comparison of regional and sex differences in movements and diving characteristics of recently-weaned pups during their first trips at sea; (Chapter III) implementation of a novel generalized hidden Markov modelling (HMM) technique to investigate the development of foraging movement patterns whilst accounting for sources of intrinsic (age, sex) and extrinsic (regional) variation; and (Chapter IV) the first analysis of grey seal pup foraging habitat preference, incorporating behavioural inferences from HMMs and investigating changes in preference through time.
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Roles for activator protein 2 (AP-2) transcription factors in zebrafish neural crest developmentLi, Wei 01 January 2008 (has links)
Neural crest is a vertebrate-specific population of embryonic precursor cells thought to have been essential in vertebrate evolution. During development, a group of naïve ectoderm cells are induced to become neural crest and then undergo series of developmental events to give rise to diverse derivatives. Failure of these events often leads to malfunction of neural crest derived tissues and organs. This thesis focuses on the genetic regulation of two events during neural crest development, induction and differentiation.
Neural crest induction refers to the specification of ectoderm cells to the neural crest lineage. It is believed that combinatorial activity of transcription factors governs neural crest induction, but the function of specific transcription factors in this process are not yet clear. The AP-2 family of transcription factors is implicated in control of neural crest development, but whether there is a cell autonomous role of AP-2 transcription factors in neural crest induction has remained uncertain. Here I show that in zebrafish, two AP-2 family members, Tfap2a and Tfap2c, are required redundantly for neural crest induction, and that this requirement is cell autonomous. Failure of neural crest induction in the zebrafish embryos that are devoid of Tfap2a and Tfap2c is not caused by defects in cell survival or cell proliferation, but rather appears to result from a failure neural crest cell fate specification. Simultaneous knockdown of Tfap2a and Tfap2c is one of the only known genetic manipulations that result in failure of neural crest induction. Thus the Tfap2a/c double knockdown embryos will be useful for further studies on the emergence of neural crest during both development and evolution.
The second section of my thesis concerns differentiation of neural crest derived zebrafish melanophores. This study reveals that Tfap2a and another AP-2 family member, Tfap2e, redundantly and autonomously regulate melanophore differentiation. This is the first report on the function of Tfap2e in any animal. Given that the expression of AP-2 transcription factors is tightly associated with the metastasis potential of human melanoma, my study reinforces the view that cancer cells co-opt regulatory pathways employed in embryonic development.
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Predator Behavior and Prey Demography in Patchy HabitatsHalstead, Brian J 28 March 2008 (has links)
Habitat loss and fragmentation are among the greatest threats to biodiversity, and these threats can be exacerbated or alleviated by the presence of interacting species. The effect of habitat loss and fragmentation on predator-prey systems has received extensive theoretical attention, but empirical studies of these systems yield few clear patterns. I examined the influence of prey abundance and spatial distribution on the foraging ecology and spatial ecology of Masticophis flagellum (Coachwhip) using capture-mark-recapture and radio telemetry techniques. I also examined the influence of saurophagous snake abundance on the survival rate of Sceloporus woodi (Florida Scrub Lizard) populations. Masticophis flagellum positively selected lizard and mammal prey, but within these categories it consumed prey species in proportion to their availability. Masticophis flagellum was vagile and constrained its movements within large home ranges. At all spatial scales examined, M. flagellum strongly selected Florida scrub habitat and avoided wetland habitats. The negative effect of saurophagous snake abundance best explained differences in S. woodi survival rates among patches of Florida scrub. Further loss and fragmentation of Florida scrub habitat will likely have a strong negative impact upon M. flagellum. Because it is precinctive to Florida scrub, Sceloporus woodi will also be negatively affected by the loss of this unique habitat. The potential positive effects of reduced predation pressure from M. flagellum that may accompany loss and fragmentation of Florida scrub is likely to be offset by increased predation rates by habitat and dietary generalist predators that incidentally prey upon S. woodi. Despite the sensitivity of these species to loss and fragmentation of Florida scrub, the prognosis is good for both M. flagellum and S. woodi on relatively large protected sites containing xeric habitats managed with prescribed fire.
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Haptoglobin: Biosynthesis and EvolutionWicher, Krzysztof B. January 2006 (has links)
<p>Haptoglobin (Hp) is a serum protein known for its ability to form a tight complex with hemoglobin (Hb) and thereby inhibiting the oxidative activity of Hb. </p><p>Mammalian Hp is synthesized as a precursor (proHp) that undergoes proteolytic cleavage by a previously unidentified enzyme in the endoplasmic reticulum (ER). In this study, a proHp-cleaving enzyme was isolated from human serum and identified as complement C1r-like protein (C1rLP). Co-expression of C1rLP with proHp in mammalian cells resulted in cleavage of the latter protein in the ER. Mutation of either the active site serine residue in C1rLP or the arginine residue in the cleavage site of Hp abolished the cleavage of proHp by C1rLP. RNAi studies in mammalian cells identified the proHp-cleaving enzyme as C1rLP.</p><p>Hp has been found in all mammals studied to date but its presence in non-mammalian species has not been unambiguously shown. By searching currently available genomic DNA and cDNA sequence databases, a gene orthologous to mammalian <i>Hp</i> was found in bony fish. Hp-like protein expressed from this gene was demonstrated to be a major Hb protein in fish serum. Surprisingly, no Hp-like gene was found in the genomes of either frog or chicken. In chicken, a protein previously described as Hp was identified as PIT54, a member of a scavenger receptor cysteine-rich family of proteins. Interestingly, ostrich serum seemed to contain two Hb-binding proteins; one similar to PIT54 and one to mammalian Hp. We are not aware of any other case where the function of one gene has been taken over by another, completely unrelated gene</p><p>Fish Hp (fHp) is composed of a serine proteinase-related domain preceded by an extension consisting of several aminoa acids and a signal peptide. The extension contains a consensus motif for cleavage by subtilisin-like proprotein convertases (SPCs). fHp was found to be cleaved by SPCs in the Golgi complex.</p><p>Collectively, this thesis presents evidence that Hp has undergone significant changes during evolution with respect to its molecular organization and to the mechanism of its proteolytic cleavage.</p>
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Bioinformatic Analysis of Mutation and Selection in the Vertebrate Non-coding GenomeBrandström, Mikael January 2007 (has links)
<p>The majority of the vertebrate genome sequence is not coding for proteins. In recent years, the evolution of this noncoding fraction of the genome has gained interest. These studies have been greatly facilitated by the availability of full genome sequences. The aim of this thesis is to study evolution of the noncoding vertebrate genome through bioinformatic analysis of large-scale genomic datasets.</p><p>In a first analysis we addressed the use of conservation of sequence between highly diverged genomes to infer function. We provided evidence for a turnover of the patterns of negative selection. Hence, measures of constraint based on comparisons of diverged genomes might underestimate the functional proportion of the genome.</p><p>In the following analyses we focused on length variation as found in small-scale insertion and deletion (indel) polymorphisms and microsatellites. For indels in chicken, replication slippage is a likely mutation mechanism, as a large proportion of the indels are parts of tandem-duplicates. Using a set of microsatellite polymorphisms in chicken, where we avoid ascertainment bias, we showed that polymorphism is positively correlated with microsatellite length and AT-content. Furthermore, interruptions in the microsatellite sequence decrease the levels of polymorphism.</p><p>We also analysed the association between microsatellite polymorphism and recombination in the human genome. Here we found increased levels of microsatellite polymorphism in human recombination hotspots and also similar increases in the frequencies of single nucleotide polymorphisms (SNPs) and indels. This points towards natural selection shaping the levels of variation. Alternatively, recombination is mutagenic for all three kinds of polymorphisms. </p><p>Finally, I present the program ILAPlot. It is a tool for visualisation, exploration and data extraction based on BLAST.</p><p>Our combined results highlight the intricate connections between evolutionary phenomena. It also emphasises the importance of length variability in genome evolution, as well as the gradual difference between indels and microsatellites.</p>
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Haptoglobin: Biosynthesis and EvolutionWicher, Krzysztof B. January 2006 (has links)
Haptoglobin (Hp) is a serum protein known for its ability to form a tight complex with hemoglobin (Hb) and thereby inhibiting the oxidative activity of Hb. Mammalian Hp is synthesized as a precursor (proHp) that undergoes proteolytic cleavage by a previously unidentified enzyme in the endoplasmic reticulum (ER). In this study, a proHp-cleaving enzyme was isolated from human serum and identified as complement C1r-like protein (C1rLP). Co-expression of C1rLP with proHp in mammalian cells resulted in cleavage of the latter protein in the ER. Mutation of either the active site serine residue in C1rLP or the arginine residue in the cleavage site of Hp abolished the cleavage of proHp by C1rLP. RNAi studies in mammalian cells identified the proHp-cleaving enzyme as C1rLP. Hp has been found in all mammals studied to date but its presence in non-mammalian species has not been unambiguously shown. By searching currently available genomic DNA and cDNA sequence databases, a gene orthologous to mammalian Hp was found in bony fish. Hp-like protein expressed from this gene was demonstrated to be a major Hb protein in fish serum. Surprisingly, no Hp-like gene was found in the genomes of either frog or chicken. In chicken, a protein previously described as Hp was identified as PIT54, a member of a scavenger receptor cysteine-rich family of proteins. Interestingly, ostrich serum seemed to contain two Hb-binding proteins; one similar to PIT54 and one to mammalian Hp. We are not aware of any other case where the function of one gene has been taken over by another, completely unrelated gene Fish Hp (fHp) is composed of a serine proteinase-related domain preceded by an extension consisting of several aminoa acids and a signal peptide. The extension contains a consensus motif for cleavage by subtilisin-like proprotein convertases (SPCs). fHp was found to be cleaved by SPCs in the Golgi complex. Collectively, this thesis presents evidence that Hp has undergone significant changes during evolution with respect to its molecular organization and to the mechanism of its proteolytic cleavage.
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