<p>Abstract</p><p>Gaining accurate population information is crucial for the conservation and management of species. The National Monitoring Program for Large Carnivores monitors the Swedish lynx population (species Lynx lynx) by surveying family groups, non-invasive sampling and genetic analysis. Ten microsatellite regions were used as genetic markers to retrieve unique individual genotypes, through polymerase chain reactions (PCR) with specific primer-pairs and capillary-electrophoresis. Complete genotypes were matched using an internal database. The aim of this degree project was to show how monitoring of lynx through genetic analysis is carried out at the Department of Evolutionary Biology at Uppsala University, and to evaluate how effective these methods are and how they might be improved.</p><p>Even though most of the methods used were fairly robust and reproducible, non-invasive sampling and microsatellite analysis posed some problems regarding DNA quality and quantity, and increased the risks of certain genotyping errors. These risks might be worth taking though, as genetic analysis, in combination with field observations, gives a more comprehensive picture of the Swedish lynx population.</p>

Abstract Gaining accurate population information is crucial for the conservation and management of species. The National Monitoring Program for Large Carnivores monitors the Swedish lynx population (species Lynx lynx) by surveying family groups, non-invasive sampling and genetic analysis. Ten microsatellite regions were used as genetic markers to retrieve unique individual genotypes, through polymerase chain reactions (PCR) with specific primer-pairs and capillary-electrophoresis. Complete genotypes were matched using an internal database. The aim of this degree project was to show how monitoring of lynx through genetic analysis is carried out at the Department of Evolutionary Biology at Uppsala University, and to evaluate how effective these methods are and how they might be improved. Even though most of the methods used were fairly robust and reproducible, non-invasive sampling and microsatellite analysis posed some problems regarding DNA quality and quantity, and increased the risks of certain genotyping errors. These risks might be worth taking though, as genetic analysis, in combination with field observations, gives a more comprehensive picture of the Swedish lynx population.

Submitted by Ronildo Prado (ronisp@ufscar.br) on 2016-09-20T14:07:22Z No. of bitstreams: 1 TeseASMCS.pdf: 3312400 bytes, checksum: c898700835e5f7333425c663a26a29d4 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-09-20T14:07:44Z (GMT) No. of bitstreams: 1 TeseASMCS.pdf: 3312400 bytes, checksum: c898700835e5f7333425c663a26a29d4 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-09-20T14:07:55Z (GMT) No. of bitstreams: 1 TeseASMCS.pdf: 3312400 bytes, checksum: c898700835e5f7333425c663a26a29d4 (MD5) / Made available in DSpace on 2016-09-20T14:08:05Z (GMT). No. of bitstreams: 1 TeseASMCS.pdf: 3312400 bytes, checksum: c898700835e5f7333425c663a26a29d4 (MD5) Previous issue date: 2015-08-14 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The intense destruction of the environment contributed to the decline and isolation of wild populations, providing an intensification of genetic drift and inbreeding effects. These factors reduce the ability of individuals to adapt to environmental changes, making them more vulnerable to extinction. The two largest predators of the Americas, the cougar (Puma concolor) and jaguar (Panthera onca) are animals which are threatened by the reduction and fragmentation of habitats, especially in the Atlantic Forest, which is one of the most degraded biomes in the world due to human actions. The present study aimed to investigate both demographic parameters and the distribution of the genetic diversity of cougar and jaguar populations within Atlantic Forest remnants. The chosen areas (Serra da Mantiqueira, Serra do Mar continuous and Iguaçu National Park) are among the most important for the conservation of these cats. Mostly non-invasive samples (feces and hair) were collected in protected areas present in those remaining. The depositor species was confirmed by amplification of the ATP6 gene and the samples were individualized using microsatellite loci, which were also employed in population analyses. The sex of the individuals was determined using a small fragment of amelogenin gene. The results suggest that at least seven individuals of jaguars (4F:3M) inhabit the Iguaçu National Park and 12 (5F:7M) are present in the Serra do Mar continuous. These populations seem to be different, with evidence of low gene flow between them (lack of migrant and mixed individuals and pairs of highly related individuals). In Iguaçu is also estimated to exist at least seven cougars (3F:4M), also four (1F:3M) in the Serra da Mantiqueira and 14 (5F:9M) in the Serra do Mar continuous. Genetic structure was detected in this species, but evidencing gene flow maintenance between two detected populations (sign of migrants and mixed individuals and predominantly non related individuals). In both species high genetic diversity could be observed. This study obtained critical information and still unknown, about the demographic and structure of jaguar and cougar populations in the Atlantic Forest remain. These data will provide substantial information that can be used in monitoring, as well as being crucial and decisive in the increase of effective strategies for the conservation of these species. / A intensa devastação no ambiente vem contribuindo significativamente para o declínio e isolamento de populações selvagens, proporcionando uma intensificação dos efeitos da deriva genética e na taxa de endogamia. Estes fatores, por sua vez, reduzem a habilidade dos indivíduos se adaptarem às mudanças ambientais, tornando-os vulneráveis à extinção. Os maiores predadores das Américas, a onça-parda (Puma concolor) e a onça-pintada (Panthera onca) estão entre os animais ameaçados pela redução e fragmentação dos habitats, principalmente na Mata Atlântica, que é um dos biomas mundiais mais antropizados. Assim, o presente trabalho teve como objetivo investigar parâmetros demográficos e a distribuição da diversidade genética de populações de onças presentes em remanescentes de Mata Atlântica. Os fragmentos elegidos (Serra da Mantiqueira, contínuo da Serra do Mar e Parque Nacional do Iguaçu) estão entre os mais importantes para a conservação desses felinos. Amostras predominantemente não invasivas (fezes e pelos) foram coletadas em Unidades de Conservação presentes nesses remanescentes. A espécie depositora das fezes foi confirmada através da amplificação do gene ATP6 e as amostras foram individualizadas por meio de locos de microssatélites, os quais também foram empregados nas análises populacionais. O sexo dos indivíduos foi determinado por meio de um fragmento do gene da amelogenina. Os resultados indicaram que ao menos sete indivíduos de onças-pintadas (4F:3M) habitam o Parque Nacional do Iguaçu e 12 (5F:7M) estão presentes no contínuo da Serra do Mar. Essas populações parecem estar diferenciadas, com evidência de baixo fluxo gênico entre elas (ausência de indivíduos migrantes e misturados, além de pares de indivíduos altamente relacionados). No Iguaçu também foi estimado existir pelo menos sete onças-pardas (3F:4M), além de quatro (1F:3M) na Serra da Mantiqueira e 14 (5F:9M) no contínuo da Serra do Mar. Estruturação genética foi detectada nessa espécie, entretanto, com indícios de fluxo gênico entre as duas populações detectadas (evidência de indivíduos migrantes e misturados, além de pares de indivíduos predominantemente não relacionados). Em ambas as espécies foram exibidos altos níveis de diversidade genética. Este estudo gerou informações primordiais, ainda desconhecidas, sobre a demografia e a estruturação de populações de onças na Mata Atlântica. Tais dados poderão ser utilizados em monitoramentos, além de serem cruciais e decisivos no incremento de estratégias efetivas para a conservação dessas espécies.

Hair follicle outer root sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells.

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Im ersten Teil der Arbeit wurden Strategien zur Analyse von Transkripten erarbeitet. Die ersten Versuche zielten darauf ab, in mit Glaskapillaren genommenen Einzelzellproben verschiedener Gewebeschichten RT-PCR durchzuführen, um spezifische Transkripte nachweisen zu können. Dies gelang für eine Reihe von Genen aus verschiedenen Pflanzenspezies. Dabei konnten sowohl Transkripte stark wie auch schwach exprimierter Gene nachgewiesen werden. <br /> Für die Erstellung von Gewebe-spezifischen Expressionsprofilen war es notwendig, die in vereinigten Zellproben enthaltene mRNA zunächst zu amplifizieren, um eine ausreichende Menge für Arrayhybridisierungen zu erhalten. Vor der Vermehrung wurde die mRNA revers transkribiert. Es wurden daran anschließend verschiedene Amplifikationsstrategien getestet: Die neben Tailing, Adapterligation und anderen PCR-basierenden Protokollen getestete Arbitrary-PCR hat sich in dieser Arbeit als einfache und einzige Methode herausgestellt, die mit so geringen cDNA-Mengen reproduzierbar arbeitet. Durch Gewebe-spezifische Array-hybridisierungen mit der so amplifizierten RNA konnten schon bekannte Expressionsmuster verschiedener Gene, vornehmlich solcher, die an der Photosynthese beteiligt sind, beobachtet werden. Es wurden aber auch eine ganze Reihe neuer offensichtlich Gewebe-spezifisch exprimierter Gene gefunden. Exemplarisch für die differentiell exprimierten Gene konnte das durch Arrayhybridisierungen gefundene Expressionsmuster der kleinen Untereinheit von Rubisco verifiziert werden. Hierzu wurden Methoden zum Gewebe-spezifischen Northernblot sowie semiquantitativer und Echtzeit-Einzelzell-RT-PCR entwickelt.<br /> Im zweiten Teil der Arbeit wurden Methoden zur Analyse von Metaboliten einschließlich anorganischer Ionen verwendet. Es stellte sich heraus, daß die multiparallele Methode der Gaschromatographie-Massenspektrometrie keine geeignete Methode für die Analyse selbst vieler vereinigter Zellinhalte ist. Daher wurde auf Kapillarelektrophorese zurückgegriffen. Eine Methode, die mit sehr kleinen Probenvolumina auskommt, eine hohe Trennung erzielt und zudem extrem geringe Detektionslimits besitzt. Die Analyse von Kohlenhydraten und Anionen erfordert eine weitere Optimierung. Über UV-Detektion konnte die K+-Konzentration in verschiedenen Geweben von A. thaliana bestimmt werden. Sie lag in Epidermis und Mesophyll mit ca. 25 mM unterhalb der für andere Pflanzenspezies (Solanum tuberosum und Hordeum vulgare) publizierten Konzentration. Weiter konnte gezeigt werden, daß zwölf freie Aminosäuren mittels einer auf Kapillarelektrophorese basierenden Methode in vereinigten Zellproben von Cucurbita maxima identifiziert werden konnten. Die Übertragung der Methode auf A. thaliana-Proben muß jedoch weiter optimiert werden, da die Sensitivität selbst bei Laser induzierter Fluoreszenz-Detektion nicht ausreichte.<br /> Im dritten und letzten Teil der Arbeit wurde eine Methode entwickelt, die die Analyse bekannter wie unbekannter Proteine in Gewebe-spezifischen Proben ermöglicht. Hierzu wurde zur Probennahme mittels mechanischer Mikrodissektion eine alternative Methode zur Laser Capture Microdissection verwendet, um aus eingebetteten Gewebeschnitten distinkte Bereiche herauszuschneiden und somit homogenes Gewebe anzureichern. Aus diesem konnten die Proteine extrahiert und über Polyacrylamidgelelektrophorese separariert werden. Banden konnten ausgeschnitten, tryptisch verdaut und massenspektrometrisch die Primärsequenz der Peptidfragmente bestimmt werden. So konnten als Hauptproteine im Mesophyll die große Untereinheit von Rubisco sowie ein Chlorophyll bindendes Protein gefunden werden.<br /> Die in dieser Arbeit entwickelten und auf die Modellpflanze Arabidopsis thaliana angewandten Einzelzellanalysetechniken erlauben es in Zukunft, physiologische Prozesse besser sowohl räumlich als auch zeitlich aufzulösen. Dies wird zu einem detaillierteren Verständnis mannigfaltiger Vorgänge wie Zell-Zell-Kommunikation, Signalweiterleitung oder Pflanzen-Pathogen-Interaktionen führen. / The subject of this thesis was the analysis of single plant cells in respect to their contents of i) transcripts, ii) inorganic cations and anions, iii) metabolites like amino acids and carbohydrates as well as iv) proteins. One task was the transfer of existing methods to single cell analysis on leaf tissues of the model plant Arabisopsis thaliana L., the second one was the refinement and the development, respectively, of new protocols for the analysis of such picoliter samples. For cell type specific sampling two different complimentary methods were applied: Using micro glass capillaries specific single cell contents could be harvested from intact plants, whereas typical sample volumes were in the picoliter range. Even the sampling of inner cell types such as companion cells could be demonstrated. Using mechanical micro dissection of embedded tissue a larger amount of homogenous tissue could be collected.<br /> Because single cell samples contain only femtogram amounts of mRNA, direct detection of transcripts is impossible. Therefore, two amplification protocols were applied to the cell samples: The first procedure makes use of specifically primed RT-PCR for amplification. Several genes derived from different plants and tissues could be detected after successful RT-PCR, including high as well as low expressed genes. The second method was developed to monitor the activity of many genes in parallel using array hybridisation with filters containing the cDNA of as many as 16.000 ESTs. For this purpose, unspecific RT-PCR as it is applied in the differential display was used to amplify different transcripts in just one reaction. However, in these tissue specific array hybridisations the expression patterns of several hundreds genes could be monitored. These included known tissue specific expression patterns (of mainly photosynthesis related genes) as well as a couple of unknown expression patterns. To verify the tissue specificity of gene activity some results were reconsidered using tissue specific northern blot hybridisations and real time RT-PCR, respectively. <br /> Secondly, metabolites (including inorganic ions) were investigated: Because gas chromatography-mass spectrometry does not reveal the sensitivity which in necessary for the analysis of even multiple pooled single cell samples capillary electrophoresis was applied for these studies. This method has a high potential as it needs only small amounts of starting material, has uncomparable low detection limits and exhibits a high number of theoretical plates.<br /> The analysis of inorganic anions and carbohydrates needs further optimisations. Using UV absorption-detection potassium could be detected in different cell types whereas the concentrations in mesophyll and epidermis were found around 25 mM each. These concentrations are lower than in other species as Solanum tuberosum or Hordeum vulgare. For investigations of amino acids the cell samples were derivatized to make the use of laser induced fluorescence-detection capable. In samples derived from pumpkin (Cucurbita maxima) mesophyll twelve amino acids could be detected and identified. The transfer of this method to A. thaliana derived samples exhibited no results which may be due to the low concentration of free amino acids in these plants.<br /> Finally, a method was developed with which the existence of known and unknown proteins in tissue specific samples could be monitored. For this, mechanical micro dissection was used to: After embedding and sectioning the tissue of interest was cut out by an vibrating steel chisel to get homogenous samples. The proteins contained in these tissue pieces were extracted and separated by one dimensional SDS polyacrylamid gel electrophoresis. Several protein bands could be detected after staining with either silver or coomassie blue stain. These bands were cut out and sequenced by mass spectrometry. The large subunit of rubisco as well as one chlorophyll binding protein could be identified as the major proteins within the mesophyll.<br /> The single cell analysis methods which were developed and applied to the model plant A. thaliana in this thesis allow a better spatial as well as temporal resolution of analysis. This will lead to a more detailed understanding of physiological processes like cell to cell communication, signalling or plant-pathogen interactions.

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Black grouse (Tetrao tetrix) is a bird species with a lek mating system found in the Palearctic boreal taiga. It is assumed that it has a continuous distribution along Scandinavia and Siberia, whereas in Central Europe it has declined during the last decades. The primary objective of this thesis was to obtain a deeper understanding of the history, systematic classification and the genetic structure of black grouse on different geographical scales using microsatellites and control region mtDNA sequences (CR). I determined how much the mating system, habitat fragmentation and historical population processes have influenced the partitioning of genetic diversity in this species. Phylogeographical results are consistent with a demographic population expansion, and the patterns of postglacial dispersal suggest that a glacial refugium was located somewhere in central Asia, and from there black grouse spread out to Europe following the retreat of glacial ice sheets. I suggest that the two European black grouse subspecies, T. t. Tetrix and T. t. britannicus correspond to only one subspecies: T. t. tetrix, and that this lineage has diverged from T.t. viridanus, a subspecies found in Kazakhstan. The British population is significantly divergent from the remaining Eurasian samples for microsatellites but it is not for mtDNA. Therefore, they should regard as a separate Management Unit and not as a subspecies. Furthermore, British black grouse occur in three independent genetic units, corresponding to Wales, northern England/southern Scotland and northern Scotland. There was also genetic structure within Sweden. Habitat fragmentation is the main cause of population genetic structure in southern Swedish black grouse. In contrast, low levels of genetic differentiation and high connectivity were found in northern Sweden due to female-biased dispersal. On a finer geographical scale, I found genetic differences between leks due to a mixture of related and unrelated individuals within leks. However, mean relatedness values hardly differed from zero. Some leks were similar to one another and I interpret this as a result of variation in local reproductive success and philopatry. These factors would cause genetic structuring but this by itself would not reveal that kin selection is operating within black grouse leks.

Avec la mise en place des politiques de conservation des espèces sauvages, l'extension de l'urbanisation et l'accroissement des populations humaines, le contact homme-faune a considérablement augmenté au cours de ces dernières décennies. Par conséquent, le nombre de maladies d'origines zoonotiques a explosé avec six apparitions d'agents infectieux par an, dont 75% sont susceptibles d’être transmises par un vecteur. La plupart de ces maladies n'ayant pas encore de vaccins, les principales méthodes d'évitement sont basées sur les stratégies de lutte anti-vectorielle adaptées à l'écologie et au comportement alimentaire des vecteurs. Au Gabon, particulièrement dans les parcs nationaux, nous avons identifié six espèces de glossines (Glossina palpalis palpalis, G. fuscipes fuscipes, G. fusca congolense, G. pallicera newsteadi, G. caliginea et G tabaniformis) vivant principalement en milieux forestiers, six espèces de stomoxes (Stomoxys calcitrans, S. inornatus, S. niger niger, S. niger bilineatus, S. omega omega et S. transvittatus) inféodées aux milieux ouverts types forêt secondaire, savane et villages. Nous avons également identifié six espèces de tabanides (Ancala sp., Atylotus sp., Chrysops sp., Haematopota sp., Tabanus par et T. taeniola), mais leur distribution n'était pas claire dans les milieux prospectés. Par ailleurs, nous constatons que ces mouches hématophages ont un régime alimentaire très diversifié, comprenant les mammifères terrestres et aquatiques, les reptiles et les oiseaux. Elles se nourrissent à 86% sur la faune, contre seulement 14% sur l'homme. Cependant, dans les milieux anthropisés les repas sanguins d'origine humaine sont très importants, notamment dans les villages (100%) et autour des camps de recherche implantés dans les parcs (24%). Ainsi en l'absence de faune dans le milieu, ces mouches hématophages se nourrissent sur l'homme. Comme 75% des maladies émergentes chez l'homme proviennent de la faune sauvage et que près de ¾ d'entre elles circulent via le sang, elles sont donc susceptibles d’être détectées dans les repas sanguins de mouches hématophages. Cette technique d'échantillonnage non-invasif de la faune sauvage semble être un bon moyen d'identifier les agents infectieux à ADN (plasmodiums et trypanosomes), mais reste encore imprécise pour les agents infectieux à ARN (arbovirus). / The contact between human and wild fauna has considerably increased during these last decades due to the increase of human population size but also to conservation policies. As a consequence, the number of zoonotic diseases soared with a mean of six new infectious diseases per year, 75% of whom being vectorially transmitted. The way to avoid the human contamination by these emergent diseases is based on the efficient vector control resulting from a deep knowledge of the ecology and the feeding behavior of the different vector species. During our work, we have identified and characterized the ecology of 6 tsetse species (Glossina palpalis palpalis, G. fuscipes fuscipes, G. fusca congolense, G. pallicera newsteadi, G. caliginea and G. tabaniformis) that live in forests and 6 stomoxe species (Stomoxys calcitrans, S. inornatus, S. niger niger, S. niger bilineatus, S. omega omega and S. transvittatus) that live in and around (anthropized places) conservation areas. We have also identified 6 tabanid species (Ancala sp., Atylotus sp., Chrysops sp., Haematopota sp., Tabanus par and T. taeniola). The feeding ecology of the tsetse species have been studied through the determination of host extracted from blood meals in the insect caught with molecular techniques. These hematophagous insects had a diversified diet that was constituted of diverse mammal species but also reptiles and birds. The food intake results mostly from wild fauna (86%) and more rarely from humans (14%). However, in anthropised habitats (villages and research’s camps within the parks), the blood intakes from human origin were important, in particular in the villages (100%), suggesting that without wild fauna the flies shift on human host. In the last part of our work, we tried to identify pathogens in the blood samples extracted from the tsetse species in order to test whether these species could be used as living sampling syringe of the wild fauna. This new proposed non-invasive sampling techniques allowed to detect the DNA of various infectious agents (plasmodiums and trypanosomes), but failed to detect the RNA of viruses (arbovirus) suggesting that this approach could be useful but need to be improved.