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The area of stem cell research is rapidly evolving. One of the recent achievements is the capture of naïve human embryonic stem cells through reprogramming of primed human embryonic stem cells. In this thesis, Gene Co-expression Networks are used to further our understanding of naïve and primed human embryonic (hESCs). We found that GCNs of naïve and primed hESCs exhibits distinct network topological structures. Rewiring analysis of naïve and primed hESC GCNs showed significant rewiring and change in networks structures and behaviors. This demonstrates that naïve and primed hESCs are distinct cellular states. In addition, KLF genes circuitry, NANOG, and SOX2 were more active in naïve GCNs and formed more edges with other genes. Those genes were significantly rewired in our GCNs. We found that KLF5 is major player in our naïve hESCs GCNs. In addition, NANOG and SOX2 interacted only in naïve hESCs GCNs..The observations in our GCNs concerning KLF circuitry activity and, NANOG and SOX2 interactions were consistent with published stem cell literature. This demonstrates the power of GCNs in unfolding cellular characteristics and understanding the underlying gene dynamics.

Deux états d'autorenouvellement des cellules souches pluripotentes (PSCs) ont été définis, à savoir les états naïf et amorcé. De nombreuses différences existent entre ces deux états dont la plus marquante est la capacité unique des PSCs à l'état naïf, de coloniser l'embryon préimplantatoire et former des chimères. L'objectif de mon projet doctoral a été d'étudier la pluripotence chez le lapin. Dans ce cadre, j'ai d'abord entrepris de fabriquer et de caractériser des cellules souches pluripotentes induites (RbiPSCs), puis d'évaluer leur capacité à coloniser l'embryon et à former des chimères. Trois lignées de RbiPSCs dépendantes du FGF2 ont été obtenues par reprogrammation de fibroblastes de lapin. Leur caractérisation moléculaire et fonctionnelle a révélé des caractéristiques mixtes, naïves et amorcées. En revanche, sur le plan fonctionnel, elles sont incapables de coloniser l'embryon de lapin, une caractéristique de la pluripotence amorcée. La seconde partie de mon projet doctoral a consisté à reprogrammer des RbiPSCs vers l'état naïf. Dans ce but, j'ai surexprimé KLF2 et KLF4, deux gènes appartenant au réseau de pluripotence naïf, et utilisé les conditions de culture des PSCs de souris. Les cellules ainsi obtenues présentent un profil d'expression génique plus proche de celui de l'ICM de lapin, dû notamment à la réactivation de marqueurs spécifiques de la pluripotence naïve. Enfin, les cellules ainsi reprogrammées présentent une capacité accrue pour la colonisation de l'embryon préimplantatoire de lapin. Mes travaux constituent le premier exemple de reprogrammation de cellules souches pluripotentes vers l'état naïf chez le lapin. Les cellules ainsi produites ouvrent la voie à la fabrication de chimères somatiques et germinales / Pluripotent stem cells (PSCs) can self-renew at two distinct states, the naive and primed states. Many differences exist between these two states, the most striking is the unique ability of PSCs naïve to colonize the preimplantation embryo and form chimeras. The purpose of my doctoral project was to study pluripotency in rabbits. In this context, I initially manufactured and characterized induced pluripotent stem cells (RbiPSCs) and then evaluated their ability to colonize the embryo and form chimeras. Three RbiPSCs lines were obtained by rabbit fibroblasts reprogramming. Their molecular characterization revealed mixed characteristics, naïve and primed. However, functionally, they are unable to colonize the rabbit embryo, a feature of primed pluripotency. The second part of my doctoral project was to reprogram RbiPSCs to the naïve state. To this end, I have overexpressed Klf2 and Klf4, two genes belonging to the naïve pluripotency network and the mouse PSCs culture conditions. These new cell lines have a gene expression profile closer to that of the rabbit ICM, particularly due to the reactivation of specific markers of naïve pluripotency. Finally, the reverted cells have an increased capacity of colonization of the preimplantation embryo rabbit. My work represents the first example of pluripotent stem cells reprogramming toward the naive state in rabbits. The cells thus produced pave the way for the production of somatic and germline chimeras

Two hundred and forty three fungal isolates were obtained from roots of four Woollsia pungens (Cav.) F. Muell plants collected from a field site in New South Wales, Australia. 175 sterile isolates were slow growing and dark-coloured on 2 percentage malt agar and were selected for further analysis. Microsatellite-primed PCR using the primers (GACA)4 and (GTG)5 separated these isolates into 50 genets. Isolates representative of 43 genets (including 168 isolates in total) formed typical ericoid mycorrhizal structures when inoculated onto roots of Vaccinium macrocarpon Ait (Ericaceae), confirming their status as mycorrhizal endophytes. It was estimated that a minimum of 43 genetically-distinct mycorrhizal mycelial genets were present in the root systems of the sampled W. pungens population with 7 to 15 distinct endophytic genets identified in each host plant, indicating that considerable genetic diversity exists within the endophyte population. While most genets were represented by less than 8 isolates, 3 genets contained up to 41 isolates, suggesting that root system colonisation by some mycelia may be extensive. While most fungal genets were shown to be confined to individual plants, 2 genets (genets 32 and 33), however, were present within the root systems of 2 adjacent plants (plants C and D), suggesting that the two root systems were interconnected by the endophyte mycelia. The ITS region of 13 mycorrhizal endophytes and a non-mycorrhizal isolate selected from the endophyte population were sequenced and compared to the sequence of Hymenoscyphus ericae as well as sequences from the GenBank database. A phylogenetic tree was generated from the nucleotide sequence data. This analysis revealed that 6 putative taxa were present in the root systems of 4 host plants. No isolates were positively identified to genus or species level. Closest matches with fungal sequences in the database indicated that most isolates probably belonged to the order Leotiales. Cluster analysis on the basis of the ITS sequences indicated that H. ericae was not clustered together with any endophytes from W. pungens, suggesting that endophytes of W. pungens are not identical to the known ericoid mycorrhizal fungus H. ericae. H. ericae had a low degree of sequence similarity with isolates from W. pungens, with similarities ranging from 68.3-80.6%. Cluster analysis based on DNA sequences of the ITS region did not fully support the groupings inferred from microsatellite-based fingerprinting. / Master of Science (Hons)

Den här studien handlar om hur svenska officerare fattar sina beslut i dynamiska situationer, det vill säga situationer som bland annat präglas av stress, oklarheter och tidspress. Även frågor kring vad som bygger förmågan till att fatta dylika beslut och hur till exempel gruppen och ledarskapet påverkar beslutsfattandet studeras. Det tycks finnas motsättningar gällande frågan om beslut fattas på ett rationellt och analytiskt sätt eller om det sker på ett intuitivt vis. Syftet med uppsatsen är att studera hur svenska officerare fattar beslut i dynamiska situationer och vad det kan innebära för deras utbildning och utveckling. Frågan studeras genom sammanlagt femton intervjuer med erfarna svenska officerare och officerskadetter och i studien har en tematisk analys med en induktiv ansats använts. Studiens resultat tyder på att det militära beslutsfattandet i dynamiska situationer kan förklaras med två teman: a) individuella och b) strukturella faktorer. Individuella faktorer handlar bland annat om intuitivt beslutsfattande, ledarskap och gruppdynamik medan strukturella faktorer avser erfarenhet och utbildning. Resultaten bekräftar teorier om att det intuitiva beslutsfattandet förutsätter att officeren är erfaren och har en hög nivå av expertis. En expertis och erfarenhet som genom år av träning har skapat en minnesbank som medger igenkänning eller mönsterförståelse i många militära dynamiska situationer. Med resultaten i åtanke diskuteras slutligen huruvida den inslagna vägen för att forma morgondagens officerare och högre officerare är den mest produktiva.

Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit. / Ph. D.

Den här studien handlar om hur svenska officerare fattar sina beslut i dynamiska situationer, det vill säga situationer som bland annat präglas av stress, oklarheter och tidspress. Även frågor kring vad som bygger förmågan till att fatta dylika beslut och hur till exempel gruppen och ledarskapet påverkar beslutsfattandet studeras. Det tycks finnas motsättningar gällande frågan om beslut fattas på ett rationellt och analytiskt sätt eller om det sker på ett intuitivt vis. Syftet med uppsatsen är att studera hur svenska officerare fattar beslut i dynamiska situationer och vad det kan innebära för deras utbildning och utveckling. Frågan studeras genom sammanlagt femton intervjuer med erfarna svenska officerare och officerskadetter och i studien har en tematisk analys med en induktiv ansats använts. Studiens resultat tyder på att det militära beslutsfattandet i dynamiska situationer kan förklaras med två teman: a) individuella och b) strukturella faktorer. Individuella faktorer handlar bland annat om intuitivt beslutsfattande, ledarskap och gruppdynamik medan strukturella faktorer avser erfarenhet och utbildning. Resultaten bekräftar teorier om att det intuitiva beslutsfattandet förutsätter att officeren är erfaren och har en hög nivå av expertis. En expertis och erfarenhet som genom år av träning har skapat en minnesbank som medger igenkänning eller mönsterförståelse i många militära dynamiska situationer. Med resultaten i åtanke diskuteras slutligen huruvida den inslagna vägen för att forma morgondagens officerare och högre officerare är den mest produktiva.

Human pluripotent stem cells (hPSCs) exist in multiple states of pluripotency, broadly categorised as naïve and primed states. These provide an important model to investigate the earliest stages of human embryonic development. Naïve cells can be obtained through primed-to-naïve reprogramming; however, there are no reliable methods to prospectively isolate unmodified naïve cells during this process. Moreover, the current isolation strategies are incompatible for enrichment of naïve hPSCs early during reprogramming. Consequently, we know very little about the temporal dynamics of transcriptional changes and remodelling of the epigenetic landscape that occurs during the reprogramming process. To address this knowledge gap, I sought to develop an isolation strategy capable of identifying nascent naïve hPSCs early during reprogramming. Comprehensive profiling of cell-surface markers by flow cytometry in naïve and primed hPSCs revealed pluripotent state-specific antibodies. By compiling the identified state-specific markers into a multiplexed antibody panel, I was able to distinguish naïve and primed hPSCs. Moreover, the antibody panel was able to track the dynamics of primed-to-naïve reprogramming, as the state-specific surface markers collectively reflect the change in pluripotent states. Through using the newly identified surface markers, I found that naïve cells are formed at a much earlier time point than previously realised, and could be subsequently isolated from a heterogeneous cell population early during reprogramming. This allowed me to perform the first molecular characterisation of nascent naïve hPSCs, which revealed distinct transcriptional changes associated with early and late stage naïve cell formation. Analysis of the DNA methylation landscape showed that nascent naïve cells are globally hypomethylated, whilst imprint methylation is largely preserved. Moreover, the loss of DNA methylation precedes X-chromosome reactivation, which occurs primarily during the late-stage of primed-to-naïve reprogramming, and is therefore a hallmark of mature naïve cells. Using the antibody panel at discrete time points throughout reprogramming has allowed an unprecedented insight into the early molecular events leading to naïve cell formation, and permits the direct comparison between different naïve reprogramming methods. Taken together, the identified state-specific surface markers provide a robust and straightforward method to unambiguously define human PSC states, and reveal for the first time the order of transcriptional and epigenetic changes associated with primed to naïve reprogramming.

Att det idag finns flera metoder och modeller för att fatta beslut är inte konstigt då beslutsfattande sker varje dag och i vissa sammanhang kan ge ödesdigra konsekvenser om det blir fel. Denna kandidatuppsats kan ses som en förstudie för hur naturligt beslutsfattande skulle kunna stödjas genom en förbättrad utbildning till beslutsfattare. Uppsatsen ämnar behandla naturligt beslutsfattande och intuition hos experter inom räddningstjänsten, närmare bestämt om de använder intuition och i så fall vad som ligger till grund för denna intuition. Intervjuer utfördes på räddningstjänsten i Motala efter critical decision method som är framtagen för cognitive task analysis. Inom räddningstjänsten använder sig befälen av intuition när de tar det flesta av sina beslut i fält. Resultaten av intervjuerna pekar på att intuition verkar ske undermedvetet vilket ger stöd för recognition primed decision model, i kombination med negativa ledtrådar och hävstångspunkter.

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