The inhibitory effects of NaCl, L- and D-asparagine, and asparagine substrate analogs, beta-aspartyl hydroxamate (AAH) and albizziin, alone or in combination on Columbia Arabidopsis seed germination and seedling survival were characterized under aseptic conditions. Germination on an agar medium supplemented with inorganic nutrients was prevented by 200 mM NaCl, 20 mM L-asparagine, 60 mM D-asparagine, 1.4 mM AAH, or 8 mM albizziin. Established seedlings were generally more tolerant to these chemicals than germinating seeds. Exogenous L- and D-asparagine partly reversed the inhibitory effects of NaCl on seed germination. L-asparagine also partly reversed AAH inhibition of germination. A M2 seed bank was created from the self-pollinated progeny of ethyl methane sulfonate treated seeds. Arabidopsis mutants having increased tolerance to NaCl and AAH, but not albizziin, were successfully selected from this seed bank.

The APOBEC3 (Apo3) family of proteins are single stranded (ss) DNA cytosine deaminases (C → U). They are grouped into two different structural groups, the single catalytic domain Apo3 enzymes (Apo3A, Apo3C, and Apo3H) and the double catalytic domain Apo3 enzymes (Apo3B, Apo3D, Apo3F, and Apo3G). Apo3G has been implicated in protection from HIV proliferation by becoming encapsidated into budding HIV virions and subsequently mutationally inactivating the synthesized provirus. This largely occurs in the absence of HIV viral infectivity factor (Vif) which mediates the ubiquitination and degradation of Apo3G. Apo3G is a processive enzyme, able to catalyze numerous deaminations in a 5'CCC motif in a single interaction with a substrate. There is a paucity of biochemical data on other Apo3 family members. We performed basic biochemical assays that determined the relative specific activities, processivity, cytosine motif preferences, and binding affinities for DNA, of Apo3A and Apo3H using synthetic DNA substrates in deamination assays. We found Apo3A to be an enzyme with low processivity and Apo3H to be a highly processive enzyme; both of which deaminate a 5'TC motif. Using a reconstituted HIV replication assay we assessed if processivity is needed for efficient restriction of HIV. We were able to demonstrate that each, Apo3G, Apo3A, and Apo3H were able to catalyze deaminations during in vitro reverse transcription. The mutation profile of both Apo3A and Apo3H showed that the 5'TC motif preference was less effective compared to Apo3G in triggering missense and nonsense mutations in the HIV protease active site coding sequence. Nuclear DNA can become deaminated by the related Apo3 family member activation-induced deaminase (AID), when it is present in the nucleus of activated B cells. Apo3A and Apo3H are located in the nucleus but the extent of the damage they cause has only recently been investigated. Here we used an in vitro transcription assay to determine the efficiency of Apo3A and Apo3H deamination during transcription and found that, like AID, they are highly capable of causing deaminations during transcription. Taken together, the results presented here demonstrate that processivity is not necessary for an Apo3 enzyme to catalyze deaminations during HIV reverse transcription and that Apo3A and Apo3H can catalyze deaminations during DNA transcription that could damage host genomic DNA. These results imply a potential cost for maintaining nuclear deaminases.

Synonymous codon usage bias has been extensively studied in Bacteria and Eukaryotes and yet there has been little investigation in the third domain of life, the Archaea. In this thesis I therefore examine the coding sequences of nearly 70 species of Archaea to explore patterns of codon bias. Heterogeneity in codon usage among genes was initially explored for a single species, Methanococcus maripaludis, where patterns were explained by a single major trend associated with expression level and attributed to natural selection. Unlike the bacterium Escherichia coli, selection was largely restricted to two-fold degenerate sites. Analyses of patterns of codon usage bias within genomes were extended to the other species of Archaea, where variation was more commonly explained by heterogeneity in G+C content and asymmetric base composition. By comparison with bacterial genomes, far fewer trends were found to be associated with expression level, implying a reduced prevalence of translational selection among Archaea. The strength of selected codon usage bias (S) was estimated for 67 species of Archaea, and revealed that natural selection has had less impact in shaping patterns of codon usage across Archaea than across many species of Bacteria. Variation in S was explained by the combined effects of growth rate and optimal growth temperature, with species growing at high temperatures exhibiting weaker than expected selection given growth rate. Such a relationship is expected if temperature kinetically modulates growth rate via its impact upon translation elongation, since rapid elongation rates at high temperatures reduce the selective benefit of optimal codon usage for the efficiency of translation. Consistent with this, growth temperature is negatively correlated with minimal generation time, and numbers of rRNA operons and tRNA genes are reduced at high growth temperatures. The large fraction of thermophilic Archaea relative to Bacteria account for the lower values of S observed. Two major trends were found to describe variation in codon usage among archaeal genomes; the first was attributed to GC3s and the second was associated with arginine codon usage and was linked both with growth temperature and the genome-wide excess of G over C content. The latter is unlikely to reflect thermophilic adaptation since the codon primarily underlying the trend appears to be selectively disfavoured. No correlations were observed with genome wide GC3s and optimal growth temperature and neither was GC3s associated with aerobiosis. The identities of optimal codons were explored and found to be invariant across U and C-ending two-fold degenerate amino acid groups. The identity of optimal codons and anticodons across four and six-fold degenerate amino acid groups was found to vary with mutational bias. As was first observed in M. maripaludis, selected codon usage bias was consistently greater across two-fold relative to four-fold degenerate amino acid groups across Archaea. This broad pattern could reflect ancestral patterns of optimal codon divergence, prevalent among four-fold but not two-fold degenerate amino acid groups. Consistent with this, the strength of selected codon usage bias was found to be reduced following the divergence of optimal codons, and implies that optimal codon divergence typically proceeds following the relaxation of selection. Finally, a method was developed to partition the strength of selection (S) into separate components reflecting selection for translational efficiency (Seff) and selection for translational accuracy (Sacc) by comparing the codon usage across conserved and nonconserved amino acid residues. While estimates of Sacc are somewhat sensitive to the designation of conserved sites, a general pattern emerged whereby accuracy-selected codon usage bias was consistently strongest across a subset of the most highly conserved sites. Several estimates of Sacc were consistently higher than the 95% range of null values regardless of the dataset, providing evidence for accuracy-selected codon usage bias in these species.

At present, species are faced with a host of human-induced impacts that have already led to a loss of biodiversity and shifts in species’ ranges. Knowledge of the ecological and evolutionary processes of dynamic species’ ranges, that are either shifting or expanding, will be key to understanding the ability of species to persist and adapt during periods of environmental change. The aim of this thesis is to investigate the effects of range expansion on the genetics and evolution of species, and understand the processes that facilitate the spatial spread of populations. Using individual-based simulation models, this work demonstrates that the unique selective environment of range expansion can have a significant effect on spatial genetics and the evolution of life-history traits. It is found that the survival and spread of mutations, of various fitness effects, is influenced substantially by landscape features encountered by the expanding range. The incorporation of more specific genetic architecture shows that a substantially increased frequency of fitness peak shifts may occur on the edge of an expanding range than would arise within a stationary population. Range expansion is shown to select for increased dispersal and reproduction at the expense of competitive ability on the front of the expanding wave which results in an accelerating spread rate. The survival and spread of a population during range expansion is also affected substantially by the specific movement behaviour of individuals. The exact nature of suitable habitat, in terms of the number of suitable habitat patches and level of fragmentation, will also greatly affect the ability of a population to survive and spread during a period of environmental change. These findings are synthesised within a conceptual framework that is proposed for the evolution of populations during range expansion, where a flattening of the fitness landscape leads to adaptive revolutions.

Cardiac contraction at the level of the sarcomere is regulated by the thin filament (TF) composed of actin, alpha tropomyosin (TPM), and the troponin (Tn) complex (cTnT: cTnC: cTnI). The "gate-keeper" protein, α-TPM, is a highly conserved α-helical, coiled-coil dimer that spans actin and regulates myosin-actin interactions. The N-terminus of one α-TPM dimer inter-digitates with the C-terminus of the adjacent dimer in a head-to-tail fashion forming the flexible and cooperative TPM-overlap that is necessary for myofilament activation. Two dilated cardiomyopathy (DCM) causing mutations in TPM (D84N and D230N) and one hypertrophic cardiomyopathy (HCM) causing mutation (E62Q), all identified in large, unrelated, multigenerational families, were utilized to study how primary alterations in protein structure cause functional deficits. We hypothesize that structural changes from a single point mutation propagate along the -helical coiled-coil of TPM, thus affecting its regulatory function. Structural effects of the mutations studied via differential scanning calorimetry (DSC) on TPM alone revealed significant changes in the thermal unfolding temperatures of both the C- and N-termini for all mutants compared to WT, indicating that mutational effects propagate to both ends of TPM, thus affecting the overlap region. Although, of note, the proximal termini to the mutation has shown more significant structural changes compared to WT. DSC analysis on fully reconstituted TF’s (Tn:TPM:Actin) revealed effects on the TPM-Actin cooperativity of activation, affecting interaction strength (thermal stability), and the rigidity of TPM moving along actin (FWHM). To characterize the resultant functional effect of these discrete changes in thermal stability and TPM rigidity, ATPase assays were used to measure actomyosin activation in the presence and absence of Ca2+. Together, these data will provide a molecular level understanding of the structural and functional deficits caused by these mutations to help elucidate the mechanisms leading to disease.

Le récepteur P2Y[indice inférieur 6] est un récepteur couplé à une protéine G responsable de l’activation de nombreuses voies de signalisation. Dans l’épithélium du côlon, il participe au maintien de l’équilibre hydrique, mais il a été montré que le récepteur P2Y[indice inférieur 6] participait à l’aggravation des symptômes inflammatoires dans la maladie de Crohn ou dans la colite ulcéreuse. Les maladies inflammatoires de l’intestin sont des facteurs pouvant mener au cancer colorectal. En effet, il existe deux types de cancers colorectaux : le cancer sporadique et le cancer associé à la colite qui se différencient notamment par la séquence d’apparition de mutations génétiques. Par exemple, le gène TP53 est muté de façon tardive dans le cancer colorectal sporadique et muté de façon précoce dans le cancer associé à l’inflammation. Puisque le récepteur P2Y[indice inférieur 6] est impliqué dans la création d’un environnement pro-inflammatoire, nous nous sommes intéressés au rôle de p53 sur l’expression du gène P2RY[indice inférieur 6] et avons formulé l’hypothèse suivante : la présence de TP53 mutant va réguler de façon différentielle l’expression du gène P2RY[indice inférieur 6] dans le cancer colorectal. L’objectif général des travaux est le suivant : caractériser les mécanismes moléculaires liés à TP53 régulant l’expression du gène P2RY[indice inférieur 6] dans le cancer colorectal. Les objectifs spécifiques pour ce projet de recherche sont donc : (1) déterminer et caractériser les régions promotrices du gène P2RY[indice inférieur 6] dans les cellules épithéliales intestinales cancéreuses et (2) étudier l’effet de la protéine p53 de type sauvage ou mutée sur l’expression du récepteur P2Y[indice inférieur 6]. Le gène P2RY[indice inférieur 6] code pour 8 variants d’ARN messagers. Les variants 1, 2, 3, 5, 6, 7 et 8 codent pour l’isoforme 1 du récepteur P2Y[indice inférieur 6], forme connue du récepteur. Le variant 9 code pour l’isoforme 2, non caractérisée. Nos travaux ont permis de mettre en évidence l’existence de quatre régions promotrices potentielles du gène P2RY[indice inférieur 6] et la présence du variant 9, codant pour l’isoforme 2 du récepteur P2Y[indice inférieur 6] dans la lignée cellulaire Caco-2. Nous avons également montré que les formes normale et mutée de p53 régulent de façon différentielle l’expression du récepteur P2Y[indice inférieur 6]. Enfin, le rôle de l’isoforme 2 reste à étudier, mais les tests effectués suggèrent qu’elle est activable par l’UDP.

<p>Olfaction is one of the most primitive of our senses, and the olfactory receptors that mediate this very important chemical sense comprise the largest family of genes in the mammalian genome. It is therefore surprising that we understand so little of how olfactory receptors work. In particular we have a poor idea of what odorous chemicals are detected by most of the olfactory receptors in the genome, and for those receptors which we have paired with ligands, we know relatively little about how the structure of these ligands can either activate or inhibit the activation of these receptors. Furthermore the large repertoire of olfactory receptors, which belong to the G protein coupled receptor (GPCR) superfamily, can serve as a model to contribute to our broader understanding of GPCR- ligand binding, especially since GPCRs are important pharmaceutical targets.</p><p>In this dissertation, I explore the relationship between olfactory receptors and their ligands, both by manipulating the ligands presented to the olfactory receptors, as well as by altering the structure of the receptor itself by mutagenesis. Here we report the probable requirement of a hydrated germinal-diol form of octanal for activation of the rodent OR-I7 receptor by ligand manipulation, and the successful in vitro modeling and manipulation of ketamine binding to MOR136-1. We also report the results of a large-scale screen of 1190 human and mouse olfactory receptors for receptors activated by volatile general anesthetics, which has lead to the identification of 32 olfactory receptor-volatile general anesthetic pairs.</p> / Dissertation

<p>The advent of next-generation sequencing, now nearing a decade in age, has enabled, among other capabilities, measurement of genome-wide sequence features at unprecedented scale and resolution. </p><p>In this dissertation, I describe work to understand the genetic underpinnings of non-Hodgkin’s lymphoma through exploration of the epigenetics of its cell of origin, initial characterization and interpretation of driver mutations, and finally, a larger-scale, population-level study that incorporates mutation interpretation with clinical outcome. </p><p>In the first research chapter, I describe genomic characteristics of lymphomas through the lens of their cells of origin. Just as many other cancers, such as breast cancer or lung cancer, are categorized based on their cell of origin, lymphoma subtypes can be examined through the context of their normal B Cells of origin, Naïve, Germinal Center, and post-Germinal Center. By applying integrative analysis of the epigenetics of normal B Cells of origin through chromatin-immunoprecipitation sequencing, we find that differences in normal B Cell subtypes are reflected in the mutational landscapes of the cancers that arise from them, namely Mantle Cell, Burkitt, and Diffuse Large B-Cell Lymphoma. </p><p>In the next research chapter, I describe our first endeavor into understanding the genetic heterogeneity of Diffuse Large B Cell Lymphoma, the most common form of non-Hodgkin’s lymphoma, which affects 100,000 patients in the world. Through whole-genome sequencing of 1 case as well as whole-exome sequencing of 94 cases, we characterize the most recurrent genetic features of DLBCL and lay the groundwork for a larger study. </p><p>In the last research chapter, I describe work to characterize and interpret the whole exomes of 1001 cases of DLBCL in the largest single-cancer study to date. This highly-powered study enabled sub-gene, gene-level, and gene-network level understanding of driver mutations within DLBCL. Moreover, matched genomic and clinical data enabled the connection of these driver mutations to clinical features such as treatment response or overall survival. As sequencing costs continue to drop, whole-exome sequencing will become a routine clinical assay, and another diagnostic dimension in addition to existing methods such as histology. However, to unlock the full utility of sequencing data, we must be able to interpret it. This study undertakes a first step in developing the understanding necessary to uncover the genomic signals of DLBCL hidden within its exomes. However, beyond the scope of this one disease, the experimental and analytical methods can be readily applied to other cancer sequencing studies.</p><p>Thus, this dissertation leverages next-generation sequencing analysis to understand the genetic underpinnings of lymphoma, both by examining its normal cells of origin as well as through a large-scale study to sensitively identify recurrently mutated genes and their relationship to clinical outcome.</p> / Dissertation

Kaffee, die in der westlichen Welt am häufigsten verwendete psychoaktive Substanz, erwies sich im Mikrokern-Testes an Maus-Lymphomzellen L 5178Y als modulierend auf bekannte mutagene Agentien. In dieser Arbeit wurde meist Instantkaffee verwendet, der gegen N-Methyl-N-Nitro-N-Nitrosoguanidin (MNNG), Mitomycin C (MMC), Genistein, und Methylmethansulfonat getestet wurde. Bei MMC und Genistein erwies sich Kaffee als antimutagen. Bei MNNG hatte Kaffee keinen klaren Einfluss auf die Gentoxizität, ebenso blieb die Kaffee-Wirkung bezüglich MMS unklar. Kaffee minderte dosisabhängig das Wachstum und die Überlebensrate von L 5178Y - Zellen. Es wurde die Frage nach den Ursachen der modulierenden Effekte diskutiert. Insbesondere wurde die Hypothese erörtert, dass für die Richtung der Modulation nicht so sehr die Konzentration des Kaffees, sondern die mutagene Potenz der koinkubierten Substanz eine entscheidende Rolle spielen könnte. Ggf. wirkt Kaffee - im Sinne eines "abhärtenden" Effektes - bei schwach mutagenen Substanzen anitmutagen, bei stark mutagenen Substanzen hingegen synergistisch mutagen. / Coffee, the most commonly used psychoactive substance in the western hemisphere, was regarded as an effect modifier of mutagenicity in L5178Y mouse lymphoma cells treated with mutages. Mainly instant-coffee was tested against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC), genistein, und methylmethanesulfonate (MMS). Coffee was antimutagen against mitomycin C (MMC) and genistein. No clearly influence was seen against MNNG or MMS. Dose-dependent Coffee reduces the growing and survival rate of L5178Y mouse lymphoma cells. The reasons for the modifying-effects of coffee were discussed. The hypothesis was found, that the concentration of the coffee ist not so influential in making antimutagene or mutagene effects, rather the mutagen power-factor of the coincubating substances. Coffee could be an “indurating” parameter: Causing antimutage effects in the presence of “gentle” mutagens, causing synergistic mutagen or zytotoxic effects in the presence of “strong” mutagens.

Die progressiven Muskeldystrophien Duchenne (DMD) und Becker (BMD) entstehen durch verschiedene Mutationstypen (Deletionen, Duplikationen, Punktmutationen) im Dystrophin-Gen, welches als größtes Gen des Menschen 79 Exons aufweist und sich auf dem kurzen Arm des X-Chromosoms befindet. Es wurden Daten von 1365 Personen bezüglich der Häufigkeit der Mutationstypen sowie der Verteilung der einzelnen Mutationen auf die Exons des Dystrophin-Gens ausgewertet. Hieraus konnte ermittelt werden, dass sich bei 780 männlichen Patienten mit gesicherter Diagnose zu 65 Prozent Deletionen, 9 Prozent Duplikationen und 26 Prozent Punktmutationen nachweisen ließen. Desweiteren wurde gezeigt, dass sich bei der Verteilung der Deletionen auf das Dystrophin-Gen zwei hot spot Regionen finden, eine größere im Bereich der Exons 45 - 54 und eine kleinere im Bereich 11 - 20. Die Duplikationen weisen eine Häufung der betroffenen Exons am Anfang des Gens auf, wobei Exon 2 am häufigsten das erste betroffene Exon darstellt. Die Punktmutationen dagegen verteilen sich zufällig über das Gen. Es konnte weiterhin gezeigt werden, dass hinsichtlich der Verteilung der gleichen Mutationen auf das Dystrophin-Gen zwischen einer Gruppe von männlichen Patienten und der Gesamtheit aller Probanden einschließlich Konduktorinnen keine Unterschiede bestehen. Dagegen unterschieden sich die verschiedenen Mutationstypen im Vergleich miteinander hinsichtlich ihrer Verteilung auf das Dystrophin-Gen. Bei der Untersuchung der geographischen Verteilung der DMD und BMD konnte lediglich bei den Duplikationen eine Gleichverteilung in Deutschland bestätigt werden. / The progressive muscular dystrophies Duchenne (DMD) and Becker (BMD) originate from different mutation types (deletions, duplications, point mutations) in the dystrophin gene, the biggest human gene, which has 79 exons and is located on the short arm of the X-chromosome. Data of 1365 people were evaluated with regard to the frequency of the mutation types as well as the distribution of mutations over the exons of the dystrophin gene. We found that in 780 male patients with secured diagnosis, there were deletions in 65 percent of the cases, duplications in 9 percent and point mutations in 26 percent. Furthermore it was shown that the distribution of deletions in the dystrophin gene shows two hot spot regions, a larger one in the area of exons 45 - 54 and a smaller one in the area of exons 11 - 20. Duplications showed an accumulation of affected exons at the beginning of the gene, with exon 2 being the first affected exon most often. Point mutations in contrast are distributed over the gene randomly. Moreover it was found out that the distribution of mutations in the dystrophin gene did not differ between a group of male patients and the group of all patients including female carriers, whereas mutation types differed regarding their dissemination over the dystrophin gene. With the analysis of the geographic distribution of DMD and BMD a uniform geographical distribution over Germany could be confirmed merely with duplications.