Дисертації з теми "Molecular delineation"

There are more than 7000 described rare genetic disorders; however, the molecular basis underlying approximately half of these disorders is unknown, and the majority are currently untreatable. Stature and growth abnormalities are a common clinical feature of many rare disorders including: Floating-Harbor syndrome (FHS), a short stature syndrome characterized by delayed osseous maturation, language deficits, and unique dysmorphic facial features; Weaver syndrome, an overgrowth syndrome characterized by advanced osseous maturation, developmental delay, and macrocephaly; and Sotos syndrome with cutis laxa, an overgrowth syndrome with marked tissue laxity in addition to the typical Sotos characteristics of developmental delay, macrocephaly, and a unique facial gestalt. The genetic basis underlying these three rare stature conditions were unknown at the outset of this study. We utilized high-throughput exome sequencing approaches to investigate the molecular etiology of these rare disorders and identified truncating mutations in the final exon of SRCAP as the genetic cause underlying FHS, missense mutations in EZH2 in Weaver syndrome, and novel mutations in the Sotos syndrome gene NSD1 in Sotos syndrome with cutis laxa. Next, we investigated the spectrum of SRCAP mutations in FHS and established the clustering of truncating SRCAP mutations in the final exon as being highly suggestive of a non-haploinsufficiency mutational mechanism in FHS. Finally, global methylation array analysis identified a unique methylation ‘epi-signature’ in FHS individuals, providing further insight into FHS disease mechanism and a diagnostic signature. These studies have delineated the molecular etiology of these three rare stature/growth disorders, furthered our understanding of the associated clinical spectrum, and provided biological insight into disease pathogenesis.

CHAPTER 2 The burgeoning shark-fishing industry has caused severe declines in many shark populations, prompting an urgent need for conservation and management of sharks on a species-specific basis. Many fisheries are managed on the basis of species groups due to difficulty in identifying the morphologically similar requiem sharks (Carcharhinidae) and their body parts. One diagnostic trait readily discernible in landed shark carcasses is the presence or absence of an interdorsal ridge, which allows shark carcasses to be categorized as either ridgeback or non-ridgeback sharks and narrows the identification process. Further identification to species, however, remains problematic and is a major impediment to management and conservation on a species-specific basis. To facilitate the goal of species-specific management, I have developed a rapid and cost-efficient DNA-based method for use in species identification. It employs a high-density, multiplex polymerase chain reaction (PCR) assay that combines seven species-specific primers and two universal primers to discriminate one ridgeback (Galeocerdo cuvier) and six non-ridgeback (Carcharhinus brevipinna, C. limbatus, C. acronotus, C. isodon, C. leucas, and Negaprion brevirostris) sharks common in U.S. Atlantic fisheries. The primers were designed based on species-specific nucleotide differences in the nuclear ITS2 locus. These seven primers were tested on 73 non-target species worldwide to assess their diagnostic utility on a global scale. Five of the seven primers are species-specific, with only the C. leucas and the C. limbatus primers each amplifying one other congener, C. perezi and C. tilstoni, respectively. This diagnostic assay was tested in a practical management context and successfully identified shark fins confiscated by NMFS law enforcement agents, indicating that it will be useful in management of the U.S. Atlantic shark fishery. Furthermore, the primers developed for G. cuvier, C. leucas and C. brevipinna successfully identified their respective target species from the Atlantic and Pacific Oceans, suggesting that they will also be useful for global applications such as monitoring international trade in shark products. CHAPTER 3 Accurate species delineation is essential to sound management and conservation of fishery resources. The species status of two butterfly rays, Gymnura marmorata and G. crebripunctata, occurring in the fisheries in the Gulf of California and on the Pacific Mexican coast, has long been debated. Direct sequence comparison and a phylogenetic analysis were carried out using a segment of the mitochondrial cytochrome b locus to elucidate the relationship between G. marmorata and G. crebripunctata and five other congeners. Sequence divergence between G. marmorata and G. crebripunctata was minimal compared to sequence divergence among the five other established congeners. A neighbor-joining analysis showed strong statistical (100%) support and a maximum likelihood analysis showed reasonable support (62%) for the monophyletic clade consisting. of G. marmorata and G. crebripunctata. Importantly, neither analysis supported the reciprocal monophyly of either species, as would be expected for true separate species. These data suggest that the current classification of G. marmorata and G. crebripunctata as two separate species needs to be revised to support their designation as a single species. Genetic analysis of the cyt b locus also revealed three single-nucleotide synapomorphies that partitioned animals from the two geographic sites sampled in Mexico (Sahuimaro, Sonora and Bahia Almejas, Baja California Sur), indicating the existence of possible population genetic structure between the two sites. By analyzing cyt b sequences from seven ofthe 10 described Gymnura species, I also provide the first phylogenetic hypothesis for interspecies relationships in the genus Gymnura.

Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia of the Western world, and despite the recent evolution in clinical treatment of the disease, it remains incurable. Although current therapies such as allogeneic hematopoietic stem cell (HSC) transplantation have been successfully used to treat CLL, this is an option for only a minority, as most CLL patients are diagnosed over the age of sixty and cannot withstand the harsh transplantation procedures. Combination chemotherapy, such as fludarabine and cyclophosphamide, has been shown to significantly improve response rate and prolonged remission in CLL patients, however, no improvement in overall survival has been observed. Patients eventually relapse due to re-emergence of minimal residual disease (MRD). Therefore it is critical that further clinical therapies are investigated in order to eliminate MRD, and offer hope to patients that are unresponsive to current treatments. CLL is marked by the presence of the accumulation of long-lived mature monoclonal B cells in peripheral lymphoid organs, bone marrow and peripheral blood with the specific phenotype of CD19hi, CD5+, CD23+ and IgMlo that resist apoptosis. The in vivo accumulation of leukaemic lymphocytes is highly facilitated by interactions of CLL cells with other cells present in their microenvironment, including stromal cells and soluble factors such as IL4. Studies have established a variety of mechanisms potentially responsible for disease progression in CLL, including chromosomal abnormalities and intrinsic defects in the apoptotic machinery due to higher levels of the anti-apoptotic protein Bcl-2 family member proteins Bcl-2 and Mcl-1, thus making this disease extremely heterogeneous. Although the apoptotic machinery is certainly dysregulated in CLL, it is not simply a disease of a clonal accumulation of B cells, rather, proliferation is occurring as well as apoptosis, accounting for up to 2% of the clone size per day. CLL B cell proliferation centres exist within lymph nodes (LN) and bone marrow (BM) where B cells receive signals from their B cell antigen receptor (BCR) to proliferate, generating a very aggressive form of the disease. In addition, evidence suggests that stimulation through the BCR plays a pivotal role in pathogenesis of CLL since CLL B cells have a phenotypic profile of B cells activated by antigen interaction and a genetic expression profile of antigen experienced B cells. During the course of our studies assessing the impact of modulating protein kinase C (PKC) signaling in B cell development in vitro or in vivo, we developed a unique model system to investigate the mechanisms underlying the induction of CLL. Introduction of full length, catalytically inactive PKCα (PKCα-KR) into HSCs derived from wild type mouse fetal liver (FL), and subsequent culture of the cells either in vitro or in vivo resulted in the generation of a population of B lymphocytes that are phenotypically similar to human CLL cells (CD19hi, CD5+, CD23+, IgMlo). PKCα-KR-expressing FL cells also expressed enhanced proliferative capacity over untransduced cells and were refractory to apoptosis. These results indicate that the subversion of PKCα signaling acts as an oncogenic trigger for developing B lymphocytes. The aim of this project was to identify similarities between our murine CLL (mCLL) model and human CLL and investigate putative translational therapeutic targets. The main findings of this study implicate PKCβII as an important survival and proliferation signal within mCLL. Cyclin D1 is also upregulated within mCLL, linked to an increase in the proliferative capacity of mCLL cells, and is regulated through transcriptional repressor 4EBP1, which appears inactive in both mCLL and human CLL. In addition, PKCα-KR transduced cells harbour the potential for lineage plasticity in a microenvironment-dependent manner, whereby PKCα-KR B cells lineage switch to T cells upon Notch ligation. The reprogramming occurs via a reduction in B cell specific genes and an upregulation of T cell specific genes, implicating the deregulation of PKCα activity/expression as a potential mechanism for lineage trans-differentiation during malignancies. Importantly, in human CLL, PKCα is downregulated at the transcript and protein levels implicating it a tumour suppressor, highlighting the translational capacity of our CLL mouse model.

This work sought to investigate the clinical phenotypes and molecular basis of cardio-facio-cutaneous syndrome (CFC), a germline disorder of the Ras-MAPK pathway, like Noonan syndrome (NS) and neurofibromatosis type I, caused by mutations in genes encoding proteins that act within this signal transduction pathway. CFC is most commonly due to mutation in BRAF, and less commonly MAP2K1, MAP2K2 or KRAS. A proportion of patients currently have no mutation identified. Mutations and clinical features of patients with a molecular diagnosis of CFC were investigated, which demonstrated a wide range of causative mutations, and some unclassified variants. Both known and novel clinical features of CFC were identified. A strong association between severe contractures and the p.(Tyr130Cys) mutation in MAP2K1 was found, which has not previously been reported. In contrast to the large number of patients with a confirmed molecular diagnosis, several with a highly suggestive clinical phenotype have been found to have no mutationin any of the known CFC genes. The molecular basis of these presentations was investigated by conventional Sanger sequencing of candidate genes. Fourteen patients with the p.(Ser2Gly) mutation in SHOC2 were identified, with clinical presentations consistent with CFC, NS or CS. Target enrichment and massively parallel sequencing of selected genes was undertaken in ten patients. Mutations in known genes were identified in four patients (including the positive control). Candidate causative variants in novel genes were suggested in two further patients, one of which was confirmed on Sanger sequencing. Whole exome sequencing of patient-parent trios was also undertaken to identify de novo variants. Three trios were analysed, and in one patient with a clinical diagnosis of CFC, a frameshift mutation in NF1 was identified, which was confirmed by Sanger sequencing to be present and de novo. The molecular effects of CFC-associated mutations in BRAF on Ras-MAPK pathway signalling were studied in cell culture systems, using Western blotting for ERK1/2 phosphorylation, in vitro kinase assays and luciferase assays, to assess activity of downstream targets of the Ras-MAPK pathway. Altered pathway activity was demonstrated for novel variants that had not previously been characterised at the molecular level, which was in keeping with the findings of the effects of previously studied mutations. The cardiac phenotype in animal models of CFC, CS and NS/CFC was explored using expression microarrays to identify potentially important genes and pathways in the pathogenesis of hypertrophic cardiomyopathy (a progressive but potentially treatable disease feature) in these conditions. A signature of increased expression of Myh7, the embryonic form of myosin, was identified in the heart of the mouse model of CFC due to a B-Raf mutation at four weeks postnatal age, but comparative analysis suggested significant differences in either the mechanisms causing cardiac phenotypes, or the timescales over which these may exert their effects, in the three models. In summary, the most significant findings of this work were that SHOC2 mutation is a frequent cause of a severe NCFC presentation, and massively parallel sequencing can be an effective means of molecular investigation of this group of disorders. Novel features of CFC syndrome that were identified include severe contractures in association with p.(Tyr130Cys) mutations in MAP2K1. The analysis of mouse models of the NCFCs was hampered by heterogeneity within the expression microarray results, and low levels of expression of the H-Ras mutant allele in the mouse model of Costellosyndrome.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The study of differential DNA replication programs in Drosophila has provided important insight into the molecular control of replication initiation and fork progression during development. We investigated the mechanisms by which binding of the origin recognition complex (ORC) and replication fork inhibition give rise to locally underreplicated regions in Drosophila polyploid tissues. We identified copy number changes genome-wide in two additional polyploid tissues and compared our results to three previously profiled larval tissues. These results revealed a high level of tissue-specificity in the number of underreplicated sites within a given tissue but also highlighted the conservation of the locations of many of these underreplicated regions across tissues. By mapping ORC binding sites in the larval fat body, we found that the repression of replication initiation is a common mechanism of underreplication in polytene tissues. Our ORC localization studies also suggest that underreplication zones are hard-wired across tissues and that differential underreplication of these zones is dependent upon variation in fork progression across these regions. We then utilized the Drosophila amplicons in follicle cells (DAFCs) as a model replication system to dissect the molecular mechanisms underlying the activation of individual replication origins. Repression of the DAFC-22B origin is not achieved through changes in subnuclear localization but rather through effects of the surrounding chromatin. We identified two novel genomic sites at which origin activity is modulated directly by the surrounding chromatin environment. At one site, the surrounding chromatin promotes one additional round of origin firing at a specific developmental time point. At the other site, origin activity is repressed by the surrounding chromatin through inhibition of the localization of the MCM2-7 helicase complex. Origin repression at this site is not correlated with the establishment of heterochromatin, raising the possibility that the activity of individual replication origins are regulated by the chromatin environment on a greater, conformational level. Finally, we dissected the requirement of transcription in the activation of the DAFC-62D origin. Surprisingly, transcription is not required in cis for origin activation. These results indicate the requirement of a trans-acting factor specifically at this site and highlight the diversity of mechanisms that control metazoan origin activation.
by Brian L. Hua.
Ph. D.

Current agents used for pharmacological reactivation of foetal haemoglobin (HbF) have limited application due to moderate therapeutic properties, variable patient response and potential cytotoxic effects. Therefore, identification of novel HbF inducing agents is still a major research goal to this day. Identification of new potential HbF inducers has been mainly based on screening of drug libraries. However, this approach has not been very successful in generating new promising agents. In the current project, I employed two approaches for identifying potential HbF inducers: 1) screening of agents that are structurally similar to compounds with known HbF inducing activity; 2) investigating molecular pathways of a known HbF inducer with the aim of identifying suitable targets for therapeutic manipulation and target-based drug design. The first approach involved screening of eleven xanthines including caffeine and nine hydroxystilbenic derivatives of resveratrol as potential HbF inducers. However, none of the agents had a potent enough HbF inducing activity in order to be considered as promising therapeutic agents. In the second approach, decitabine was chosen based on its high HbF inducing activity and moderate cytotoxicity in K562 cells and primary human erythroid cultures. Chromatin immunoprecipitation was used to characterise epigenetic changes in the β-globin gene locus, and quantitative real-time PCR for investigation of changes in gene expression levels of ten erythroid-related genes, in the presence of the agent. A quantitative iTRAQ proteomic approach coupled with mass spectrometry was used for identification of changes in the proteome of decitabine-treated and un-treated primary human erythroid cultures. The findings suggest that decitabine induces HbF production through activation of signal transduction pathways rather than through hypomethylation of gene promoters. One such possible pathway is the NF-κB pathway. Among the differentially expressed proteins, twenty-seven proteins were associated with the action of decitabine. Two of those proteins, ARHGAP4 and EGLN2, were previously implicated in hydroxyurea-mediated induction of γ-globin gene expression and hypoxia-mediated erythropoiesis, respectively. In addition, the de-ubiquitinating enzyme USP11 was substantially modulated in the presence of decitabine. The exact role of these proteins in γ-globin expression remains to be established.

The Malacothamnus alliance consists of three genera, Iliamna, Malacothamnus, and Phymosia. The genera are considered taxonomically complex since hybridization freely occurs, polyploidy levels vary, and there is a lack of distinct morphological characters to delineate taxa. Several taxonomic treatments have been prepared for each genus, but relationships within the genera and the relationship of the Malacothamnus alliance to others in the Malvaceae remains unknown. This multifaceted study aimed to (a) examine the monophyly of the Malacothamnus alliance and its position in the Malvaceae, (b) determine the relationships between genera in the alliance, (c) compare variation of nuclear and chloroplast genes in the alliance, (d) prepare revised taxonomic treatments for Iliamna and Malacothamnus, and (e) examine the probability of successful hybridization in Iliamna. The monophyly of the Malacothamnus alliance was not confirmed using DNA sequences of both nuclear and chloroplast regions. In Iliamna, little sequence variation was detected among taxa in the Rocky Mountains; however, the nuclear and chloroplast regions conflicted with regard to the relationships of the western and eastern taxa. An ancestral copy of the chloroplast genome is shared between the two eastern U.S. Iliamna species and Phymosia (Bahamas and Mexico). The nuclear ITS sequences indicated the western U.S. Iliamna species were more closely related to Phymosia and Malacothamnus than to other species in Iliamna. Neither data set provided sufficient variation to resolve relationships of species in Malacothamnus. Genetic variation and the feasibility of hybridization in Iliamna supported the results of the broader phylogenetic studies. Iliamna corei and I. remota are recently derived from I. rivularis. Hybrid offspring of I. corei and I. remota had higher viability and fecundity than did hybrids between crosses of either species and I. rivularis. The Virginia populations of I. corei and I. remota are more genetically similar than either is to Illinois populations of I. remota. However, the species are morphologically distinct and can easily be distinguished from others in the genus. Revised taxonomic treatments for Iliamna and Malacothamnus based on surveys of herbarium material are presented. Taxonomic revisions include the new combinations of Iliamna grandiflora subsp. grandiflora and I. grandiflora supsp. crandallii and the resurrection of Malacothamnus hallii and M. orbiculatus.
Ph. D.

Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas.

Avec une surface de plus de six millions de kilomètres carrés, l'Amazonie abrite la plus grande forêt tropicale humide au monde, ainsi qu'une grande partie de la diversité terrestre. Cependant, les origines spatiales et temporelles de cette diversité demeurent mal comprises et nécessitent d'être plus étudiées pour identifier les causes de cette impressionnante diversification. Les amphibiens sont un groupe taxonomique particulièrement adapté à l'étude de la biogéographie historique en Amazonie car ils ont diversifié de manière importante dans la région et présentent d'importante variations d'utilisation de l'habitat ainsi que de capacités de dispersion entre groupes. Nous avons d'abord reconstruit l'histoire biogéographique du genre Allobates qui se rencontre principalement dans des forêts de terra-firme et pour lequel l'Amazonie de l'Ouest a joué un rôle important de source de diversification il y a entre 14 et 10 Millions d'années. Ce patron spatio-temporel coïncide avec une période marquée par la présence d'un système de méga-marécage qui recouvrait la plupart de l'Amazonie de l'Ouest il y a entre 23 et 10 Millions d'années. La décharge progressive de ce système marécageux a été suivie par une expansion des forêts de terra-firme en Amazonie de l'Ouest, une disponibilité en habitat favorable qui a probablement contribué à la diversification d'Allobates. Nos résultats suggèrent également que les rivières de l'Ouest Amazonien ont pu promouvoir la diversification au cours des 10 derniers Millions d'années en agissant comme une barrière semi-perméable à la dispersion, permettant la spéciation par dispersion suivie d'isolation géographique. Dans un second temps, nous avons reconstruit l'histoire biogéographique du groupe Pristimantis conspicillatus qui a diversifié de manière continue au cours du temps. Ce groupe présente un patron spatial de diversification particulier, avec quatre clades anciens ayant diversifié dans différentes aires simultanément, avec relativement peu d'évènements de dispersion comparé à Allobates. Ces importantes différences suggèrent que les espèces d'amphibiens ont des capacités de dispersion très variables qui sont probablement liées à leurs traits d'histoire de vie. Pour finir, nous avons comparé les histoires biogéographiques de six clades d'amphibiens partageant des temps d'origines comparables et qui sont représentatifs de la diversité Amazonienne en termes de taxonomie, de traits d'histoire de vie, d'utilisation de l'habitat et de type de reproduction. L'Amazonie de l'Ouest a été identifiée comme la principale source de diversification pour les amphibiens Amazonien, bien qu'elle ne se soit comportée comme tel qu'à partir d'il y a 10 Millions d'années pour les groupes présentant de l'adaptabilité dans la disponibilité de l'habitat ; et seulement entre il y a 10 et 5 Millions d'années pour les groupes conservatifs dans la disponibilité de l'habitat. Cela suggère que les groupes conservatifs dans la disponibilité de l'habitat remplissent la niche écologique plus rapidement que les espèces présentant de l'adaptativité, ce qui se traduit par des phases de diversification plus courtes. Nos résultats suggèrent également que les rivières agissent comme des barrières à la dispersion uniquement pour les groupes conservatifs dans l'utilisation de l'habitat. Nous tenons à souligner que ces résultats doivent être considérés avec prudence car ils ont été obtenus en étudiant une petite fraction de la diversité Amazonienne, ils apportent néanmoins de nouveaux éléments l'influence du conservatisme de niche sur les trajectoires évolutives en Amazonie, ce qui permettra peut-être d'inciter un plus grand effort de recherche sur cette thématique
With more than six million square kilometers, Amazonia hosts the largest tract of lowland tropical rainforest in the world and a large portion of the global terrestrial diversity. However, the temporal and spatial origins of this diversity remain poorly understood and need to be better comprehended to identify the processes responsible for this tremendous diversification. Amphibians are a particularly adequate group for investigating patterns of biogeographical history within Amazonia because they extensively diversified within the region and present important disparities in habitat use and dispersal abilities across groups. We first investigated the historical biogeography of the terra-firme genus Allobates and identified western Amazonia as an important source of diversification between 14 and 10 million years ago (Mya). This spatio-temporal pattern was coinciding with the existence of the Pebas system, a mega-wetland system that occupied most of western Amazonia during this period, that was unsuitable for terra-firme species. The Pebas system discharge was likely followed by an extension of terra-firme forests that likely fostered Allobates diversification. Our results also suggested that western Amazonia rivers might have subsequently (after 10 Mya) promoted diversification, by acting as semi-permeable barriers allowing speciation by dispersal and isolation. Secondly, we investigated the biogeographical history of the Pristimantis conspicillatus group which, instead, presented a continuous diversification throughout Neogene. This group displays a striking spatial pattern of diversification with four ancient clades that have diversified concomitantly in distinct areas in Amazonia and the Atlantic Forest, with much fewer dispersal events between areas than in Allobates. These differences suggest that amphibian species display differences in dispersal abilities that can be related to their life history traits. Finally, we compared the biogeographic histories of six frog clades, including the two aforementioned ones, that share comparable crown ages and span the Amazonian frog diversity in terms of life history traits, taxonomy, habitat use and reproduction modes. We identified western Amazonia as the principal source of diversification for Amazonian amphibians, although it acted as such only after 10 Mya for the groups that have adapted to various types of habitats; and only between 10 and 5 Mya for the ecologically conservative groups. This suggest that species with lower habitat availability reach niche filling more rapidly than ecologically adaptive species, resulting in shorter diversification phases. Our results also suggest that riverine barrier effect seems to have affected solely conservative groups particularly when the river course is stable over time. While these results were obtained by considering only a fraction of Amazonian diversity, they provide interesting insights on the influence of niche conservatism upon Amazonian evolutive trajectories, which will hopefully foster further and more ample research in this direction

Root system architecture of plant plays a key role in water and nutrient uptake from the soil, provides anchorage and acts as a storage organ. In this current research, we have focused on the molecular and physiological basis of root system variation in canola (Brassica napus L.). Genome wide association mappings in a diverse canola germplasm panel with ~37,500 and ~30,200 single nucleotide polymorphism (SNP) markers were conducted under greenhouse and field conditions, respectively. A total of 52 significant SNP markers associated with different root architectural traits were identified in the greenhouse study. Majority of the markers were distributed on five chromosomes, A01, A02, A04, C03 and C06, of B. napus. Twenty-two candidate genes related to root growth and development were detected within 50 kbp upstream and downstream of the significant markers. Three of these candidate genes, P-glycoprotein 6 (PGP6), Tetraspanin 7 (TET7) and ARABIDILLO-2, were co-localized with three markers on chromosome C03, A01 and A04, respectively. In the field study, 31 significant SNP markers associated with different root traits were detected. A total of 15 root related candidate genes were identified within 100 kbp upstream and downstream of different significant markers. We also analyzed and compared the transcriptomes from the root systems of spring (weak root system) and winter (vigorous root system) growth habits at two different time points, 30 and 60 days. A total of 169,646 transcripts were analyzed, of which, 582 and 555 transcripts were found to be significantly differentially expressed between spring and winter types at 30 and 60 days, respectively. Several cytokinin and gibberellin associated genes and genes sets were found to be upregulated in spring type compared to winter type at 60 days. Cytokinin has proven inhibitory effect on root system architecture in different crops, whereas, gibberellin promote root elongation but inhibit lateral root growth. Therefore, we suggest that cytokinin and gibberellin may play an important role in root system variation between spring and winter growth habits. Significant marker loci, candidate genes and transcriptome profile identified in this research will assist future research to understand the root system variation in rapeseed/canola.
North Dakota State University. Center of Excellence for Agbiotechnology
National institute of Food and Agriculture (U.S.)
Northern Canola Growers Association

Dopamine mediates its effects through the interaction with five distinct receptors that make up the D1-like (D1 and D5) and D2-like (D2, D3, and D4) families. Dopamine receptors are members of the heptahelical G protein-coupled receptor (GPCR) family. D1-like and D2-like receptors couple to the activation and inhibition of adenylyl cyclase, respectively. Dysfunction in dopaminergic signaling has been shown to contribute, among others to the etiology of Parkinson's disease, schizophrenia, and hypertension. The high degree of structural identity between D1 and D5 receptors has hampered the development of subtype-selective drugs. Despite the structural similarities, D1 and D5 receptor subtypes exhibit distinct ligand binding and G protein coupling properties. The objective of this thesis is to delineate the structural determinants involved in the distinct ligand binding and G protein coupling properties of D1 and D5 receptors Using chimeric and mutagenesis studies I demonstrate that differences in the primary sequence within the terminal receptor locus (a region encompassing TM6, third extracellular loop (EL3), TM7, and the cytoplasmic tail) are responsible for the functional differences of D1 and D5 receptors. I describe the EL3 domain as a key determinant in the binding of antipsychotic drugs (inverse agonists) and the agonist-mediated maximal activation of adenylyl cyclase. This study highlights a novel domain (EL3) regulating binding of inverse agonists at GPCRs. Furthermore, I describe a molecular interplay between TM6 and EL3 which mediates the subtype-specific phenotypes and activation of D1 and D5 receptors. In addition, I demonstrate that in spite of structural and functional similarities, D1-like receptors undergo a different regulatory pathway upon agonist stimulation. Specifically I demonstrate that the D5 receptor can undergo phosphorylation-independent desensitization and endocytosis. Overall, the work described in this thesis provides insight into the molecular basis of D1-like receptor signaling.

博士
國立成功大學
基礎醫學研究所
95
Angiostatin, a circulating inhibitor of angiogenesis, is an internal fragment of plasminogen that contains the first four kringle domains (K1-4) of plasminogen. Further studies showed that angiostatin-like molecules consisting of only the first three kringle domains (K1-3) was more antiangiogenic than K1-4, and that there is the existence of a naturally occurring isoform, K1-4.5. It is not clear whether different isoforms of angiostatin utilized similar or distinct pathways to mediate anti-angiogenesis. We first examined the signaling pathway mediated by proapoptotic angiostatin, K1-4. Inductions of p53-mediated intrinsic and FasL-mediated extrinsic death signaling pathways are involved in anti-angiogenic action of K1-4. We then compared the signaling pathways and mRNA expression profiles modulated by K1-3, K1-4, and K1-4.5. Although the extent of anti-angiogenic potency might vary among different isoforms, K1-3 or K1-4.5 shared similar death pathways with those mediated by K1-4. Moreover, all three forms of angiostatin induced a similar subset of mRNA expression with some variations. The common pathways shared by K1-3, K1-4, and K1-4.5 might be used as new therapeutic targets for anti-angiogenic therapy. Among the deregulated genes, the expression of E-selectin, an adhesion molecule, was not only induced by all isoforms but also previously implicated in antiangiogenic action of endostatin. Since K1-3 had the highest ability to induce E-selectin, we then used K1-3 to study the underlying mechanism responsible for the K1-3-induced expression of E-selectin. RT-PCR and western blotting analyses confirmed the time-dependent increase of E-selectin mRNA and protein induced by K1-3. Moreover, subcellular fraction and immunofluorescence microscopy confirmed the predominant presence of K1-3-induced E-selectin in lipid raft. Promoter driven reporter assays demonstrated both AP1 and Ets-1 binding sites on the E-selectin promoter were crucial for the induction of E-selectin. EMSA and ChIp assays confirmed the in vitro and in vivo binding of AP1 complex and Ets-1. Repression of JNK, a N-terminal kinase for c-Jun, significantly suppressed the K1-3 induced expression of E-selectin, suggesting a requirement of JNK activation for induction of E-selectin. The positive involvement of E-selectin in the anti-angiogenic action of K1-3 was, respectively, confirmed by overexpression and knockdown of E-selectin. In summary, we have identified the E-selectin as a novel target for the antiangiogenic action of angiostatin.

The Corticotropin Releasing Factor (CRF) peptides are an evolutionarily conserved peptide family integral to the stress response. In 2002, an investigation was undertaken into possible homologues of CRF, which led to the discovery of the Teneurin C-terminal Associated Peptide (TCAP) family located on the C-terminal end of the teneurin proteins. This peptide family is involved in neuroprotection against stress. TCAP possesses a number of biological actions independent of teneurin, however, evidence of how TCAP is processed from its proprotein had not been determined. I hypothesize that TCAP is transcribed independently and has independent functionality in promoting filopodia elongation and outgrowth. Here, I show that TCAP-1 is independently expressed as an mRNA separate from the teneurin gene. Moreover, a key step in the TCAP-1 mediated molecular mechanism of filopodia outgrowth may require an interaction between Elongation Factor-1α (EF-1α) and the actin cytoskeleton, where this mechanism is not modulated by CRF.

A subset of 199 Melissotarsus insularis and 130 Diaspididae specimens were analyzed to 1) determine the species status of M. insularis and 2) to explore the relative intimacy of the relationship between M. insularis and Diaspididae. An analysis of molecular variance and the observed lack of association between clades and distinct habitats on the M. insularis phylogeny suggested that while M. insularis exhibits isolation by distance, it does not apparently diversify by habitat. When cryptic COI pseudogenes were accounted for, the majority of the genetic diversity exhibited by M. insularis was limited to a divergence of 3% or less suggesting that M. insularis represents a single, albeit broadly distributed, species. A cophylogenetic reconstruction of the relationship between M. insularis and Diaspididae yielded 14 “cospeciation” events but was not significant unlike reconstructions of host-parasite relationships. Analyses of reduced datasets suggested that incomplete taxon sampling may significantly affect cophylogenetic reconstruction results.
National Science Foundation (grants No. DEB-0072713, DEB-0344731 to BLF and DEB-0842395 to BLF and MAS), a Natural Sciences and Engineering Research Council of Canada Discovery Grant to MAS and a Leaders Opportunity Fund grant from the Canada Foundation for Innovation to MAS

In 2018, it is estimated that 55,440 Americans will be diagnosed with pancreatic cancer and this figure is expected to continue to rise with increased life expectancy. Despite some measurable progress over the past few decades, pancreatic cancer remains one of the most lethal malignancies with five-year survival rate of 8.7%. Novel therapies, and their timely translation to the clinic, are urgently needed. As part of an effort to identify and characterize novel therapeutic strategies for pancreatic ductal adenocarcinoma, we began a study of the role of Bmi1 in tumor maintenance and progression. While Bednar and colleagues showed that Bmi1 is critical for the development of pancreatic cancer, and that its pancreas-specific deletion impairs PanIN formation, we were interested in assessing its function in established tumors. During the course of this work, we acquired a novel compound, PTC596, developed by PTC Therapeutics as a post-translational inhibitor of BMI1. Treatment with PTC596 leads to hyperphosphorylated BMI1, and this modification is associated a loss of protein activity. We planned to study this compound, in vitro and in vivo, as a complement to genetic perturbations of Bmi1. Initial characterizations of the effects of PTC596 on human and murine-derived pancreatic cancer cell lines revealed a potent anti-proliferative effect, accompanied by BMI1 hyperphosphorylation, and followed by polyploidy and cell death after prolonged treatment. Further analysis showed a clear G2/M arrest and elevated levels of phospho-histone H3. Bmi1 is known to play a role the cell cycle, but its inhibition in pancreatic cancer cell lines has been shown to induce G1 arrest. We decided to further explore the mechanism of PTC596’s antiproliferative effects by carrying out RNA sequencing on Aspc1 cells treated with PTC596. We found that 8 of the ten most down-regulated genes were members of the tubulin family and began to study this compound’s effect on microtubules. Compelling results from a cell-free tubulin polymerization assay support inhibition of tubulin polymerization as the mechanism of action for PTC596. These data are further supported by evidence that PTC596 increases the fraction of free-tubulin in treated cells, as well as dramatically alters the cell’s microtubule network. Given our laboratory’s interest in identifying novel therapies for pancreatic cancer, and the fact that PTC596 has already begun clinical trials, we continued to characterize this compound in vivo. We found PTC596 to have properties favorable for in vivo administration. PTC596 is orally available, has a plasma half-life of approximately 22 hours following oral administration, and accumulates in tumor tissue where it has an expected pharmacodynamic effect. Furthermore, it is well tolerated in vivo in combination with gemcitabine. We carried out a four-arm intervention study in tumor-bearing KPC mice, examining PTC596 alone and in combination with gemcitabine. We found that PTC596 synergizes with gemcitabine to significantly reduce tumor growth rates and provide a 3-fold extension of survival as compared to vehicle. These findings are, to our knowledge, the first evidence of in vivo synergy between a microtubule-destabilizing agent and gemcitabine for the treatment of pancreatic cancer. Importantly, this study identifies an alternative mechanism for PTC596 and implicates its efficacy in a novel treatment regimen for pancreatic ductal adenocarcinoma.

Mass spectrometry (MS) has become one of the most powerful and versatile tools for chemical analysis due to its ultra-high sensitivity, high throughput, ease of automation, and the large amount of information obtained. Nowadays, MS is extensively used in many tasks, such as identification and quantitation of drug metabolites, analysis of the products of biomass pyrolysis, and study of reactive intermediates, to name a few. However, these mass spectrometric analyses are not without challenges. For example, the requirement for quantifying trace amounts of substances in a complex mixture constantly pushes the detection limit of mass spectrometers, and the increased sample complexity demands higher and higher mass resolution. Therefore, MS is constantly evolving to address more difficult analytical challenges. A variety of MS techniques have been developed over the years, including soft ionization methods that facilitate mass spectrometric analysis of macromolecules, such as proteins and antibodies that enables the development of new therapeutic agents, benchtop high-resolution mass spectrometers, such as the orbitraps that can be used to analyze some of the most complex mixtures, and portable mass spectrometers which can be used in the home and garden and even in cancer surgery. Besides its applications in chemical analysis, MS can serve as a unique tool for the fundamental study of gas-phase ion/molecule reactions, these gas-phase reactions can be used to better understand the reactivities of many reactive intermediates and to obtain structural information for unknown analytes.

This thesis is aimed at addressing challenges involved in mass spectrometric analyses of isomeric drug metabolites (Chapter 4), quantitation of drug metabolites by using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) (Chapter 5), delineating cellulose depolymerization mechanisms upon fast pyrolysis by using pyrolysis-tandem mass spectrometry (py-MS/MS) (Chapter 6), and studying the reactivities of vinyl cation intermediates (Chapter 7). An overview of the dissertation research is given in Chapter 1, the instrumentation and principles of linear quadrupole ion trap (LQIT) mass spectrometer are discussed in Chapter 2, and the organic synthesis performed for several studies is detailed in Chapter 3.