Functional Diversity of Fibroblast Growth Factor Homologous Factor Family of Proteins
Year of Dissertation:
2010
Advisor:
Mitchell Goldfarb
Abstract
Colletotrichum gloeosporioides s.l. in North America: Sex, Host, and Habitat-mediated Diversity in a Plant-associated Ascomycete
Year of Dissertation:
2012
Determining the factors that drive the evolution of pathogenic fungi is central to revealing the mechanisms of virulence and host preference, as well as developing effective disease control measures. Prerequisite to these pursuits is the accurate delimitation of species boundaries. Colletotrichum gloeosporioides s.l. is a species complex of plant pathogens and endophytic fungi for which reliable species recognition has only recently become possible through a multi-locus phylogenetic approach. Through intensive regional sampling that encompasses multiple hosts within and beyond agricultural zones associated with cranberry (Vaccinium macrocarpon Aiton), we have integrated North American strains of Colletotrichum gloeosporioides s.l. from these habitats into a broader phylogenetic framework and characterized some of the factors that influence species diversity. We have developed polymorphic microsatellite markers for C. fructivorum, a species determined to be responsible for cranberry fruit-rot in agricultural areas throughout North America, in order to understand the biotic and abiotic factors that shape populations within the species complex. These markers amplify across several species within the C. gloeosporioides species complex and some are variable within two species, C. rhexiae and C. kahawae, that are closely related to C. fructivorum. Broad geographical and fine-scale hierarchical sampling of C. fructivorum and C. rhexiae coupled with multilocus genotyping has allowed us to gain insight into the forces that shape populations of these species. Human-mediated dispersal is an important factor dissipating the population structure of C. fructivorum throughout its range in commercial cranberry bogs. In contrast, limited evidence suggests C. rhexiae is geographically structured within a more restricted range, implying distinct patterns of diversity between Colletotrichum species associated with wild versus agricultural hosts. We also investigate the reproductive mode of C. fructivorum using estimates of haploid disequilibrium and genotypic diversity, inferring a mixed (sexual and asexual) mode of reproduction in field populations. We discuss the importance of sexual and asexual reproduction on population dynamics and speciation within the C. gloeosporioides species complex.
Novel Insights into Vascular Endothelial Growth Factor Receptor 2-Mediated Signaling to the Mammalian Target of Rapamycin/Akt Network in SK-N-SH Neuroblastoma Cells
Year of Dissertation:
2011
Advisor:
Patricia Rockwell
Mammalian target of rapamycin (mTOR) is a central regulator of cell growth and division that exerts many of its effects through regulating protein synthesis. The kinase Akt is a substrate and regulator of mTOR. These proteins are integral to pathological and physiological function in neuronal cells and the Akt/mTOR network is the focus of pharmaceutical interventions. Muscarinic acetylcholine receptors and vascular endothelial growth factor receptor 2 (VEGFR2) can signal protein synthesis but whether they cooperate to mediate mTOR activation has not been demonstrated. Using serum-starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of the mTOR substrate p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2-dependent manner. Protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid (OA) increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, OA elicited a far greater increase in phosphorylation, implicating phosphatase 2A (PP2A) as a critical determinant of their function. Furthermore, PP2A inhibition induces the appearance of novel, high molecular weight, ubiquitinated forms of Akt. The accumulation of phosphorylated Akt induced by PP2A dysfunction causes depletion of total Akt. Rapamycin potentiates Akt phosphorylation and depletion in response to OA through a mechanism regulated by a previously unknown function of VEGFR2. Although hyperactivation of Akt is a common survival mechanism in cancer cells, Akt hyperphosphorylation is associated with induction of a caspase-independent cell death mediated by oxidative stress. Taken together, these results show that the critical role of PP2A in regulating Akt activation also affects Akt ubiquitination, cleavage and removal from the cell. Furthermore, these data indicate the importance of reactive oxygen species in eliciting cell death and that PP2A promotes survival through a suppression of oxidative stress. Finally, VEGFR2 can stimulate mTOR when stimulated by ligand binding, transactivation or an unknown mechanism induced by rapamycin.
Systematics and biogeography of the New World scorpion genus Centruroides Marx, 1890 (Scorpiones: Buthidae)
Year of Dissertation:
2011
Background: The New World scorpion genus Centruroides Marx, 1890 (family Buthidae Koch, 1837) is a morphologically diverse and highly venomous taxon. Centruroides is among the most complex scorpion genera in the New World, comprising 71 described species and 5 subspecies in addition to several undescribed species. These scorpions are sexually dimorphic, the males typically exhibiting elongation of the metasoma and telson and longer, more slender pedipalp chelae. The greatest diversity of Centruroides occurs in Mexico; however the genus is distributed from the southern United States into northern South America and the Galapagos, and throughout the Caribbean. The genus includes the only scorpions of medical importance in North America, with six species that are potentially lethal to humans.
Multifunctional Roles of APL-1 in C. elegans
Year of Dissertation:
2011
Alzheimer's disease is an age-dependent disorder and the most common type of dementia. The most prevalent mutations associated with familial Alzheimer's Disease are found in the gene encoding the amyloid precursor protein (APP) or in the presenilin genes, which encode proteases that cleave APP. In mice, knockout of the APP gene family leads to lethality and type II lissencephaly, while overexpression of APP causes a shortening in lifespan and learning defects. However, the cellular function of APP and the pathways in which APP acts are unknown.
Evolution of Song Culture in the Zebra Finch
Year of Dissertation:
2009
Advisor:
Ofer Tchernichovski
Cumulative cultural evolution is when behavior in subsequent generations of learners builds on the accumulated information of previous generations to such an extent that no individual learner can produce the behavior on its own. Many examples exist in humans, but in nonhuman animals there are only a handful of suspected cases. Here, we provide the first demonstration of cumulative cultural evolution in the laboratory in nonhuman animals. We raised zebra finches in complete acoustic and social isolation to create "uncultured" animals. Isolate zebra finches sing unstructured songs that are different from wild-type songs in many aspects such as spectral details of syllables and syntactic organization. We developed an automated procedure to quantify the differences between isolate and wild-type song at different timescales of song structure: spectral features, duration of sounds, and song rhythm. We then used the isolate birds to teach their songs to juveniles who became the tutors for the next generations of learners and so on recursively. We followed the evolution of isolate song over multiple generations. We found that isolate song was gradually transforming into wild-type song over 3-4 learning generations. In addition to this experiment where we trained young birds in individual tutor-pupil pairs, we established a semi-natural colony with an isolate founder and tracked song changes over multiple generations of learners. In the colony, the song also progressed towards wild-type song in a few generations, but some of the details of the changes differed between the two conditions. The rapid evolution indicates that wild-type song culture is encoded in every bird, but it takes multiple generations to surface. The young birds used imitation biases to change isolate song features into wild-type features.
C. elegans ADAMTS ADT-2 regulates body size and cuticle collagen organization
Year of Dissertation:
2010
Advisor:
Cathy Savage-Dunn
The regulation of body size is a fundamental feature of animals critical to their survival and fitness, yet its underlying mechanisms remain poorly understood. In C. elegans, the DBL-1 signaling pathway plays a major role in growth control. The mechanisms by which other pathways regulate body size function, however, are less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma 21. I now demonstrate that sma 21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix (ECM) and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with known size regulatory pathways. I further demonstrate that ADT-2 activity is required for normal cuticle collagen fibril organization and adt-2 regulatory sequences drive expression in glial-like cells. ADT-2::GFP fusion protein is localized in the alae and the annuli of the cuticle. We therefore show that ADT-2 is secreted into the cuticle where it may act to proteolytically process secreted collagen or other ECM molecules required for normal cuticle structure and body size.
EVOLUTION, PHYLOGEOGRAPHY AND SPECIES BOUNDARIES OF THE RINGNECK SNAKE GENUS DIADOPHIS
Year of Dissertation:
2009
EVOLUTION, PHYLOGEOGRAPHY AND SPECIES BOUNDARIES OF THE RINGNECK SNAKE GENUS DIADOPHIS
Phylogenetics, biogeography and co-adaptation between a Batesian model (Micrurus fulvius) and mimic (Lampropeltis triangulum)
Year of Dissertation:
2009
The coral snake, Micrurus fulvius (Elapidae), and the scarlet king snake, Lampropeltis triangulum (Colubridae), members of a Batesian mimicry complex in the southeastern and southern United States were subjects of a study of model-mimic co-evolution. Inferring co-evolution required discovery of distinct terminals within each group, for posterior phylogenetic inference and analysis of co-variation of color patterns among sympatric populations of each species. In addition, haplotype and population differentiation were assessed. Also, hypotheses regarding selection on color patterns were tested. The latter included model-mimic tracking, environmental effects and frequency dependence of mimetic color pattern variation. Population Aggregation Analysis failed to diagnose terminals for either group. Haplotype and population genetic analyses showed both groups to be largely panmictic, with some north/south differentiation. Morphometric analyses of means and variations in proportions of color pattern components failed to reveal geographic tracking by mimics of models, but manifested possible independent geographic or environmental effects on each of the species. Frequency-dependent selection was falsified for color pattern variation in mimics.
Islet-brain-2: a novel postsynaptic density protein linked to an autism spectrum disorder
Year of Dissertation:
2010
Advisor:
Mitchell Goldfarb
Islet-brain-2 (IB2) is a neuronal protein, whose functions are not well understood. Based upon its sequence homology to JNK-interacting protein 1 (JIP1) and biochemical studies, IB2 has been described as a putative scaffold for mitogen activated protein kinase (MAPK) signaling. IB2 has been documented to interact with a wide array of functionally unrelated proteins, which has complicated efforts to confirm its biochemical role in vivo. In order to investigate the IB2 function in the nervous system, we have generated Ib2 null mice. The mutants are viable and their expression of genes neighboring Ib2 is unaffected. Ib2 knockout mice display developmental delay in grip strength until 5 weeks (P35) of age. Their behavioral analysis following disappearance of this defect shows significant deficits in their motor learning abilities. In addition, mutant mice exhibit marked reduction in social interactions, delayed fear induced learning and unresponsiveness to the environment in various behavioral tasks. These complex atypical behaviors are reminiscent of autism spectrum disorders (ASDs). Interestingly, the human Ib2 gene resides within the deleted chr22qter region in Phelan-McDermid syndrome and patients with this disorder manifest similar deficits as observed in our Ib2 null mice, suggesting that Ib2 loss-of-function is a promising candidate model for this disorder. Recent developments in the ASDs field implicate as a major culprit defective synaptic function. Using brain fractionation and co-immunoprecipitation methods, we show that IB2 is an integral component of the postsynaptic density (PSD). Immunofluorescence shows IB2 concentrated within dendritic spine heads. Ib2 mutation did not alter expression levels of many other common PSD components such as PSD95 and AMPA-type and NMDA-type receptors subunits. Signaling analyses conducted in Ib2 null cultured cortical neurons failed to reveal deficits in NMDA-evoked signaling through known IB2-interacting partners TIAM1 and p38MAPK, suggesting that IB2 modulates synaptic and behavioral functions through as yet unknown molecular mechanisms.