The purpose of my research was to analyze the effect flexible grouping had on science retention scores. Education faces so many challenges today. The old way of teaching, traditional lecture is being challenged. For centuries, traditional lecture was the primary way of teaching and most colleges still use this teaching method. In this thesis traditional lecture and flexible grouping are compared. Through the use of pretests and posttests in my traditional and honors chemistry classes, I tested both strategies. Presentation of seven chapters were used for data collection and through the research, several things were discovered. In this study, honor students had more knowledge of subjects going into each subject than my traditional students. Data, collected for a year, showed no statistically significant differences between traditional lecture and flexible grouping. Through the normalized gain, it was discovered that flexible grouping did have a positive influence on student learning. The posttest scores on average were better for flexible grouping than traditional lecture.

Cells that are migrating in a chemical gradient display a polarized distribution of signaling and cytoskeletal components. We have investigated the mechanism of polarity establishment by using the model system Dictyostelium discoideum. We used both vegetative cells that are unpolarized and developed cells that have a distinct front and back and display a polarized morphology. In order to gain insight into the role of polarity during migration, we developed an open microfluidic device (OMD) that confines cells in narrow channels. The first version of the OMD was used to study the migration of unpolarized and polarized cells by luring them into microchannels with one micropipette generating a gradient of either folic acid (for vegetative cells) or cAMP (for developed cells). The second version used two micropipettes that were placed on either side of the microchannels and permitted gradient switching. We were able to observe the redistribution of signaling and cytoskeletal components as the cells reestablished their polarity after the gradient was switched. These experiments revealed that Ras activity and PTEN are reciprocally regulated, and suggest that high PI(4,5)P2 levels block Ras activity at the new leading edge. Similar findings were found in randomly moving and in dividing cells. F-actin polymerization occurs simultaneously with front extension, while microtubules redistribute to the rear and orient the microtubule organizing center (MTOC) and nucleus in polarized cells. Treated cells lacking an actin cytoskeleton had signaling responses, but did not re-localize the MTOC/nucleus in spatial gradients of chemoattractant. PTEN, which binds PI(4,5)P2 is not necessary for initial establishment of the cell rear but helps stabilize polarity by increasing backness at the rear of the cell.

Genetic data are frequently responsible for biological insight and recent advances in sequencing technology (high-throughput sequencing; HTS) have created massive DNA-sequence based datasets. While these technologies are invaluable, there are many analytical and application issues that need to be addressed. With these data we can ask and answer novel biological questions that were previously inaccessible. One major challenge in applying HTS to biological questions is data management: the file formats and sizes are foreign to many primary researchers. In the second and third chapters of this dissertation, I introduce two pieces of software that allow researchers to utilize HTS with minimal time investment. PRGMATIC (Chapter 2) is a pipeline that collates raw HTS data into a more traditional and useable format: two diploid alleles for a given locus. LOCINGS (Chapter 3) uses these loci, the alignments from which the loci were called and demographic data to display and output important summary statistics. This program also reformats appropriate loci into three widely used biological file formats. Chapters 4 and 5 focus on a novel application of HTS to phylogeographic inference. The collective set of microbial organisms on and inside vertebrates (the microbiota) is a vast genetic resource that is poorly understood. What factors shape these communities? Chapter 4 uses an avian brood parasite (Brown-Headed Cowbird) to naturally decouple parental genetics and early environment. Cowbird gut microbiota do not cluster with each other in multivariate space. They also do not strongly affiliate with host species. Age and sampling locality are most strongly associated with the gut microbiota. Chapter 5 looks for host taxonomic and spatial signals in a more broadly sampled dataset of 60 species sampled across Costa Rica. Here, host taxonomy is most significantly associated with gut microbiota and ecological variables like host diet and foraging strata are secondarily important. Together, these chapters present novel tools and uses of HTS for evolutionary inference. The two programs, PRGMATIC and LOCINGS, allow primary researchers to utilize HTS easily. The two bird datasets, cowbirds and Costa Rican birds, demonstrate how analyzing the microbiota with HTS can provide and address novel evolutionary questions.

Understanding biodiversity is one of the driving foundations of evolutionary biology and researchers use a myriad of tools to uncover and understand the processes contributing to it. The evolutionary and ecological dynamics in a group of smooth-toothed pocket gophers, the <i>Thomomys umbrinus</i> species complex, is studied for this dissertation. This complex is distributed from south-central Arizona and southwestern New Mexico south to Veracruz, Mexico. The genetic complexity of <i>T. umbrinus</i> was initially discovered via allozymes and karyotypes, resulting in five genetic clades: three with one diploid number of chromosomes (2n = 76; two clades distributed in the Sierra Madre Occidental and 1 along the Pacific Coast) and two with a different diploid number (2n = 78; one in the Northern Desert and one in the Central Plateau). Analyses of DNA sequences from 8 genes and genotype assignment tests for 21 allozyme loci establish the Sierra Madre clade within what was formerly <i>T. umbrinus</i> as a genetically isolated taxon. Accordingly, <i>Thomomys sheldoni</i> Bailey, 1915 is resurrected to recognize this divergent clade of pocket gophers with a diploid number of 2n = 76. A synonymy is provided for two subspecies within <i>T. sheldoni</i> based on a concordant genetic and morphological break. Multi-locus genetic analyses reveal a previously undescribed species of pocket gopher (2n = 76) apparently restricted to the Sierra del Nayar of northeastern Nayarit. Molecular, chromosomal, and cranial morphometric data distinguish this new species from other members of the <i>T. umbrinus</i> species complex. This new taxon, <i>T. nayarensis</i>, is described and a key to distinguishing the 3 species of <i>Thomomys</i> in northeastern Nayarit is provided. Subspecies relationships within <i>T. umbrinus</i> (2n = 78) are reevaluated using phylogenetic analyses, species tree analyses, allozymes, and morphology. Phylogenetic analyses confirm three genetic clades (Northern Desert, Central Plateau, and the Trans-Mexico Volcanic Belt [TMVB]). Reanalysis of published allozyme data shows no evidence of nuclear discordance among the three clades. Species tree analyses reveal four divergent lineages (two within the TMVB clade), which are recognized herein at the subspecies level. Species distribution models were used to assess biotic and climatic factors that may influence how members of the <i>T. umbrinus</i> complex are distributed. <i>T. sheldoni</i> and <i>T. atrovarius</i> had well-predicted niches and climatic variables that differentiated them from the <i>T. umbrinus</i> clades. Niche equivalency tests were rejected and evidence of niche conservatism was found between some, but not all, members of the species complex, indicating a complex history of niche evolution, competition, and genetic differentiation in the <i>T. umbrinus</i> species complex.

BIOLOGICAL SCIENCES INSIGHTS INTO THE ROLES OF HUMAN CDC6 AND REPLICATION PROTEIN A IN INITIATION OF EUKARYOTIC DNA REPLICATION VITALY VLADIMIROVICH KLIMOVICH Dissertation under the direction of Professor Ellen H. Fanning Since the discovery of the structure of DNA as an information-bearing molecule of the cell in 1953 by Watson and Crick, considerable effort was directed towards elucidating the mechanism by which DNA replication occurs. Simian virus 40 (SV40) provides a powerful model system for study of eukaryotic DNA replication in which a viral protein, large T antigen (Tag), marshals the hosts replication machinery to replicate the viral mini-chromosome. SV40 replication requires interaction of Tag with the host ssDNA binding protein, replication protein A (RPA). The C-terminal protein interaction domain of the RPA32 subunit (RPA32C) facilitates initiation of SV40 DNA replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR were used to demonstrate physical interaction between RPA32C and the origin DNA binding domain of Tag. The structures of these domains were docked together using NMR data. Based on electrostatic complementarity in the complex, point mutations were designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact RPA impaired initiation of SV40 DNA replication and primosome activity, implying a critical role for this interaction in assembly and progression of the SV40 replisome. Cell division cycle 6 (Cdc6) protein plays an essential role in initiation of DNA replication by loading the minichromosome maintenance (MCM) complex of proteins onto chromatin. In order to accomplish its function, human Cdc6 (hCdc6) must also be phosphorylated by cyclin-dependent kinases. Phosphorylation of mammalian Cdc6 is also required for its export from the nucleus to the cytoplasm for replication to ensue. Analysis of several GFP-tagged phosphorylation deficient mutants of hCdc6 for sub-cellular localization in microinjected human cell lines suggests that phosphorylation alone is not sufficient for hCdc6 export from the nucleus. We propose that in human cells an additional mechanism, that could involve human prolyl isomerase, must be activated to initiate replication leading to the release of hCdc6 from the nucleus. Approved_____________________________________Date_______________________

PSF (PTB-associated splicing factor) has been implicated in both early and late steps of pre-mRNA splicing as well as multiple nuclear events including transcription regulation, nuclear RNA retention/export, topoisomerase activity, and DNA recombination. We found that PSF and its closely related protein p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with the U4/U6.U5 tri-snPNP. Further analysis of the association of PSF with U5 snRNP resulted in the interesting discovery that a portion of PSF is contained within a large multi-snRNP complex (PCC complex). The formation of the PCC complex occurs when HeLa cell nuclear extracts are adjusted to splicing conditions but surprisingly does not require the addition of pre-mRNA nor ATP hydrolysis. Sedimentation analyses demonstrated that the complex contains all of the five splicing snRNPs and has a size close to the spliceosome. Approximately 70 proteins were identified in the PCC complex by mass spectrometry analysis, and 70% of them are either known spliceosomal proteins or proteins related to splicing. This suggests that penta-snRNPs and/or pre-formed spliceosomes may exist in mammalian cells.

The temperature-sensitive (TS) paralyzed and blind Drosophila mutant rolling blackout (rbo) identifies a gene encoding a protein that pioneers a novel family of membrane-associated putative lipolytic enzymes highly conserved from yeast to human. RBO protein is enriched in synapse-dense neuropil and predominantly localized in presynaptic boutons at neuromuscular junction synapses, but undetectable in central neuronal cell bodies. The protein is also abundant throughout sensory neurons, including retinal photoreceptors. RBO protein is acutely required for both phototransduction and synaptic transmission. rbo TS mutants show a reversible loss of phototransduction in an activity-dependent manner, in correlation with an activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2) within minutes at non-permissive temperature. These results suggest a rapid down-regulation of phospholipase C (PLC) activity. rbo TS mutants also show reversible TS paralysis and complete block of both central and peripheral synaptic transmission within minutes at non-permissive temperature. This loss of synaptic transmission, at least partially, is due to a block of synaptic vesicle (SV) exocytosis downstream of SV docking. The conclusion is that RBO putative lipase may regulate the PLC-PIP2 signaling in both phototransduction and synaptic transmission.

During vertebrate gastrulation, massive cell movements shape the basic body plan. Key components of gastrulation are convergence and extension (C&E) movements, which narrow and lengthen the embryonic tissues, respectively. By studying knypek;trilobite non-canonical Wnt mutants with impaired C&E, I demonstrate crucial roles of C&E gastrulation movements in multiple aspects of zebrafish somite development. Combining time-lapse analyses and computational simulations, I show that C&E movements of the medial presomitic mesoderm are achieved by the cooperation of planar and radial cell intercalations that preferentially separate anterior and posterior neighbors. In knypek;trilobite double mutants, the anteroposterior bias of cell intercalations is reduced and the asymmetric localization of non-canonical Wnt components is lost, revealing that non-canonical Wnt signaling defines distinct properties of anterior and posterior cell edges to bias the orientation of cell intercalation. Using cell tracing analyses and genetic manipulations, I demonstrate that C&E mediates the specification and fate maintenance of slow muscle precursors, the adaxial cells. During gastrulation, C&E regulates the number of specified adaxial cells by defining the size of the interface between the inducing axial and target presomitic tissues. After somite forms, convergence movements ensure the tight apposition of adaxial cells to the notochord, which is necessary for their continuous Hedgehog reception and fate maintenance. I further demonstrate that in knypek;trilobite double mutants, impaired C&E disrupts notochord development, which in turn impedes the adaxial cell shape changes and interferes with their lateral migration during later somitogenesis. <p> Additionally, in a genetic screen for genes essential for zebrafish early development, we isolated the calamityvu69 mutation that affects pigmentation and notochord development. We determined that calamityvu69 inactivates a zebrafish homolog of the human Menkes atp7a gene, which is critical for copper homeostasis in humans. Genetic mosaic analyses indicate that atp7a acts cell-autonomously in melanocytes to ensure melanin synthesis. Identification of the calamityvu69 mutant establishes a novel vertebrate model for studying the human Menkes disease and copper metabolism. <p>

Drs2p, a phospholipid translocase required for generation of membrane asymmetry, plays a role in the clathrin-coated vesicle formation from the trans-Golgi network (TGN) and endosomal membranes. The mechanism for coupling Drs2p to specific transport pathways requires its appropriate localization. To maintain its steady-state residence at the TGN, Drs2p primarily cycles between the TGN and the early endosome, although it occasionally traffics to the plasma membrane where it is rapidly endocytosed and efficiently retrieved to the TGN from early endosomes. Endocytosis of Drs2p is mediated by multiple signals, including two NPFXD motifs near the C-terminus and PEST-like sequences near the N-terminus that likely mediate ubiquitin (Ub)-dependent endocytosis. The NPFXD motifs are specifically recognized by Sla1p, part of an endocytic coat/adaptor complex with clathrin, Pan1p, End3p, and Sla2p. When both ubiquitin- and NPFXD-dependent endocytic mechanisms are abrogated, Drs2p accumulates on the plasma membrane. Surprisingly, the pan1-20 temperature-sensitive mutant is constitutively defective for Ub-dependent endocytosis but is not defective for NPFXD-dependent endocytosis at the permissive growth temperature. To sustain viability of pan1-20, Drs2p must be internalized through the NPFXD/Sla1p pathway. Thus, Drs2p is an essential endocytic cargo for the NPFXD/Sla1p system in cells compromised for Ub-dependent endocytosis. These discoveries provide a novel example for the critical role of endocytosis in retrieving Golgi proteins that escape to the plasma membrane. The clathrin adaptor AP-1 interacts with Drs2p and disruption of AP-1 dramatically increases the rate of Drs2p transport to the plasma membrane. Retrieval of Drs2p back to the TGN seems to be unaffected in AP-1 mutants, since the steady-state localization is not perturbed. Therefore, Drs2p depends on AP-1 for anterograde transport from the TGN to the endosome, but not for endosome to TGN retrograde transport. Importantly, Drs2p is required for AP-1 function, but does not appear to contribute significantly to the GGA/clathrin pathway. AP-1 and clathrin are recruited to TGN and endosomal membranes in the absence of Drs2p but fail to support transport of AP-1/clathrin-dependent cargo. Based on these observations, we propose that Drs2p plays an essential role in budding vesicles by pumping phospholipid to the cytosolic leaflet to generate positive membrane curvature that is captured by AP-1/clathrin coats. Drs2p actually embarks within these AP-1/clathrin-coated vesicles for delivery to the early endosome, but uses an AP-1 independent pathway for retrieval back to the TGN.

The cerebellum, which forms from anterior hindbrain, coordinates motor movements and balance. Sensory input from the periphery is relayed and modulated by cerebellar interneurons, which are organized into layers. The mechanisms that specify the different neurons of the cerebellum and direct its layered organization remain poorly understood. Drawing from investigations of spinal cord, we hypothesized that the embryonic cerebellum is patterned on the dorsoventral axis by opposing morphogens. We tested this using zebrafish. Here we show that expression of olig2, which encodes a bHLH transcription factor, marks a subset of PNs. In combination with other markers, olig2 reveals a dorsoventral organization of cerebellar neurons in embryos. Disruption of Hedgehog signaling, which patterns the ventral neural tube, produced a two-fold increase in the number of olig2+ PNs. By contrast, olig2+ PNs did not develop in embryos deficient for Wnt signaling, which patterns dorsal neural tube, nor did they develop in embryos deficient for both Hedgehog and Wnt signaling. Our data indicate that Hedgehog and Wnt work in opposition across the dorsoventral axis of the cerebellum to regulate formation of olig2+ PNs. Specifically, we propose that Hedgehog limits the range of Wnt signaling, which is necessary for olig2+ PN development.