Biology Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/2749

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Now showing 1 - 7 of 7
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    Myosins IIIa and IIIb regulate stereocilia length by transporting espin 1 to the polymerizing end of actin filaments
    (2009) Merritt, Raymond Clyde; Kachar, Bechara; Popper, Arthur N; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Mutations in the myosin IIIa gene are linked to DFNB30 late onset deafness, in which those affected individuals can hear for the first twenty years of life. Mutations in the espin gene are linked to DFNB36 congenital deafness. Both myosin IIIa and espin 1 are expressed in the inner ear hair cells and colocalize at stereocilia tips, the site of actin polymerization. Overexpression of these proteins in hair cells produce an increase in stereocilia length, and when both are co-expressed together they produce a length increase greater than when each one are overexpressed individually. These results suggest that these proteins interact and influence stereocilia length regulation. We confirmed the interaction of these two proteins in heterologous COS-7 cells. We observed that when co-expressed, these proteins promote elongation of filopodial actin protrusions in a synergistic manner. Mutational analyses show that myosin IIIa3THDI binds to the ankyrin repeats of espin 1. Live and fixed cell imaging shows that myosin IIIa transports espin 1 to the filopodia tips where espin 1 promotes actin polymerizations through its WH2 domain. Because of the late onset of deafness associated with myosin IIIa, it has been speculated that the lack of myosin IIIa function is partially compensated by the paralogous protein, myosin IIIb. Myosin IIIb, encoded by a distinct gene, lacks a C-terminal actin-binding domain shown to be essential for myosin IIIa filopodia tip localization. We observed that myosin IIIb localizes at stereocilia tips and is expressed at an earlier stage than myosin IIIa. We confirmed that myosin IIIb transports espin 1 to stereocilia tips and promotes actin polymerization, consistent with the hypothesis that it partially compensates for myosin IIIa. We found that binding to espin 1 is required for myosin IIIb movement and localization. We observed that myosin IIIb can downregulate the myosin IIIa localization in vestibular, but not in cochlear, hair cells. The interplay of myosins IIIa, IIIb, and espin 1 and their influence on stereocilia length unravels a novel molecular complex at the polymerizing end of F-actin and a framework to understand the cause of DFNB30 and DFNB36 deafnesses.
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    Breaking the A-P axis: Evolution of diverse asexual reproduction strategies in Convolutriloba acoels
    (2009) Sikes, James M.; Bely, Alexandra E.; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The defining characteristic of the Bilateria is the presence of a distinct head end and tail end, which defines the anterior-posterior (A-P) axis, a feature that is established during embryogenesis and generally remains unaltered during the lifetime of an organism. While a few bilaterians have evolved asexual reproduction strategies that allow them to subdivide the A-P axis, acoels in the genus Convolutriloba have an unparalleled ability to alter the A-P axis during modes of transverse fission, longitudinal fission, and reversed polarity budding. Convolutriloba acoels thus offer an exceptional opportunity to investigate the mechanisms that allow for the radical modification of an already established A-P body axis and to explore the evolution and development of diverse asexual reproduction strategies among related species. In this study, I reconstruct the evolutionary history of asexual reproduction in the Convolutriloba and compare the diverse modes of asexual reproduction at the level of body-wall musculature, nervous system development, and cell proliferation while also exploring the regenerative potentials of tissues across species with different modes of asexual reproduction. In addition, I further explore the unusual process of A-P axis reversal that occurs during reversed polarity budding in C. retrogemma through studies of body patterning and regeneration. The results of these analyses suggest that a rich developmental toolkit of regenerative abilities, including the ability to utilize both epimorphosis and morphallaxis, to regenerate all parts of its body even from a small fragment, and to produce bifurcated A-P axes were present in the ancestor of the Convolutriloba allowing for the evolution of A-P axis modifications unlike any other bilaterian group. This toolkit along with the evolution of a seemingly unpatterned zone of tissue within the body of C. retrogemma capable of generating new anterior axes appear to have allowed this species to evolve the ability to form reversed A-P axes during budding.
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    Differential requirements of the hindbrain and mesenchyme on inner ear patterning
    (2009) Liang, Jennifer Kimiko; Lee, Hey-Kyoung; Wu, Doris K; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Microsurgical manipulations were performed in ovo to identify the tissues that are required for conferring inner ear patterning. Our results show that the hindbrain, namely rhombomeres 5 and 6, are required for the formation and patterning of the cochlear duct (basilar papilla). Rhombomere 5 and its underlying notochord appear to be important for the growth of the cochlear duct, whereas rhomobomere 6 and its respective notochord are required for cochlear patterning. Rotating the segment of hindbrain from rhombomere 5 to rhombomere 6 along the anteroposterior axis affects cochlear duct formation but has no effect on the development of vestibular structures. The signaling molecules intrinsic to these tissues are distinct from Sonic Hedgehog, which has been shown to be required for cochlear duct outgrowth. In contrast, otic mesenchyme adjacent to the developing inner ear provides anteroposterior axial information to pattern the anterior and posterior canals and ampullae.
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    Ceramide Metabolism and Transport: Implications on the Initiation of Apoptosis
    (2006-12-19) Stiban, Johnny; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Apoptosis is a process by which unwanted cells are eliminated in a controlled manner. Early in apoptosis, ceramide levels rise and the mitochondrial outer membrane becomes permeable to proteins. The permeability of the outer membrane is attributed to the self-assembly of ceramide in form of channels. In the only direct structural study, to date, ceramide channels were visualized in liposomes using transmission electron microscopy. Those channels were of various sizes, averaging 10 nm in diameter. In concert, using electrophysiological techniques, the estimated diameter of ceramide channels was also around 10 nm. These channels are large enough to release all the pro-apoptotic intermembrane space proteins to initiate apoptosis. Dihydroceramide desaturase converts the inactive precursor, dihydroceramide to ceramide. Both long and short chain dihydroceramides inhibit ceramide channel formation in mitochondria. The inhibition is strong as one tenth as much dihydroceramide inhibited the outer membrane permeabilization by 95% (C2) and 51% (C16). Other mitochondrial components are not required for such inhibition as comparable amounts prevented the permeabilization of liposomes. Hence, the apoptogenic activity of ceramide may depend on the ceramide to dihydroceramide ratio perhaps resulting in a more abrupt transition from the normal to the apoptotic state. The location of the desaturase is the endoplasmic reticulum (ER). Only minimal activity was measured in mitochondria. However, newly synthesized ceramide from 14C-C8-dihydroceramide or 3H-sphingosine (in the ER) can transfer rapidly to mitochondria (40 % in 10 min) and permeabilize them to cytochrome c and adenylate kinase. The transfer of sphingolipids is bidirectional and non-specific. The transfer mechanism is consistent with direct membrane contact, since reducing the organellar concentrations by half resulted in a four-fold reduction of the transfer rate. Thus this ceramide exchange obviates the need for a complete ceramide de novo pathway in mitochondria in order for cells to use ceramide to activate mitochondria-mediated apoptosis. These results demonstrate the ability of ceramide to form large channels capable of releasing proteins from mitochondria. Ceramide can rapidly reach mitochondria and there are mechanisms to control the propensity for ceramide channel formation. Clearly ceramide channels play a central role in the decision to undergo apoptosis.
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    Unconventional Myosins in Fish Ears
    (2005-04-21) Coffin, Allison; Popper, Arthur N; Kelley, Matthew W; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Unconventional myosins are critical motor proteins in the vertebrate inner ear. Mutations in both myosins VI and VIIa cause multiple forms of human hereditary deafness but the precise function of these proteins is unknown. This dissertation uses a comparative approach to better understand the role of myosins VI and VIIa in vertebrate ears. Gene expression and protein distribution for these two myosins is examined in the ears of evolutionarily diverse fishes. RT-PCR data shows that myo7a is expressed in the ears of all taxonomically diverse fish species examined here, and immunofluorescence reveals that myo7a protein is distributed throughout the sensory hair bundles of all inner ear regions. Myosin VI expression and distribution is more complex. Studies in other laboratories show that zebrafish (Danio rerio) have two myo6 paralogs with differing gene expression patterns. This dissertation extends previous findings by showing that all teleost fishes have two myo6 genes while non-teleost fishes and tetrapods have one, suggesting that myo6 duplication occurred in an ancestral teleost, probably during a genome-wide duplication. RT-PCR experiments suggest that both myo6 paralogs are expressed in teleost ears. mRNA localization with in situ hybridization shows, however, that myo6a is not expressed in sensory epithelia. Immunocytochemical data shows that myo6 protein is distributed throughout hair bundles in all inner ear end organs of the sea lamprey (Petromyzon marinus) and the zebrafish but is not found in utricular hair bundles in other fishes. While protein expression studies find that the myo6 antibody used in this dissertation binds to both myo6 proteins in the zebrafish, the gene expression studies suggest that only myo6b is expressed in hair cells, and therefore that this is differential distribution of a single protein. This dissertation adds depth to current studies of myo6-associated hereditary deafness and suggests that comparative studies between zebrafish and other fishes such as shad (Alosa sapidissima) that differ in myo6 protein distribution will help elucidate the function of this critical hair cell protein. Comparisons between the two myo6 paralogs will further aid in functional studies and shed light on evolutionary processes during the teleost radiation.
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    REGULATION AND DISTRIBUTION OF TRANSPOSONS IN HYPERTHERMOPHILIC ARCHAEA
    (2004-04-30) Kanoksilapatham, Wirojne; Robb, Frank T; Biology
    The genus Pyrococcus consists of approximately 12 described species that are Archaea with optimal growth temperatures near to 100 oC. Pyrococcus furiosus and Pyrococcus woesei were both isolated from marine sediments in the same Italian Vulcano Island site. Insertion sequences (ISs) were identified in the genomes of P. furiosus and P. woesei. Specific primers to differentiate between the types of IS elements were developed. Nucleotide sequence of the rDNA operon (AY519654) from P. woesei was sequenced for the first time. Extensive co-linearity between the genomes of the P. woesei and P. furiosus was demonstrated using radio-labeled IS element sequence probes. A recent transposition event that disrupted the napA gene of P. woesei was revealed. The presence of a type I IS element was observed in the same context in the genomes of P. furiosus and P. woesei. The strains were proposed to be sub sp. of P. furiosus, based on their identical rDNA operon sequence and the presence of IS element markers. The ISs have putative archaeal promoters with a 5' TATA box "boxA". Full length IS mRNAs were detected. Evidence for programmed translational frameshift sites that might limit the translation of the full length transposase was found. Two functional programmed translational frameshift sites were identified in E. coli. The mechanism of frameshifting identified in context 79 in the tnp-I sequence appears to be a novel -2 mechanism, in that the shift site is part of a -1 frameshifting motif. The possible role of a putative ribosome binding site (rbs) observed in context 79, that correlates with high frameshift levels, is discussed.
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    Activity-dependent regulation of Schwann cell development by extracellular ATP
    (2003-12-05) Stevens, Beth; Davenport, Roger W; Fields, Douglas; Biology
    During development, the importance of activity-dependent plasticity in neurons is widely appreciated, but comparatively less is known of the role of electrical activity in controlling glial development. Schwann cells (SCs)--the myelinating cells in the peripheral nervous system--are critically dependent on axons during the perinatal period, but axonal signals controlling SC development and myelination have remained elusive. The onset of high frequency action potential activity along developing peripheral nerves corresponds to the period when SCs are exiting the cell cycle and initiating myelination. We postulated that neural impulse activity could play an instructive role in regulating SC gene expression and function during development. To address these questions, a neuron/SC co-culture system equipped with stimulating electrodes was used to evoke action potentials in dorsal root ganglion neurons (DRGs), and study the ensuing effects in pre-myelinating SCs. We found that SCs can detect neural impulse activity in pre-myelinated axons, and the activity-dependent axon-Schwann cell signaling molecule was identified as extracellular ATP. Activity-dependent release of ATP activated multiple intracellular signaling pathways in SCs, and increased levels of several transcription factors, including CREB, c-fos, and krox-24. Importantly, we found that ATP has profound effects on SC development. Activity-dependent ATP release significantly inhibited SC proliferation, arrested SC differentiation, and completely prevented the formation of myelin. Extracellular ATP can activate multiple types of purinergic receptors; therefore we explored the specific purinergic receptors and signaling pathways that could mediate this form of activity-dependent neuron-SC communication. Using a combination of pharmacological and molecular approaches, we found that pre-myelinating SCs express a far more complex array of ATP receptors (P2X and P2Y) that previously thought. Surprisingly, we discovered that pre-myelinating SCs also express a class of functional adenosine receptors (A2), which are positively coupled to cAMP. Extracellular adenosine, a breakdown product of ATP, regulated MAP Kinase signaling and proliferation in SCs independently of ATP. Collectively, our findings suggest that ATP and adenosine released from electrically active axons activate a complex intracellular signaling network in SCs, whereby ATP and adenosine act together to regulate SC function during development and nervous system plasticity.