No description available.

<p>Dynamic processes such as morphogenesis and tissue patterning require the precise control of many cellular processes, especially cell migration. Historically, these processes are thought to be mediated by genetic and biochemical signaling pathways. However, recent advances have unraveled a previously unappreciated role of mechanical forces in regulating these homeostatic processes in of multicellular systems. In multicellular systems cells adhere to both deformable extracellular matrix (ECM) and other cells, which are sources of applied forces and means of mechanical support. Cells detect and respond to these mechanical signals through a poorly understood process called mechanotransduction, which can have profound effects on processes such as cell migration. These effects are largely mediated by the sub cellular structures that link cells to the ECM, called focal adhesions (FAs), or cells to other cells, termed adherens junctions (AJs).</p><p>Overall this thesis is comprised of my work on identifying a novel force dependent function of vinculin, a protein which resides in both FAs and AJs - in dynamic process of collective migration. Using a collective migration assay as a model for collective cell behavior and a fluorescence resonance energy transfer (FRET) based molecular tension sensor for vinculin I demonstrated a spatial gradient of tension across vinculin in the direction of migration. To define this novel force-dependent role of vinculin in collective migration I took advantage of previously established shRNA based vinculin knock down Marin-Darby Canine Kidney (MDCK) epithelial cells.</p><p>The first part of my thesis comprises of my work demonstrating the mechanosensitive role of vinculin at AJ’s in collectively migrating cells. Using vinculin knockdown cells and vinculin mutants, which specifically disrupt vinculin’s ability to bind actin (VinI997A) or disrupt its ability to localize to AJs without affecting its localization at FAs (VinY822F), I establish a role of force across vinculin in E-cadherin internalization and clipping. Furthermore by measuring E-cadherin dynamics using fluorescence recovery after bleaching (FRAP) analysis I show that vinculin inhibition affects the turnover of E-cadherin at AJs. Together these data reveal a novel mechanosensitive role of vinculin in E-cadherin internalization and turnover in a migrating cell layer, which is contrary to the previously identified role of vinculin in potentiating E-cadherin junctions in a static monolayer.</p><p>For the last part of my thesis I designed a novel tension sensor to probe tension across N-cadherin (NTS). N-cadherin plays a critical role in cardiomyocytes, vascular smooth muscle cells, neurons and neural crest cells. Similar to E-cadherin, N-cadherin is also believed to bear tension and play a role in mechanotransduction pathways. To identify the role of tension across N-cadherin I designed a novel FRET-based molecular tension sensor for N-cadherin. I tested the ability of NTS to sense molecular tension in vascular smooth muscle cells, cardiomyocytes and cancer cells. Finally in collaboration with the Horwitz lab we have been able to show a role of tension across N-cadherin in synaptogenesis of neurons.</p> / Dissertation

The relationships between vegetation architecture and spider vii community attributes were examined in a big sage (Artemisia tridentata) community. Spiders were separated into guilds using similarities of species' hunting behavior. Shrub architecture was experimentally manipulated in the field by either clipping 50% of a shrub's foliage to decrease foliage density or tying together a shrub's branches to increase foliage density. Temporal patterns of spider species density, diversity (H') and evenness (J') showed midsummer peaks in both 1974 and 1975. Seasonal spider guild trends reflected the temporal prominence of a member species or genus. These temporally abundant species appeared to play a major functional role in this community. Shrub perturbations resulted in changes in spider species and guild densities. Spider species and guild density in the tied shrubs were significantly higher than that in the clipped or control shrubs sampled. Spider species diversity, density and guild density were also positively correlated with indicators of shrub volume and shrub foliage diversity. This suggests that structurally more complex shrubs (tied) can support greater spider species densities and diversity. Spider guild densities and IV's were significantly altered by changes of shrub architecture. The observed guild distributions. were in agreement with known hunting behavior and life history data of the member species. The data suggest that architectural properties of habitat may be an important determinant of predatory invertebrate species diversity and distribution. Guild analysis may be useful in examining the roles of species groups in community studies.

Across animals, embryos regulate hatching timing in response to environmental cues. This is well-documented in frogs, where some species accelerate hatching to escape threats such as egg-predators, pathogens, flooding, and dehydration, while others delay hatching until cues indicate favorable conditions for larvae. The diversity of reproductive modes and cued hatching responses make anurans excellent subjects for the study of environmentally cued hatching (ECH). Nonetheless, the mechanisms by which frogs alter hatching timing were unknown. The hatching process has been described for only a few aquatic-breeding species: hatching gland cells (HGCs) secrete hatching enzyme (HE) that gradually digests the vitelline membrane. I investigated hatching mechanisms and their regulation in two anurans with well-documented hatching plasticity. Obligately terrestrial embryos of Agalychnis callidryas hatch early to escape flooding, drying, and fungus, and within seconds in predator attacks. I discovered that their hatching mechanism is novel: two types of HGCs mediate hatching at different developmental stages and differ in their distribution, morphology, and timing of appearance and regression, but produce the same HE, storing it for acute release. The vitelline membrane is digested locally at hatching, with no prior degradation. Facultatively terrestrial Dendropsophus ebraccatus embryos hatch early to escape drying and ant attacks. We found that HGC abundance and HE gene expression peak early, then decline before hatching, and that vitelline membranes degrade gradually, as described in aquatic eggs. Drying-induced acceleration of hatching is not mediated by changes in HGC development, HE gene expression, or embryo behavior. However, in simulated attacks, rapid enzyme release leads to local vitelline membrane rupture and embryos escape. Thus, both species use acute HE release to escape acute threats, despite other differences in hatching mechanisms. Agalychnis callidryas shows greater control of the timing of HE-release and a novel HGC type that extends the developmental period when hatching is possible. Mechanisms that enable and regulate hatching shape embryos’ capacities for adaptive shifts in hatching timing. Ancestral flexibility may be elaborated or further modified in species with more dynamic plastic responses. The diversity of anuran hatching mechanisms revealed here will facilitate comparative work to understand the evolutionary diversity of hatching strategies. / 2020-02-14T00:00:00Z

Plant defense responses vary depending on the pathogen and intensity of attack. These responses are mediated through two levels of defense, with the first level being pathogen-triggered immunity (PTI) that is triggered by host recognition of microbe-associated molecular patterns (MAMPs). Successful pathogens are able to evade PTI by secreting effector molecules into host cells. These effectors are designed to suppress host defenses. In turn, effectors are inhibited by the second level of plant defense called effector-triggered immunity (ETI). In ETI, intracellular resistance proteins recognize and block effector dampening of host defenses. ETI results in gene expression changes that can lead to localized cell death known as the hypersensitive response (HR) as well as a plant-wide systemic acquired resistance. The Arabidopsis thaliana mutant alf3-1 (aberrant lateral root formation 3-1) was characterized as the first and only case of HR in the root system. The alf3-1 mutant’s primary and lateral roots die unless they are grown in auxin-supplemented medium or at elevated ambient temperature. This thesis describes further characterization of the mutant phenotype and identifies a candidate gene for ALF3. Consistent with an autoimmune response, we found that the alf3-1 mutant has increased production of phenylalanine- and tryptophan-derived defense compounds, as well as increased production of salicylic acid (SA), a plant hormone that mediates innate immunity. Based on gene expression profiling, we found that many immune and defense response genes were expressed highly in alf3-1 compared to wild type (WT). These genes include the SA-responsive PR1 and PBS3 as well as several WRKY transcription factors, a gene family implicated in plant defense. Importantly, we found that the vast majority of defense-related phenotypes dysregulated in alf3-1 returned to WT levels when the mutant was grown at elevated temperatures or in medium supplemented with auxin, conditions that suppress innate immunity. To determine the identity of ALF3, we used whole genome re-sequencing to identify a candidate gene that encodes an uncharacterized TIR domain protein. Because characterized plant TIR domain proteins have been shown to function in plant innate immunity, we hypothesize that alf3-1 is a gain-of-function mutation that causes an autoimmune phenotype in roots. / 2020-07-12T00:00:00Z

OBJECTIVE: Ankylosing spondylitis (AS) is an inflammatory disorder within the spondyloarthritis family of rheumatic diseases. AS is characterized by chronic inflammation at entheses, the attachment sites of ligaments or tendons to bone, most prominently in the spine. Chronic enthesitis leads to pathological new bone formation, fusion of vertebral bodies and loss of spinal mobility. Studies in mice identified discrete subsets of enthesis-resident lymphocytes that function within the IL-23/IL-17A signaling pathway and are thought to play a critical role in mediating inflammation and new bone formation. Little is known about such enthesis-resident lymphocytes in humans. The goal of this project was therefore to develop methods to isolate lymphocytes from spinal entheses and analyze them in vitro. METHODS: Cell preparation and staining conditions were optimized using peripheral blood mononuclear cells (PBMCs). We compared surface marker staining of PBMCs exposed to collagenase D, Liberase TL, and dispase II. In addition, we compared the staining of multiple antibody clones specific for CD56 and CD4. To measure cytokine expression, cells were stimulated with PMA/ionomycin for 4 hours in the presence of monensin and brefeldin A. Following stimulation, the cells were stained with a 17-color staining panel enabling the identification of 8 lymphocyte subsets, 3 killer immunoglobin-like receptors (KIRs), and 4 cytokines implicated in AS pathology. Cell were analyzed on a 5 laser LSRFortessa flow cytometer. Spine tissue was obtained from laminectomy surgeries performed on patients with degenerative disease of the spine. Interspinous ligament and spinous process bone were cut into 5 mm pieces and cryopreserved in CryoStor CS10, a serum free cryopreservation medium. Samples were stored at -80° C until analysis. After thawing, samples were digested with collagenase D to generate single cell suspensions for analysis by flow cytometry. RESULTS: Incubation of lymphocytes with collagenases resulted in variable signal attenuation for CD56, CD4, and KIR3DL2. Loss of signal for CD56 occurred with all enzymes and anti-CD56 clones tested but correlated with duration of enzyme exposure. For the anti-CD4 clone OKT4, fluorescence remained stable after digestion. KIR3DL2 signal was lost after incubation with Liberase TL but was not affected by collagenase D. Spine tissue samples contained the same lymphocyte subsets present in peripheral blood: natural killer (NK) cells, CD4+ T cells, CD8+ T cells, double negative αβ T cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells including vδ1 and vδ2 cells. PMA/ionomycin-stimulated CD4+ T cells from a spine tissue donor had higher fractions of IL-17A and IL-22 positive cells compared to PBMCs from unmatched donors. Granulocyte-macrophage colony-stimulating factor (GM-CSF) production was also increased in spine tissue compared to unmatched PBMCs. CONCLUSION: Here we describe an optimized method for releasing lymphocytes from cryopreserved spine tissue specimens with minimal impact on surface marker detection. Similar populations of lymphocytes could be detected in spine tissue and peripheral blood. Stimulation of tissue-resident lymphocytes induced them to produce inflammatory cytokines. The methods described will be useful to analyze a larger sample size and further characterize the lymphocytes resident to spinal entheses.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015. / Cataloged from PDF version of thesis. "September 2015." / Includes bibliographical references. / Throughout organismal development, precise three-dimensional organization of tissues is required for proper tissue function. These three-dimensional forms are generated by coordinated cell shape changes that induce global tissue shape changes, such as the transformation of an epithelial sheet into a tube. A model for this transformation occurs early in Drosophila development where approximately 1,000 cells on the ventral side of the embryo constrict their apical sides. Apical constriction drives the formation of a furrow that invaginates, forming a tube, and consequently, a new cell layer in the embryo. Constriction of ventral cells is driven by cycles of assembly and disassembly of actin-myosin networks at the cell apex, called pulses. Pulsatile myosin leads to phases of cellular contraction and cell shape stabilization that result in step-wise apical constriction. While many of the key components of the pathway have been identified, how pulsatile myosin is regulated was previously not well understood. The results presented in this thesis identify mechanisms of regulation of these myosin pulses. First, we demonstrated that cycles of phosphorylation and dephosphorylation of the myosin regulatory light chain are required for myosin pulsing and step-wise apical constriction. Uncoupling myosin from its upstream regulators resulted in loss of pulsatile myosin behavior and continuous, instead of incremental, apical constriction. A consequence of persistent, non-pulsatile myosin is a loss of myosin network integrity as the tissue invaginated. Thus, pulsatile myosin requires tight coordination between its activator and inactivator to generate cycles of myosin assembly, coupled to cellular constriction, and myosin disassembly, associated with cell shape stabilization. Second, we demonstrated that myosin motor activity is required for efficient apical constriction and for effective generation of tissue tension. This work defines essential molecular mechanisms that are required for proper cellular constriction and tissue invagination. / by Claudia G. Vasquez. / Ph. D.

Blood at a crime scene is not always detected by the naked eye, and hence requires the use of special reagents and alternate light sources. Two of these reagents – luminol and fluorescein – have been in use in forensic science both in their original forms and in the form of commercially produced reagents, Bluestar® and Hemascein™. The manufacturer of Hemascein™, the relatively newer product, states that the reagent exhibits lower amounts of cross-reactivity with bleach and is less detrimental to DNA as compared to Bluestar®. They also say that the reagent does not require complete darkness to function, unlike Bluestar®. These parameters, along with the impact of Bluestar® and Hemascein™ on pattern detail, were evaluated in this study. It was found that both reagents exhibited some positive reactions with bleach; however, Bluestar® had a less intense reaction with lower concentrations of bleach, while Hemascein™ showed a lower intensity for cross reactions with higher concentrations. A distinct difference was observed for the reagents with respect to retention of pattern characteristics in bloodstains, with Hemascein™ causing considerable diffusion of the pattern. The results also demonstrated that there was a dependence of the color of the substrate on the performance of Hemascein™, with lighter colored substrates far outperforming their darker counterparts. Hemascein™ performed better than Bluestar® in well-lit conditions, with a positive luminescent reaction observed in ambient lighting conditions. Additionally, neither of the reagents showed inhibition during quantitative PCR, thus deeming them both appropriate for use when subsequent recovery of DNA is desired. There were differences in the amounts of DNA recovered from the treated blood samples, however, further studies and a larger sample size would be required to determine if these variations are related to the application of Bluestar® and Hemascein™. All DNA recovered was sufficient in quantity to expect successful DNA profiling if such analysis had been carried out.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-01

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019 / Cataloged from PDF version of thesis. "February 2019." / Includes bibliographical references (pages 118-128). / DNA replication is a highly regulated process across all organisms. Improper regulation of DNA replication can be detrimental. I identified an overinitiating, conditional synthetic lethal mutant of Bacillus subtilis. I isolated suppressors of this mutant and uncovered novel genes associated with DNA replication. These suppressors acted both at the steps of initiation and elongation to overcome the detrimental replication initiation of the synthetic lethal [delta]yabA dnaA 1 mutant. One class of suppressors decreased levels of the replicative helicase, DnaC. I showed that decreased levels of helicase are sufficient to limit replication initiation under fast growth conditions. I also explored the regulation of DnaA as a transcription factor. The replication initiation inhibitor, YabA, binds to DnaA and prevents its cooperative binding at the origin. In addition to its role in replication initiation, DnaA also directly regulates expression of several genes. YabA has been shown to inhibit DnaA binding at several promoters but its effect on DnaA-mediated gene expression is unclear. I found that YabA inhibits sda activation by DnaA but does not significantly affect repression of ywlC by DnaA. Lastly, I showed that YabA appears to stimulate autoregulation of dnaA. This preliminary data illustrates a role for YabA regulation in DnaA-mediated gene expression. / by Mary E. Anderson. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biology