Biology Department Dissertations Collection

Regulation of the hypothalamic progenitor cells by Hh/Gli signaling in post-embryonic zebrafish

Ozacar, Ayse Tuba — Wed, 16 Jan 2013 12:24:58 PST

The major goals of my research were to characterize the hypothalamic neural progenitors and to understand how Hh/Gli signaling plays a role in regulating cell proliferation in the hypothalamic neurogenic zone. In contrast to mammals, the zebrafish brain has a life-long potential to grow continuously. Thus, for comparative neurogenesis studies, zebrafish become an indispensible model organism to understand adult neurogenesis and regulatory signaling pathways. Identification of the regulatory mechanisms underlying the controlled cell proliferation in adult zebrafish brain will pave the way to manipulate the healing potential of the mammalian brain. Using immunohistochemistry and in situ hybridization techniques to label known markers for neural stem/ and progenitor cells I have identified three different populations of cells with radial glia (RG) like morphology in the adult zebrafish hypothalamic ventricular zone. In adult zebrafish, cells with RG-like morphology in the ventricular regions are thought to be the neurogenic population. ^ The first population of cells I identified was positive for the neural stem cell marker NESTIN and showed additional characteristics of neural stem cells. Using a label retention assay we showed that Nestin(+) cells are slow cycling. The second population of RG-like cells was Hh responsive, and expressed markers of neural progenitor/transit amplifier cells. Double labeling experiments reveal that the Hh responsive cells were distinct from the Nestin(+) cells These cells were proliferative and cycled faster compared to nestin(+) neural stem cells. The third population of cells with RG morphology in the hypothalamic ventricular zone expressed shh ligand, indicating a regulatory role for Hh signaling in the hypothalamic ventricular zone. Down-regulation of Hh signaling at larval and adult stages reduced proliferation in the hypothalamic ventricle, indicating that Hh acts as a positive regulator of proliferation, as in the dorsal brain. According to our working model, nestin(+) cells are slow cycling, and/or quiescent neural stem cell population in the hypothalamic ventricular zone, whereas Hh responsive cells are the fast cycling transit amplifier cells which proliferate and give rise to new neurons and glia in the adult. My comprehensive analysis of the neural stem/progenitors in the adult zebrafish hypothalamic ventricular zone provides a starting point for the continued study of the mammalian hypothalamic ventricular zone. This study also demonstrates Hh signaling functions as a positive regulator of cell proliferation in the post-embryonic zebrafish hypothalamus consistent with its role in the dorsal brain. (Abstract shortened by UMI.)^

Gradients and ranges of visually selective attention based on location, objects, color, and size: Gradients are universal, but range is uniquely spatial

Bush, William S — Wed, 16 Jan 2013 12:24:32 PST

Two interesting properties of the distribution of spatially selective attention have been noted in the behavioral and electrophysiological literature. First, there is a graded field of attention that expands from the center of the attended area. Second, the size of the attended area can be adjusted to be either larger or smaller in order to match the demands of the current task. Five event-related potential (ERP) studies are presented that extend these findings in several important ways; 1) The time frame of these two distribution properties is different. Results are consistent with a two stage model of spatial attention in which visual processing is initially enhanced for all stimuli presented near the center of the attended area as indexed by the amplitude of the first negative peak in the waveform (N1). Subsequently, the effects of narrowing or expanding the attentional field to the relevant size affects visual processing as indexed by the amplitude of the second negative peak (N2). 2) Object boundaries had limited impact on either the spread of the initial gradient of spatial selection or the scale of attention. 3) When selecting visual stimuli for attentive processing based on features such as color and size there is also a gradient of facilitation, but the impact of this graded selection on visual processing is not observed until later in processing, and is indexed by the amplitude of the selection negativity (SN). Furthermore, similar to the lack of interaction between object boundaries and the range of cued locations, the gradients of feature-based selection are not affected by the range of cued features.^

Tree-Based Methods and a Mixed Ridge Estimator for Analyzing Longitudinal Data With Correlated Predictors

Eliot, Melissa Nicole — Mon, 05 Dec 2011 12:51:24 PST

Due to recent advances in technology that facilitate acquisition of multi-parameter defined phenotypes, new opportunities have arisen for predicting patient outcomes based on individual specific cell subset changes. The data resulting from these trials can be a challenge to analyze, as predictors may be highly correlated with each other or related to outcome within levels of other predictor variables. As a result, applying traditional methods like simple linear models and univariate approaches such as odds ratios may be insufficient. In this dissertation, we describe potential solutions including tree-based methods, ridge regression, mixed modeling, and a new estimator called a mixed ridge estimator with expectation-maximization (EM) algorithm. Data examples are provided. In particular, flow cytometry is a method of measuring a large number of particle counts at once by suspending them in a fluid and shining a beam of light onto the fluid. This is specifically relevant in the context of studying human immunodeficiency virus (HIV), where there exists a great potential to draw from the rich array of data on host cell-mediated response to infection and drug exposures, to inform and discover patient level determinants of disease progression and/or response to anti-retroviral therapy (ART). The data sets collected are often high dimensional with correlated columns, which can be challenging to analyze. We demonstrate the application and comparative interpretations of three tree-based algorithms for the analysis of data arising from flow cytometry in the first chapter of this manuscript. Specifically, we consider the question of what best predicts CD4 T-cell recovery in HIV-1 infected persons starting antiretroviral therapy with CD4 count between 200-350 cell/μl. The tree-based approaches, namely, classification and regression trees (CART), random forests (RF) and logic regression (LR), were designed specifically to uncover complex structure in high dimensional data settings. While contingency table analysis and RFs provide information on the importance of each potential predictor variable, CART and LR offer additional insight into the combinations of variables that together are predictive of the outcome. Specifically, application of tree-based methods to our data suggest that a combination of baseline immune activation states, with emphasis on CD8 T cell activation, may be a better predictor than any single T cell/innate cell subset analyzed. In the following chapter, tree-based methods are compared to each other via a simulation study. Each has its merits in particular circumstances; for example, RF is able to identify the order of importance of predictors regardless of whether there is a tree-like structure. It is able to adjust for correlation among predictors by using a machine learning algorithm, analyzing subsets of predictors and subjects over a number of iterations. CART is useful when variables are predictive of outcome within levels of other variables, and is able to find the most parsimonious model using pruning. LR also identifies structure within the set of predictor variables, and nicely illustrates relationship among variables. However, due to the vast number of combinations of predictor variables that would need to be analyzed in order to find the single best LR tree, an algorithm is used that only searches a subset of potential combinations of predictors. Therefore, results may be different each time the algorithm is used on the same data set. Next we use a regression approach to analyzing data with correlated predictors. Ridge regression is a method of accounting for correlated data by adding a shrinkage component to the estimators for a linear model. We perform a simulation study to compare ridge regression to linear regression over various correlation coefficients and find that ridge regression outperforms linear regression as correlation increases. To account for collinearity among the predictors along with longitudinal data, a new estimator that combines the applicability of ridge regression and mixed models using an EM algorithm is developed and compared to the mixed model. We find from a simulation study comparing our mixed ridge (MR) approach with a traditional mixed model that our new mixed ridge estimator is able to handle collinearity of predictor variables better than the mixed model, while accounting for random within-subject effects that regular ridge regression does not take into account. As correlation among predictors increases, power decreases more quickly for the mixed model than MR. Additionally, type I error rate is not significantly elevated when the MR approach is taken. The MR estimator gives us new insight into flow cytometry data and other data sets with correlated predictor variables that our tree-based methods could not give us. These methods all provide unique insight into our data that more traditional methods of analysis do not offer.

Conservation while under invasion: Insights from a rare hemiparasitic plant, swamp lousewort (Pedicularis lanceolata Michx.)

Record, Sydne — Thu, 16 Jun 2011 13:15:09 PDT

Competition with non-native invasive species is considered a major threat to many rare native species. As such, invasives removals are a common management strategy. Rare native species that interact uniquely with other organisms in their community (e.g., hemiparasitic plants) may be adversely affected by removing invasives. A management plan for a regionally rare hemiparasitic plant in Massachusetts, Pedicularis lanceolata Michx., identified invasives as a threat, but more quantitative evidence is needed to determine how P. lanceolata's persistence is influenced by its co-occurrence with native or invasive hosts. This research asks how P. lanceolata is affected by growth with native versus invasive hosts. Chapter I describes the species associated with P. lanceolata throughout its range, comparing areas where it is considered common and rare. Relative abundances of natives, non-native invasives, non-native non-invasives, and species with both native and non-native genotypes growing with P. lanceolata did not differ significantly at sites where the species is considered common in the Midwest compared to sites where the species is considered rare in the east. Chapter II outlines greenhouse and field removal experiments in which the types of host plants growing with P. lanceolata were manipulated. In the greenhouse, P. lanceolata growth, survival, and flowering were lower when it was growing with invasive compared to native graminoids. However, differences in P. lanceolata growth and survival when natives versus non-native were removed in the field varied from year to year due to succession of native shrubs at the site during the study. Chapter III asks how the population growth of P. lanceolata differs in uninvaded and invaded patches using an Integral Projection Model to perform population projections, sensitivity and elasticity analyses, and a life table response experiment. The population growth rate of P. lanceolata in uninvaded patches was lower than in invaded patches due to the succession of native shrubs in uninvaded patches. Chapter IV describes a metapopulation model for the invaded population of P. lanceolata in Massachusetts. The quasi-extinction probability was significantly affected by probabilities of dispersal, positive correlations in vital rates between sites, and catastrophes. These data will be used to update the management plan for P. lanceolata.^

The role of bacteriocins in mediating interactions of bacterial isolates from cystic fibrosis patients

Bakkal, E. Suphan — Tue, 24 May 2011 15:18:37 PDT

Cystic Fibrosis (CF) is a common autosomal genetic disorder in Caucasian populations. CF is caused by mutations in the cftr gene, which encodes the CF transmembrane conductance regulator (CFTR). CFTR regulates chloride and sodium ion transport across the epithelial cells lining the exocrine organs. Mutations in the cftr result in a failure to mediate chloride transport, which leads to dehydration of the mucus layer surrounding the epithelial cells. The mucus coating in the lung epithelia provides a favorable environment for invasion and growth of several opportunistic bacterial pathogens resulting in life threatening respiratory infections in CF patients. ^ Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) are associated with chronic lung infections and are responsible for much of the mortality in CF. Little is known about interactions between these two, often co-infecting, species. When in competition, it is not known whether Bcc replaces the resident Pa or if the two species co-exist in the CF lung. ^ Bacteriocins are potent toxins produced by bacteria. They have a quite narrow killing range in comparison to antibiotics and have been implicated in intra-specific and inter-specific bacterial competition brought on by limited nutrients or niche space. Both Pa and Bcc produce bacteriocins known as pyocins and cepaciacins, respectively. More than 90% of Pa strains examined to date produce one or more of three pyocin types: R, F, and S. A limited number of phenotypic surveys suggest that approximately 30% of Bcc also produce bacteriocins. ^ The goals of my thesis study were to determine if clinical strains of Pa and Bcc produce bacteriocins and to determine whether these toxins play a role in mediating intra- and inter-specific bacterial interactions in the CF lung. The final goal was to identify novel bacteriocins from clinical Pa and Bcc strains. ^ First, I designed a phenotypic bacteriocin survey to evaluate bacteriocin production in 66 clinical Pa (38) and Bcc (28) strains procured from CF patients. This study revealed that 97% of Pa strains and 68% of Bcc strains produce bacteriocin-like inhibitory activity. Further phenotypic and molecular based assays showed that the source of inhibition is different for Pa and Bcc. In Pa, much of the inhibitory activity is due to the well known S- and RF-type pyocins. S- and RF pyocins were the source of within species inhibitory activity while RF pyocins were primarily implicated in the between species inhibitory activity of Pa strains. In contrast, Bcc inhibition appeared to be due to novel inhibitory agents. Finally, I constructed genome libraries of B. multivorans, B. dolosa, and B. cenocepacia to screen for genes responsible for the inhibitory activity previously described in Bcc. ∼10,000 clones/genome were screened, resulting in fifteen clones with the anticipated inhibition phenotype. Of these fifteen, only five clones had stable inhibitory activity. These clones encoded proteins involved in various metabolic pathways including bacterial apoptosis, amino acid biosynthesis, sugar metabolism, and degradation of aromatic compounds. Surprisingly, none of Bcc clones possessed typical bacteriocin-like genes. These data suggest that, in contrast to all bacterial species examined in a similar fashion to date, Bcc may not produce bacteriocins. Instead, Bcc may be using novel molecular strategies to mediate intra- and inter-specific bacterial interactions. ^

Characterization of the zebrafish umleitung (uml) mutant: Essential roles for the transmembrane protein brother of Cdo (Boc) in regulating hedgehog (Hh) mediated cell differentiation and neural patterning

Bergeron, Sadie A — Tue, 24 May 2011 15:18:19 PDT

The secreted Hedgehog (Hh) molecule acts as a morphogen, mitogen, and cell survival factor during embryogenesis and during later growth and tissue homeostasis periods in vertebrates. Hh plays these diverse roles by regulating the transcription of a number of genes that promote cellular processes including differentiation, proliferation, and resistance to programmed cell death. At the cellular level, the response to Hh signaling appears to change depending on the period during development that the cells are exposed to Hh, potentially allowing the switch, particularly in the brain, from early morphogen to a late mitogen. Mutations that reduce Hh signaling lead to congenital disorders in humans (e.g. holoprosencephaly), while mutations that inappropriately activate Hh signaling can lead to cancer (e.g. medulloblastoma and basal cell carcinoma). ^ One goal of my research was to identify the major transcriptional targets of this important signaling pathway, work that may identify new genes that are targets of mutation in human disease. A collaborative effort using gene profiling techniques on zebrafish embryos with either increased or decreased Hh signaling was undertaken to identify genes regulated by the Hh/Gli signaling cascade. Our microarray approach led us to both known and novel zebrafish genes that are regulated by Hh signaling, including several with putative Gli transcription factor binding sites in their regulatory regions. We have confirmed the regulation of some known and novel genes by Hh in embryos with increased and decreased Hh signaling. This now published work has identified a large number of direct and indirect targets of the Hh signaling pathway. ^ The major goal of my research was to both phenotypically and genetically characterize the zebrafish umleitung (uml) (meaning "detour" in German) mutation. The uml^ty54 mutant was identified due to its aberrant retinal tectal axon projections, and here I show that these mutants also have defects in Hh-mediated ventral neural patterning. These defects are accompanied by reduced expression of Hh-regulated genes in the forebrain and spinal cord, similar to known mutations that affect the Hh signaling pathway. Consistent with impaired Hh signaling, uml mutants have fewer endocrine cell types in the developing anterior pituitary, a reduced number of differentiated slow-muscle fibers in the tail, and defects in development of the jaw. To further understand how uml affects Hh signaling, I isolated the gene disrupted in uml by positional cloning. I have now shown that the defects seen in uml are due to a loss of function mutation in the gene encoding an immunoglobulin (Ig) and fibronectin type III (FNIII) domain containing cell-surface Hh-receptor, Brother of Cdo (Boc). Mosaic analysis indicates that Boc exerts its affects on Hh signaling cell-autonomously and may have a very early role in establishing the dose dependent response of cells along the dorsal/ventral axis of the central nervous system (CNS) to Sonic Hedgehog (Shh). This study demonstrates a previously unknown necessity for Boc in multiple Hh-mediated vertebrate developmental processes including, forebrain patterning, endocrine cell type specification, and jaw development. My analysis also reveals intriguing new ways in which Hh signaling is regulated within the vertebrate embryo and further implicates boc as a candidate gene in the treatment of disease caused by aberrant Hh signaling.^

Identification and functional characterization of zebrafish gene Technotrousers (tnt)

McKeown, Kelly Anne — Tue, 21 Dec 2010 10:21:19 PST

Neural networks in the hindbrain and spinal cord require a proper balance between excitation and inhibition. Identification of zebrafish mutants that have defects in motor output mediated by these networks can allow entrée into underlying network mechanisms. techno trouser ( tnt) mutants demonstrate abnormal motor behavior. Two days after fertilization, wild-type larvae perform an escape response consisting of a large-amplitude body bend away from touch stimuli followed by smaller amplitude body bends to swim away. tnt mutants perform an initial large amplitude body bend away from touch stimuli, but the following smaller amplitude body bends are interrupted by several, abnormal, large amplitude body bends. Four days after fertilization, wild-type larvae exhibit faster escape behavior, whereas tnt mutants are nearly paralyzed and shorter along the rostral-caudal axis. We used meiotic mapping and candidate gene analysis to reveal that the tnt mutation disrupts slc1a2b , which encodes EAAT2, a glutamate transporter expressed in glial cells. Lesion analysis, in situ hybridization, and in vivo electrophysiological recordings all support a model in which reduced slc1a2b function results in exuberant excitation of neurons, initially in the hindbrain and later in development in the spinal cord, to produce the large-amplitude body bends and subsequent paralysis of tnt mutants. Since disruption of human EAAT2 is thought to promote several different neurological diseases, including epilepsy and amyotrophic lateral sclerosis, tnt mutants provide a new tool to understand these disorders.^

Delayed Anesthetic Preconditioning and Metallothioneins I+II: Novel Mediators of Anesthetic-Induced Protection

Edmands, Scott David — Thu, 12 Nov 2009 06:54:51 PST

Ischemic injury is a common and debilitating outcome of natural illness and as a complication of commonly performed medical procedures. Whereas naturally occurring ischemic insults are often the result of unpredictable events, such as in the case of stroke or heart attack, the risk of operative and perioperative ischemia is somewhat better characterized in the clinical setting. Given the prevalence and severity of outcomes in ischemic injury, there is significant interest in developing better pharmacological and procedural approaches to improve patient outcomes. One approach that has shown significant promise in the laboratory setting, particularly in the context of planned medical procedures, is the use of delayed anesthetic preconditioning. Delayed anesthetic preconditioning is a phenomenon whereby a prior exposure to clinical concentrations of commonly used inhaled anesthetics, including isoflurane, induces the production of endogenous protective proteins that are able to provide robust protection against subsequent, potentially toxic, ischemic insults. Although many aspects of delayed anesthetic preconditioning have been previously described, a complete understanding of preconditioning mechanism has yet to emerge. The studies described in this dissertation aim to further our understanding of molecular mechanisms involved in delayed anesthetic preconditioning. In the first project, I used DNA microarray to identify genes that were differentially expressed in adult rat liver, kidney and heart following a clinically relevant exposure to the inhaled anesthetic isoflurane. By selecting those genes that were differentially expressed in multiple tissues, I was able to identify a small group of interesting genes for further study. In my second study, I chose from our list two related genes, metallothioneins I + II, to analyze for a role in anesthetic-mediated protection. Using a combination of approaches, I was able to establish that metallotioneins I + II play an essential role in delayed anesthetic preconditioning. In the final study of this dissertation I explore a possible role for metallothioneins I + II as sensor molecules, involved in detecting cellular oxidative stress. Taken together, these three studies represent an important contribution to our understanding of the mechanisms of delayed anesthetic preconditioning and how they might contribute to protecting against ischemic stroke.

The Conformational Gymnastics of the Escherichia Coli SecA Molecular Machine and its Interactions with Signal Sequences

Maki, Jenny Lynn — Thu, 12 Nov 2009 06:53:17 PST

Protein secretion is a selective and regulated process that is essential in all organisms. In bacteria the preprotein translocase SecA, either free in the cytosol or associated with the SecYEG translocon, recognizes and binds most post-translational secretory proteins containing an N-terminal signal sequence. In Gram-negative bacteria, the molecular chaperone SecB binds many of the preproteins to keep them in a translocation-competent state. Subsequently, SecB delivers the preproteins to the translocon-associated SecA, which binds the signal sequence and also interacts with mature regions of the preprotein. After the preprotein/SecA/SecYEG complex has formed, the energy derived from ATP hydrolysis by SecA coupled with the proton motive force drives the insertion of the preprotein through the translocon pore. During the translocation reaction, the conformation of SecA dramatically changes from an inactive closed form (c-SecA) to one more active and open states. The various crystal structures of SecA have provided many structural details about c-SecA. The recent low resolution crystal structure of a fragment of SecA bound to SecYEG (Zimmer et al., 2008) has provided a starting point for structural analysis of the active and open conformation of SecA. Previous work in our laboratory demonstrated that an N-terminal proteolytic fragment of SecA, SecA64, is an activated form of SecA that with higher affinity signal peptides better than c-SecA (Triplett et al., 2001). To correlate the SecA64 results with full-length SecA, we determined that SecA in the presence of low concentrations of urea has an enhanced ATPase activity similar to translocation level, which is comparable to what was observed with SecA64. Analysis by CD and Trp fluorescence indicates the presence of an intermediate at 2.2 M urea at 22ºC (termed u-SecA). Using limited proteolysis, we determined that u-SecA is in an protease-sensitive conformation that mimics the translocation-active form of SecA. These structural rearrangements occur primarily in the C-terminal one-third of the protein. Next, we sought to understand the signal sequence interactions with c-SecA and translocation-active u-SecA. Using a photoactivatable cross-linking approach along with limited proteolysis, two-dimensional gels, and domain mapping with region-specific antibodies, the signal sequence-binding site was mapped to the interface of NBF II, PPXD, and HSD. The site is the same in both forms of SecA but in our data suggests u-SecA that the binding groove as expanded.

Spatial ecology, population structure, and conservation of the wood turtle, Glyptemys insculpta, in central New England

Jones, Michael T — Thu, 24 Sep 2009 10:20:32 PDT

Wood turtles (Glyptemys insculpta) are of conservation interest rangewide. Anecdotal accounts demonstrate that some populations have been decimated since 1850, and recent studies demonstrate that declines are still underway. From 2004–2008 I investigated the ecology of wood turtles in Massachusetts and New Hampshire. I obtained between one and five years of annual home range data for 150 turtles, and evaluated population structure at 31 sites in five major watersheds. ^ Seasonal floods displaced 7% of wood turtles annually in one watershed, and accounted for elevated mortality. Twelve wood turtles were displaced < 16.8 km, and two were displaced over a 65-foot dam. Several turtles overwintered at their displacement site and two returned successfully, indicating that floods are a mechanism of population connectivity. Several homing turtles ended up in new areas. Turtles occupied stream segments with gradient < 1%, lower than generally available. ^ Agricultural machinery accounted for most observed mortality, followed by automobiles and mammals. Female turtles exhibit smaller home ranges in agricultural areas. Older turtles move farther from the river than do young turtles, possibly reflecting their familiarity with a former landscape. ^ Population density ranged from 0–40.4 turtles/river-kilometer. The highest densities occur in central New Hampshire and lower densities occur in the Housatonic watershed. Population density is negatively correlated with agriculture at both riparian and watershed scales, and responds unimodally to forest cover. Wood turtle populations in western Massachusetts are declining by 6.6–11.2% annually. ^ I estimated ages of turtles by assessing shell-wear rates from photographs. Wood turtles regularly achieve ages over 80 years, and like related species, do not exhibit clear signs of senescence. Old wood turtles are reproductively dominant, and their survival rates are twice as high as young turtles. Carapace scutes appear to require 80 years to become worn. ^ Population modeling indicates that wood turtle populations are declining in New England due to anthropogenic and natural factors. Conservation efforts must address the effects of agriculture on adult survival. Climate change may negatively affect northeastern wood turtles through increased flooding. Populations in mountainous areas may be likely candidates for conservation because they don't occupy prime agricultural land, but may be more susceptible to floods.^