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Thesis advisor: David R. Burgess / Centration of the nucleus after fertilization is an essential step for setting-up cell division and proper embryonic development in many proliferating cells such as the sea urchin. The sperm aster must capture the female pronucleus for fusion as well as the nucleus becoming positioned at the center of the cell. Microtubules (MTs) are known to play a role in this centration but the exact mechanism remains unknown. This begins to investigate current models of nuclear centration and the role of various interactions. Three phases of migration were observed as the male aster migrated with support in independent movements of the male and female pronuclei. Dimpling affects present that altered the morphology of the cell were observed when engagement occurred between the male and female pronuclei. It was discovered that this dimpling effect was a result of an interaction between MTs and the cortex, as confirmed by visualization of sheared cells in which only the cortex remained. Stemming from previous and current research in the lab, the role of post-translational modifications (PMTs) in nuclear centration was investigated for the different forces exerted due to various factors. Tyrosinated and detyrosinated populations were observed with and without the presence of parthenolide (PTL), an agent that inhibits detyrosination. PTL was observed to not only prevent the proper migration, but also that it expanded tyrosination of tubulin – which would further disrupt the force vectors created through the PMTs promotion of dyneins and kinesins. The results have lead to a new hypothesis to be furthered in order to gain an in-depth understanding in the mechanism(s) for pronuclear migration. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

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Cladistic analyses of the interclass relationships of the phylum Echinodermata have not provided a phylogeny that is separately supported by both larval and adult characters. Similar to the reported incongruence with cladistic analyses, molecular analyses of ribosomal RNA (rRNA) genes have also given ambiguous results, which could be due to a number of factors. The use of short sequences, systematic errors such as long branch attraction, and mis-alignments of the data that are introduced by programs which are unsuitable for non-protein coding genes, have resulted in a controversy as to the true nature of echinoderm relationships. Historically, it is the position of the ophiuroids among the five extant classes of echinoderms that has been the most poorly understood, and the most recently published proposal is that there are three plausible relationships, albeit none of these are sufficiently supported. Re-analysis of 28S and 18S rRNA gene sequence data, with the addition of more phylogenetically informative sites as well as new taxa, the use of an alignment procedure that is based on rRNA secondary structure, and the testing of a myriad of evolutionary models have resulted in some new findings of ancestry. Interestingly, it is the phylogenetic position of the ophiuroids that proves to be among the more solid results from this analysis, while the historically supported sister group relationship between the echinoid and holothuroid classes are not greatly corroborated.

The phosphagen kinases are a family of enzymes that catalyze the reversible phosphorylation of specific phosphagens using ATP as the phosphate donor. The evolutionary relationships between the enzymes in this family have been studied for over 30 years and yet aspects of the relationships remain unclear. Here, arginine kinase from Strongylocentrotus purpuratus eggs was purified to homogeneity and analyzed for physical and kinetic characteristics as well as its sequence homology with other phosphagen kinases. The results indicate that dimeric arginine kinase from S. purpuratus eggs evolved from a dimeric creatine kinase after dimeric creatine kinase evolved from a monomeric arginine kinase. The molecular weight/subunit composition and sequence of dimeric arginine kinase from S. purpuratus eggs is more comparable with dimeric creatine kinases than monomeric arginine kinases. However, the kinetic characteristics of dimeric arginine kinase from S. purpuratus eggs, including the absence of substrate cooperativity, are more comparable with other arginine kinases than creatine kinases. These results indicate a unique evolution for the dimeric arginine kinase, as well as the importance sequence composition can have on the three dimensional structure of an enzyme and its kinetic characteristics.

Arginine kinase catalyzes the reversible phosphorylation of arginine using ATP. This phosphotransferase is found throughout invertebrate species; whereas a homologous enzyme, creatine kinase, is found in both vertebrate and invertebrate species. Arginine kinases are found as monomers of 40 kDa or 80 kDa and dimers of 80 kDa while creatine kinases are found as dimers of 80 kDa, monomers of 150 kDa, or octamers of 320 kDa. The significance or advantage of the dimeric state or various quaternary structures is still not understood for this family of enzymes. Here, arginine kinase from Isostychopus badonotus muscle was purified to homogeneity and analyzed for physical, kinetic, and immunological characteristics. The results indicate that arginine kinase from the sea cucumber, I. badonotus, is a dimer with a molecular weight of 87 kDa that displays physical and kinetic characteristics similar to other arginine kinases regardless of their weight or subunit composition. However, immunological cross-reactivity using I. badonotus polyclonal antibodies shows that dimeric arginine kinase from the sea cucumber can react with dimeric arginine and creatine kinases but not with monomeric arginine or creatine kinases. Comparable results are seen with polyclonal antibodies raised against purified monomeric arginine kinase from the American cockroach, Periplaneta americana. Monomeric arginine kinase from the cockroach reacted with monomeric arginine kinases but not with dimeric arginine or creatine kinases or monomeric creatine kinases. Arginine kinase from the sea cucumber and the cockroach is substantially inhibited by the anion nitrate which mimics the transferable phosphoryl group in the assumed rapid equilibrium, random addition reaction. Here, nitrate has been shown to inhibit both the initial velocity and percent of product formed from arginine kinase in I. badonotus and P. americana. Difference spectra for each enzyme in the presence of varying components of the transition state analog suggest that nitrate has an effect on the enzyme itself and inhibits through a mechanism beyond that of stabilization of the dead-end complex. Further characterization of the dimeric state in these enzymes on a structural level included the elucidation of the protein sequence from the American cockroach and a comparison with dimeric arginine and creatine kinases.

Animal chemosensation involves several families of G protein-coupled receptors (GPCRs) and, though some of these families are well characterized in vertebrates and nematode worms, receptors have not been identified for most metazoan lineages. In this dissertation, I use a combination of bioinformatics approaches to identify candidate chemosensory receptors in three invertebrates that occupy key positions in the metazoan phylogeny. In the sea urchin Strongylocentrotus purpuratus, I uncovered 192 candidate chemosensory receptors many of which are expressed in sensory structures including pedicellariae and tube feet. In the cephalochordate Branchiostoma floridae, my survey uncovered 50 full-length and 11 partial odorant receptors (OR). No ORs were identified in the urochordate Ciona intestinalis. By exposing conserved amino acid motifs and testing the ability of those motifs to discriminate between ORs and non-OR GPCRs, I identified three OR-specific amino acid motifs that are common in cephalochordate, fish and mammalian ORs and are found in less than 1% of non-ORs from the rhodopsin-like GPCR family. To further investigate the antiquity of vertebrate ORs, I used the OR-specific motifs as probes to search for orthologs among the protein predictions from 12 invertebrates. My search uncovered a novel group of genes in the cnidarian Nematostella vectensis. Phylogenetic analysis that included representatives from the major subgroups of rhodopsin-like GPCRs showed that the cnidarian genes, the cephalochordate and vertebrate ORs, and a subset of genes S. purpuratus from my initial survey, form a monophyletic clade. The taxonomic distribution of these genes indicates that the formation of this clade began at least 700 million years ago, prior to the divergence of cnidarians and bilaterians. Furthermore, my phylogenetic analyses show that three of the four major subgroups of rhodopsin-like GPCRs existed in the ancestor of cnidarians and bilaterians. The utility of the new genes I describe here is that they can be used to identify candidate olfactory cells and organs in cnidarians, echinoderms and cephalochordates that can be tested for function. These genes also provide the raw material for surveys of other metazoans as their genomes become available. My sequence level comparison between chordates, echinoderms and cnidarians exposed several conserved amino acid positions that may be useful for understanding receptor mediated signal transduction. ORs and other rhodopsin-like GPCRs have roles in cell migration, axon guidance and neurite growth; therefore duplication and divergence in the rhodopsin-like gene family may have played a key role in the evolution of cell type diversity (including the emergence of complex nervous systems) and in the evolution of metazoan body plan diversity. / Graduate

abstract: Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Les voies de signalisation Wnt jouent un rôle important dans divers processus de l’embryogenèse des métazoaires. Par exemple, les signaux WNT sont requis pour l’établissement de la polarité cellulaire, les mouvements de tissus ou la mise en place de la polarité antéropostérieure. La signalisation Wnt tire son nom des protéines WNTs qui sont des protéines secrétées capables de signaler via leurs récepteurs membranaires FRIZZLED (FZD). Une interaction WNT-FZD peut déclencher au moins trois cascades de signalisation distinctes, selon le contexte cellulaire et les couples WNT-FZD impliqués. Chez l'homme, la dérégulation des voies de signalisation Wnt conduit, au cours du développement, à des maladies congénitales, ou chez l'adulte, à des cancers. La compréhension de la diversité numéraire des wnt et des fonctions biologiques qu’ils exercent représente donc un challenge contemporain qu’il est nécessaire de relever, non seulement au titre de l’acquisition de nouvelles connaissances, mais aussi en vue du développement de nouvelles approches à but de diagnostic ou de thérapie. Lors de ma thèse, je me suis dans un premier temps attaché à décrire les patrons d’expression temporels et spatiaux de l’ensemble des gènes wnt et fzd présents chez l’oursin Paracentrotus lividus. L’oursin, comme l’homme, appartient au phylum des deutérostomiens. De plus, son développement est simple et ses embryons se prêtent à l’expérimentation, du fait de multiples approches disponibles pour ce modèle. Par ailleurs, contrairement au lignage des vertébrés, celui de l’oursin n’a pas subi de duplications complète de génome, exhibant ainsi la même diversité de familles de gènes, mais avec moins de redondance au sein de ces dernières, ce qui facilite leur analyse. Etudier les mécanismes qui régissent l'embryogénèse de l’oursin permet donc d’apporter des informations quant aux mécanismes régissant celle des deuterostomes. Mes résultats mettent en évidence que les ligands wnt et les récepteurs fzd sont exprimés de façon dynamique au cours de l’embryogenèse de P. lividus. Fait intéressant, jusqu’à la gastrulation, les gènes codant les récepteurs FZD, présentent des patrons d’expressions distincts, leur somme couvrant néanmoins la très vaste majorité des territoires embryonnaires. Ainsi, cette étude a permis d’établir un catalogue des compatibilités spatio-temporelles potentielles entre les protéines WNT et FZD durant l’embryogenèse d’un deutérostomien, ainsi que des hypothèses quant aux rôles possibles jouées par ces protéines durant ce processus. Dans un second temps, je me suis intéressé à retracer l’évolution des protéines WNT, et à déterminer s’il existait une fonction intrinsèque ancestrale des WNT qui soit conservée, parmi les métazoaires. Pour cela, j’ai d’une part analysé la composition des répertoires wnt de dix-sept espèces de métazoaires, la séquence primaire des protéines qu’ils encodent, la position relative des wnt au sein de leurs génomes respectifs et, pour certaines espèces, l’environnement génomique des wnt. D’autre part, j’ai contribué à vérifier si trois ligands WNT d’éponge et une séquence ancestrale, calculée à partir d’un échantillon de séquences de métazoaires, étaient capable d’activer les voies de signalisation Wnt lorsqu’exprimées chez trois animaux planulozoaires (cnidaires et bilatériens). Les données obtenues au cours de cette étude ont permis d’établir un modèle de diversification de la famille de gènes wnt chez les métazoaires et ont révélé l’existence de fonctions intrinsèques conservées parmi les ligands WNT. / The Wnt signaling pathways play crucial roles during several processes of metazoan embryogenesis. For instance Wnt signaling is involved in morphogenetic movements as well as cell polarity and anteroposterior polarity establishment. Wnt signaling takes its name from the WNT proteins, which are secreted molecules that signal through their cognate receptors, the FRIZZLED (FZD) proteins. A WNT-FZD interaction may trigger several distinct intracellular signaling cascades, depending on the cellular context and the WNT-FZD couples involved. In humans, a deregulation of WNT signaling during embryonic development or adult life leads to congenital diseases and cancers, respectively. Understanding the great diversity of wnt genes and their biological functions is thus a modern-day challenge, which needs to be addressed, not only to foster general knowledge, but also to allow the identification of novel therapeutic targets and approaches towards future new medical applications.In the course of my Ph.D., I first aimed at describing the spatial and temporal expression patterns of all wnt and fzd genes present in the sea urchin, Paracentrotus lividus. Like humans, sea urchins belong to the deuterostome phyla. In addition, the early development of the sea urchin is relatively simple and is easy to interfere with, thanks to the experimental approaches available for this model. Moreover, the sea urchin lineage did not undergo whole genome duplications, unlike humans, exhibiting the same diversity of gene families, but with less redundancy within the subfamilies, thereby facilitating functional analyses. Thus, studying the mechanisms that drive sea urchin embryogenesis represent a valuable asset to provide insights into the conserved mechanisms controlling the development of deuterostomes. My results show that wnt and fzd genes are expressed in a dynamic fashion throughout P. lividus embryogenesis. Interestingly, until the gastrulation, the genes encoding FZD receptors are expressed in distinct territories that, taken together, cover almost the entire embryo. Accordingly, this work allowed me to establish a catalog of possible WNT-FZD couples likely to form during the embryogenesis of a deuterostome animal, based on the compatibility of their respective spatiotemporal distribution, and to further provide insights into the potential biological functions of these WNT and FZD proteins during this process. The second goal of my Ph.D. was to reconstruct the evolution of the WNT proteins and to determine whether an ancestral intrinsic function of WNTs is conserved within metazoans. To this end, I analyzed the composition of the wnt repertoires of seventeen metazoan species, the primary sequences of the WNT proteins they encode, the relative positions of the wnts within their respective genomes, and, for selected species, the genomic environment of their wnts. In addition, I investigated whether three sponge WNT ligands and an ancestral WNT sequence, which was calculated from a sample of metazoan WNT sequences, are able to trigger Wnt signaling pathways, when expressed in three planulozoan (cnidarian and bilaterian) species. The data obtained during this study allows me to propose a model for the diversification of wnt genes in metazoans and revealed conserved intrinsic functional capabilities for the WNT ligands.