Characterizing alternative splicing and long non-coding RNA with high-throughput sequencing technology

Indiana University-Purdue University Indianapolis (IUPUI)Several experimental methods has been developed for the study of the central dogma since late 20th century. Protein mass spectrometry and next generation sequencing (including DNA-Seq and RNA-Seq) forms a triangle of experimental methods, corresponding to the three vertices of the central dogma, i.e., DNA, RNA and protein. Numerous RNA sequencing and protein mass spectrometry experiments has been carried out in attempt to understand how the expression change of known genes affect biological functions in various of organisms, however, it has been once overlooked that the result data of these experiments are in fact holograms which also reveals other delicate biological mechanisms, such as RNA splicing and the expression of long non-coding RNAs. In this dissertation, we carried out five studies based on high-throughput sequencing data, in an attempt to understand how RNA splicing and differential expression of long non-coding RNAs is associated biological functions. In the first two studies, we identified and characterized 197 stimulant induced and 477 developmentally regulated alternative splicing events from RNA sequencing data. In the third study, we introduced a method for identifying novel alternative splicing events that were never documented. In the fourth study, we introduced a method for identifying known and novel RNA splicing junctions from protein mass spectrometry data. In the fifth study, we introduced a method for identifying long non-coding RNAs from poly-A selected RNA sequencing data. Taking advantage of these methods, we turned RNA sequencing and protein mass spectrometry data into an information gold mine of splicing and long non-coding RNA activities.2019-05-0

SDPs and Robust Satisfiability of Promise CSP

For a constraint satisfaction problem (CSP), a robust satisfaction algorithm is one that outputs an assignment satisfying most of the constraints on instances that are near-satisfiable. It is known that the CSPs that admit efficient robust satisfaction algorithms are precisely those of bounded width, i.e., CSPs whose satisfiability can be checked by a simple local consistency algorithm (eg., 2-SAT or Horn-SAT in the Boolean case). While the exact satisfiability of a bounded width CSP can be checked by combinatorial algorithms, the robust algorithm is based on rounding a canonical Semidefinite programming(SDP) relaxation. In this work, we initiate the study of robust satisfaction algorithms for promise CSPs, which are a vast generalization of CSPs that have received much attention recently. The motivation is to extend the theory beyond CSPs, as well as to better understand the power of SDPs. We present robust SDP rounding algorithms under some general conditions, namely the existence of particular high-dimensional Boolean symmetries known as majority or alternating threshold polymorphisms. On the hardness front, we prove that the lack of such polymorphisms makes the PCSP hard for all pairs of symmetric Boolean predicates. Our method involves a novel method to argue SDP gaps via the absence of certain colorings of the sphere, with connections to sphere Ramsey theory. We conjecture that PCSPs with robust satisfaction algorithms are precisely those for which the feasibility of the canonical SDP implies (exact) satisfiability. We also give a precise algebraic condition, known as a minion characterization, of which PCSPs have the latter property.Comment: 62 pages, to appear in STOC 202

Los receptores para el reconocimiento de patrones moleculares: aportaciones de la química computacional para el diseño de fármacos y la modulación de la inmunidad innata

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Orgánica y Farmacéutica, leída el 18/11/2019In this Thesis we have aimed the study of the molecular recognition processes of receptors involved in the innate immunity. More concretely, we have focused in two different types of lectins, Galectins and DC-SIGN, and in Toll-like receptor 4. We have made use of computational techniques, including docking and virtual screening, molecular dynamics simulations, conformational analysis and quantum mechanics calculations. The work has been organized into several chapters that are summarized as follows: Chapter 1 corresponds to the current knowledge and perspectives about receptors related to immunity, in particular: galectins, DC-SIGN, and Toll-like receptor 4, corresponding to the molecular recognition events and modulation by small molecules. Chapter 2 describes the state-of-the-art methods in molecular modeling and computational chemistry applied to the study of molecular recognition processes and drug design...En esta tesis hemos estudiado los procesos reconocimiento molecular de receptores involucrados en la inmunidad innata. Más concretamente, nos hemos centrado en dos tipos diferentes de lectinas, Galectinas y DC-SIGN, y en el receptor Toll-like 4 (TLR4). Hemos utilizado técnicas computacionales, incluyendo docking y cribado virtual, simulaciones de dinámica molecular, análisis conformacional y cálculos de mecánica cuántica. El trabajo se ha organizado en diferentes capítulos que se resumen como sigue: El capítulo 1 corresponde al estado del arte y las perspectivas relacionadas con los estudios de reconocimiento molecular proteína-carbohidrato y diseño de nuevos moduladores con actividad biológica en receptores de la inmunidad, en particular galectinas, DC-SIGN y el receptor Toll-like 4. El capítulo 2 describe el estado actual de los métodos en modelado molecular y química computacional aplicados al estudio de los procesos de reconocimiento molecular y diseño de fármacos...Fac. de FarmaciaTRUEunpu

Modulación del TLR4 : estudios de reconocimiento molecular y diseño de fármacos por modelado molecular

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Orgánica y Farmacéutica, leída el 04/05/2018The heterodimeric complex, formed by Toll-Like Receptor 4 (TLR4) and its accessory protein Myeloid Differentiation factor 2 (MD-2) is responsible of activating the innate immune system when sensing the presence of particular pathogen-associated molecular patterns (PAMPs) from bacteria. The outer membrane of Gram-negative bacteria is primarily populated by lipopolysaccharides (LPS) which are essential for their growth and survival. These LPSs are specifically recognized by the TLR4/MD-2 complex as follows: an LPS binds to MD-2 inside a deep molecular hydrophobic pocket causing molecular rearrangements of the receptorial complex resulting in the dimerization of another TLR4/MD-2 unit. TLR4 ectodomains dimerization event brings together the TLR4 intercellular domains initiating the activation of innate immune system signaling pathways. Interestingly, this activation is not only modulated by naturally occurring LPSs from many different Gram-negative bacteria but also by non-naturally occurring glycolipids and other non-LPS like molecules...El complejo heterodimérico, formado por el receptor Toll-like 4 (Toll-like receptor 4, TLR4) y su proteína accesoria, el Myeloid Differentiation factor 2 (MD-2), es responsable de activar la respuesta del sistema inmune innato cuando detecta la presencia de patrones moleculares asociados a patógenos (pathogen associated molecular patterns, PAMPs), que provienen de bacterias y virus. En concreto, la membrana externa de bacterias Gram-negativas está poblada principalmente por lipopolisacáridos (lipopolisaccharides, LPS), compuestos que son esenciales para su crecimiento y supervivencia. Estos LPS son reconocidos de forma específica por el complejo TLR4/MD-2 de la siguiente manera: una molécula de LPS se une a la proteína MD-2 dentro de un profundo bolsillo hidrofóbico dando lugar al reordenamiento molecular del complejo resultando en la dimerización de otra unidad de TLR4/MD-2. El evento de dimerización de los ectodominios del TLR4 hace que se acercan los dominios intracelulares que inician la activación de las vías de señalización del sistema inmune innato. Curiosamente, esta activación no sólo está modulada por LPS naturales de muchas bacterias Gram-negativas distintas, sino también por glicolípidos no naturales y otras moléculas de estructura química diferente a los LPS...Fac. de FarmaciaTRUEunpu

Estudios computacionales de mecanismos moleculares de la inmunidad innata

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 20-12-2022Antimicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity, which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of bacterial lipopolysaccharide (LPS), and the phospholipid composition of the membrane, inflecting the membrane permeability to antibiotics. We have applied coarse-grained molecular dynamics simulations to capture the role of the phospholipid composition and lipid A structure in the membrane properties and morphology of ESKAPE Gram-negative bacterial vesicles. Moreover, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf were used to unveil their implications for membrane disruption. This study opens a promising starting point for understanding the molecular keys of bacterial membranes and promoting the discovery of new antimicrobials to overcome AMR...La resistencia a los antimicrobianos (AMR) es una emergencia sanitaria mundial. Los patógenos ESKAPE incluyen las familias bacterianas más resistentes a antibióticos y son altamente virulentas. En particular, las bacterias Gram negativas destacan por la complejidad de su pared celular, que presenta una fuerte resistencia frente a los antibióticos. Un elemento clave para la AMR es la estructura química del lipopolisacárido bacteriano (LPS) y la composición de los fosfolípidos de la membrana bacteriana, que influyen en su permeabilidad a los antibióticos. Se han empleado simulaciones de dinámica molecular de grano grueso para captar el papel de la composición de los fosfolípidos y la estructura del LPS en las propiedades y morfología de modelos de vesículas bacterianas Gram negativas ESKAPE. Además, se han empleado los péptidos antimicrobianos Cecropin B1, JB95 y PTCDA1-kf para desvelar su mecanismo disrupción de la membrana bacteriana. Este estudio abre un prometedor punto de partida para comprender las claves moleculares de la resistencia en membranas bacterianas y acelerar el descubrimiento de nuevos antibióticos para hacer frente a la AMR...Fac. de FarmaciaTRUEunpu

McNair Research Journal - Summer 2015

Journal articles based on research conducted by undergraduate students in the McNair Scholars Program Table of Contents Biography of Dr. Ronald E. McNair Statements: Dr. Neal J. Smatresk, UNLV President Dr. Juanita P. Fain, Vice President of Student Affairs Dr. William W. Sullivan, Associate Vice President for Retention and Outreach Mr. Keith Rogers, Deputy Executive Director of the Center for Academic Enrichment and Outreach McNair Scholars Institute Staf

Study of the interaction between sialic acid-binding immunoglobulin-type lectins (Siglec) and sialylated glycans for the development of a new generation of immunomodulators.

Glycans and complementary glycan-binding proteins represent essential components in the control of both innate and adaptive immunity. Sialic acids are a family of sugars found on the terminal end of mammalian glycoconjugates; they able to act as marker of self in the immune system, as such residues are absent in most microbes. Sialic acid-binding immunoglobulin-like lectins, or Siglecs, are cell surface receptors that recognize sialic acids and are known to modulate immune responses, influencing almost every cell in the hematopoietic system. Siglecs are involved in events like cell adhesion and signaling, inhibition or regulation of the immune cell activation, all mediated by the interaction with sialylated ligands. Sialic acid-Siglec interactions have been associated with a broad spectrum of diseases, stretching from autoimmunity to neurodegeneration and cancer. Thus, strategies for a rational modulation of the interactions between Siglecs and sialylated glycans in pathophysiological processes exhibit a great therapeutic potential. In this context, the present thesis project aimed at the study of the interaction between Siglecs and their cognate sialic acid containing ligands, to disclose the key recognition events underlining host immune suppression or activation. To this end, a multidisciplinary approach combining advanced technologies as ligand-based NMR techniques, including STD-NMR and tr-NOESY, biophysical binding assays and computational methodologies, such as homology modelling docking and MD simulations, was applied to provide an atomistic depiction of the interaction interfaces between various sialoglycans and their receptors. The described strategy has been employed to characterize the binding features of several receptors of the Siglecs family, namely CD22/Siglec-2, Siglec-10 and Siglec-7. CD22 is a B-cell surface inhibitory protein capable of selectively -(2,6) linked sialylated glycans, thus dampening autoimmune responses against self-antigens. The characterization of complex-type N-glycans by CD22 allowed to describe the conformational behavior of the flexible ligands; the formation of CD22 homo-oligomers on the B-cell surface was also addressed. Furthermore, it was provided a global vision of how the most diffuse neuraminic acid forms of sialylated N-glycans are accomodated in the binding pocket of CD22. Moreover, the elucidation of the binding epitope of a synthetic sialo-mimetic upon CD22 interaction afforded new hints for the design and synthesis of high-affinity ligands of therapeutic relevance against B-cell derived malignancies. Then, the Siglec-10, an inhibitory receptor that recognize 2,3 and -linked sialoglycans was studied, thus providing the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. After that, Siglec-7, a well-established inhibitory receptor that is primarily located on natural killer where it acts as inhibitor of cancer cells cytotoxicity via sialylated ligands binding, has been characterized in the interplay with the oncogenic pathogen F. nucleatum. Indeed, the presence of sialylated lipopolysaccharide (LPS) on certain F. nucleatum strains, hinted that it may have a significant role at the immune interface. The interaction between Siglec-7 and the O-polysaccharide chain from the LPS of F. nucleatum 10953 strain has been depicted, thus delineating a structural binding model that might contribute to explain the etiological role of F. nucleatum in carcinogenesis. A similar approach was employed to other sialoglycan- related systems, i. e. to dissect the mechanism of sialic acid recognition and hydrolysis by mumps virus hemagglutinin neuraminidase, a viral glycoprotein that plays key roles in virus entry and infection; and to assess the binding of the human macrophage galactose-type lectin (MGL) in the interplay with lipooligosaccharide of E. coli strain R1. In conclusion, the structural and functional characterization of Siglec- sialylated glycans interaction have allowed the analysis, at a molecular level, of crucial feature of 3D complexes, highlighting the molecular determinants involved in recognition and binding events, that will aid for the development or optimization of molecules for therapeutic targeting of the Siglecs