756 research outputs found

    Production, Functional Characterization and Inhibition of Carbonic Anhydrases of Parasites

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    Hiilihappoanhydraasit (carbonic anhydrase, CA) ovat metalloentsyymejä, joita esiintyy kaikkialla elollisessa luonnossa. Niiden tehtävä on katalysoida elintärkeää käänteistä reaktiota: hiilidioksidin hydraatiota hiilihapoksi (CO2 + H2O ⇋ H2CO3 ⇋ HCO3- + H+). Reaktio on osana monissa fysiologisissa ja metabolisissa prosesseissa, kuten happo-emästasapainon säätelyssä, glukoneogeneesissa ja yhteyttämisessä. Hiilihappoanhydraasit jaetaan yhteensä kahdeksaan perheeseen, joista ihmisillä esiintyy vain ensimmäisenä löydettyä α-entsyymiperhettä. Monilla taudinaiheuttajilla, kuten bakteereilla ja loisilla, on myös muiden hiilihappoanhydraasiperheiden proteiineja. Schistosoma mansoni on suolistotulehdusta aiheuttava halkiomato, jota esiintyy endeemisenä monilla trooppisilla ja subtrooppisilla seuduilla. Entamoeba histolytica ja Acanthamoeba castellanii ovat ihmisille infektioita aiheuttavia ameboja. E. histolytica aiheuttaa suolistotulehdusta ja A. castellanii pääasiassa sarveiskalvon tulehdusta, sekä immuunipuutteisille henkilöille myös syviä infektioita. Yhteistä näille loisille on, että niiden diagnostiikassa tai hoidossa on puutteita, ja ne ovat globaalisti erittäin yleisiä. Niiden aiheuttamat sairaudet voivat olla tappavia, ja aiheuttavat lievimmilläänkin merkittävää elämänlaadullista haittaa. Väitöskirjatutkimukseni tavoitteena on löytää nopeampi menetelmä tunnistaa A. castellanii biologisesta näytteestä sekä löytää uusia potentiaalisia lääkeainemolekyylejä näiden kolmen loisen aiheuttamien sairauksien tehokkaammaksi hoitamiseksi. Väitöstutkimuksessani tuotin E. histolytican ja S. mansonin β-hiilihappoanhydraasit Escherichia coli -bakteereissa. Tutkimme hiilihappoanhydraasien inhibiittorien vaikutusta tuotettuihin entsyymeihin, ja löysimme useita lupaavia inhibiittoreita. Lisäksi kehitimme uuden nopean diagnostisen menetelmän A. castellaniin havaitsemiseksi näytteestä, sillä nykyinen ameban viljelyyn perustuva menetelmä on kliiniseen diagnostiikkaan liian hidas. Nykyiset A. castellaniin aiheuttamien infektioiden hoitoon käytetyt lääkkeet ovat vain kohtalaisen tehokkaita ja niihin liittyy useita haittavaikutuksia, joten kehitimme uuden menetelmän uusien lääkeaineiden etsimistä varten ja testasimme useita, jo kliinisessä käytössä olevia hiilihappoanhydraasin estäjiä menetelmällämme. Havaitsimme, että dorsolamidi ja asetatsoliamidi ovat lupaavia uusia vaihtoehtoja akantamebakeratiitin hoitamiseksi. Niiden etuna on pitkään jatkunut käyttö ihmispotilaiden muiden sairauksien hoidossa, joten niiden haitta- ja sivuvaikutukset ovat tunnettuja. Valitettavasti asetatsoliamidi on vain kohtalaisen tehokas inhiboimaan E. histolytican ja S. mansonin β-hiilihappoanhydraaseja. Dorsolamidi on tehokas inbiittori S. mansonin β-hiilihappoanhydraasia vastaan, toisin kuin E. histolytican β-hiilihappoanhydraasia se inhiboi huonosti. Onneksi moni muu inhibiittori on tehokas estämään niiden molempien toimintaa. Tämä väitöskirja lisää tietoa ja mahdollisuuksia hyödyntää hiilihappoanhydraaseja loisinfektioiden diagnostiikassa ja hoidossa.Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that can be found in almost all living organisms. They catalyze a vital chemical reaction: the reversible hydration of carbon dioxide (CO2 + H2O ⇋ H2CO3 ⇋ HCO3− + H+). It is part of multiple physiological and metabolic processes, such as p regulation, gluconeogenesis and photosynthesis. CAs are divided into eight families, from which the human genome only involves α-CAs. Many pathogens, such as bacteria and parasites, also have CA enzymes from other families. Schistosoma mansoni is a parasitic blood fluke that causes intestinal infection in many tropical and subtropical areas. Entamoeba histolytica and Acanthamoeba castellanii are amoebae that cause different infections in humans. E. histolytica causes intestinal infection, and A. castellanii mainly infects the cornea but also causes invasive infections in immunocompromised people. The infections of these parasites are common globally, and unfortunately, the diagnosis and treatment are often delayed. The infections can be lethal, and at their mildest, they cause a significant reduction in the quality of life. My study aims to find a faster and more effective way to detect A. castellanii from a biological sample and discover new potential drug candidates to treat these parasitic infections. In my thesis, I produced the β-CAs of E. histolytica and S. mansoni using Escherichia coli as a protein production organism. We studied the inhibitory effects of many known CA inhibitors and identified several promising candidates or potential leads for further development. In addition, we developed a novel rapid diagnostic method to detect A. castellanii from a biological sample. The novel method could replace the current gold standard, sample culture, which is slow in clinical diagnostics. We established a new method to investigate potential drug candidates for treating A. castellanii, as the current treatment options are only moderately effective and have many adverse side effects. By this method, we tested several clinically used CA inhibitors with interesting outcomes. Dorzolamide and acetazolamide were found to be new promising options for treating Acanthamoeba keratitis. The advantage of these drugs is also the long use history with human patients in treating other conditions, which has made the side effects and risks very familiar to medical staff. Unfortunately, acetazolamide was only moderately effective against the β-CAs of S. mansoni and E. histolytica, as was dorzolamide against the β-CA of E. histolytica. In contrast, dorzolamide was an effective inhibitor against the β-CA of S. mansoni. We also found many other effective inhibitors against the β-CAs of E. histolytica and S. mansoni. This thesis increases the knowledge and options to use the CAs of parasites to diagnose and treat infections caused by parasites

    Computational Analyses of Metagenomic Data

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    Metagenomics studies the collective microbial genomes extracted from a particular environment without requiring the culturing or isolation of individual genomes, addressing questions revolving around the composition, functionality, and dynamics of microbial communities. The intrinsic complexity of metagenomic data and the diversity of applications call for efficient and accurate computational methods in data handling. In this thesis, I present three primary projects that collectively focus on the computational analysis of metagenomic data, each addressing a distinct topic. In the first project, I designed and implemented an algorithm named Mapbin for reference-free genomic binning of metagenomic assemblies. Binning aims to group a mixture of genomic fragments based on their genome origin. Mapbin enhances binning results by building a multilayer network that combines the initial binning, assembly graph, and read-pairing information from paired-end sequencing data. The network is further partitioned by the community-detection algorithm, Infomap, to yield a new binning result. Mapbin was tested on multiple simulated and real datasets. The results indicated an overall improvement in the common binning quality metrics. The second and third projects are both derived from ImMiGeNe, a collaborative and multidisciplinary study investigating the interplay between gut microbiota, host genetics, and immunity in stem-cell transplantation (SCT) patients. In the second project, I conducted microbiome analyses for the metagenomic data. The workflow included the removal of contaminant reads and multiple taxonomic and functional profiling. The results revealed that the SCT recipients' samples yielded significantly fewer reads with heavy contamination of the host DNA, and their microbiomes displayed evident signs of dysbiosis. Finally, I discussed several inherent challenges posed by extremely low levels of target DNA and high levels of contamination in the recipient samples, which cannot be rectified solely through bioinformatics approaches. The primary goal of the third project is to design a set of primers that can be used to cover bacterial flagellin genes present in the human gut microbiota. Considering the notable diversity of flagellins, I incorporated a method to select representative bacterial flagellin gene sequences, a heuristic approach based on established primer design methods to generate a degenerate primer set, and a selection method to filter genes unlikely to occur in the human gut microbiome. As a result, I successfully curated a reduced yet representative set of primers that would be practical for experimental implementation

    Advanced sequencing technologies applied to human cytomegalovirus

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    The betaherpesvirus human cytomegalovirus (HCMV) is a ubiquitous viral pathogen. It is the most common cause of congenital infection in infants and of opportunistic infections in immunocompromised patients worldwide. The large double-stranded DNA genome of HCMV (236 kb) contains several genes that exhibit a high degree of variation among strains within an otherwise highly conserved sequence. These hypervariable genes encode immune escape, tropism or regulatory factors that may affect virulence. Variation arising from these genes and from an evolutionary history of recombination between strains has been hypothesised to be linked to disease severity. To investigate this, the HCMV genome has been scrutinised in detail over the years using a variety of molecular techniques, most looking only at one or a few of these genes at a time. The advent of high-throughput sequencing (HTS) technology 20 years ago then started to enable more in-depth whole-genome analyses. My study extends this field by using both HTS and the more recently developed long-read nanopore technology to determine HCMV genome sequences directly from clinical samples. Firstly, I used an Illumina HTS pipeline to sequence HCMV strains directly from formalin-fixed, paraffin-embedded (FFPE) tissues. FFPE samples are a valuable repository for the study of relatively rare diseases, such as congenital HCMV (cCMV). However, formalin fixation induces DNA fragmentation and cross-linking, making this a challenging sample type for DNA sequencing. I successfully sequenced five whole HCMV genomes from FFPE tissues. Next, I developed a pipeline utilising the single-molecule, long-read sequencer from Oxford Nanopore Technologies (ONT) to sequence HCMV initially from high-titre cellcultured laboratory strains and then from clinical samples with high HCMV loads. Finally, I utilised a direct RNA sequencing protocol with the ONT sequencer to characterise novel HCMV transcripts produced during infection in cell culture, demonstrating the existence of transcript isoforms with multiple splice sites. Overall, my findings demonstrate how advanced sequencing technologies can be used to characterise the genome and transcriptome of a large DNA virus, and will facilitate future studies on HCMV prognostic factors, novel antiviral targets and vaccine development

    Design of new algorithms for gene network reconstruction applied to in silico modeling of biomedical data

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    Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Ingeniería, Ciencia de Datos y BioinformáticaClave Programa: DBICódigo Línea: 111The root causes of disease are still poorly understood. The success of current therapies is limited because persistent diseases are frequently treated based on their symptoms rather than the underlying cause of the disease. Therefore, biomedical research is experiencing a technology-driven shift to data-driven holistic approaches to better characterize the molecular mechanisms causing disease. Using omics data as an input, emerging disciplines like network biology attempt to model the relationships between biomolecules. To this effect, gene co- expression networks arise as a promising tool for deciphering the relationships between genes in large transcriptomic datasets. However, because of their low specificity and high false positive rate, they demonstrate a limited capacity to retrieve the disrupted mechanisms that lead to disease onset, progression, and maintenance. Within the context of statistical modeling, we dove deeper into the reconstruction of gene co-expression networks with the specific goal of discovering disease-specific features directly from expression data. Using ensemble techniques, which combine the results of various metrics, we were able to more precisely capture biologically significant relationships between genes. We were able to find de novo potential disease-specific features with the help of prior biological knowledge and the development of new network inference techniques. Through our different approaches, we analyzed large gene sets across multiple samples and used gene expression as a surrogate marker for the inherent biological processes, reconstructing robust gene co-expression networks that are simple to explore. By mining disease-specific gene co-expression networks we come up with a useful framework for identifying new omics-phenotype associations from conditional expression datasets.In this sense, understanding diseases from the perspective of biological network perturbations will improve personalized medicine, impacting rational biomarker discovery, patient stratification and drug design, and ultimately leading to more targeted therapies.Universidad Pablo de Olavide de Sevilla. Departamento de Deporte e Informátic

    2023-2024 Catalog

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    The 2023-2024 Governors State University Undergraduate and Graduate Catalog is a comprehensive listing of current information regarding:Degree RequirementsCourse OfferingsUndergraduate and Graduate Rules and Regulation

    Undergraduate Catalog of Studies, 2022-2023

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    Genomic epidemiology and antimicrobial resistance of Klebsiella pneumoniae in the Comunitat Valenciana

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    La resistencia antimicrobiana (RAM) es una importante amenaza para la salud pública a nivel mundial. El mal uso de los antibióticos ha llevado al surgimiento y propagación de infecciones resistentes a los antibióticos. Entre los patógenos que más muertes causan asociadas a la RAM bacteriana se encuentra Klebsiella pneumoniae, uno de los patógenos más preocupantes. DE hecho, en 2019, K. pneumoniae se clasificó como la segunda causa de muertes atribuible a la RAM. Entre las resistencias adquiridas en K. pneumoniae, las que mayor preocupación causan son las cepas que han desarrollado resistencia a cefalosporinas de tercera generación (3GC) y carbapenems (CRKp). En esta tesis se ha investigado la epidemiología de K. pneumoniae resistente a 3GC y carbapenems utilizando la información genómica recopilada en el proyecto Vigilancia de Klebsiella pneumoniae en la Comunitat Valenciana (SKPCV). Bajo el proyecto SKPCV, se recolectaron casi 2200 aislados de K. pneumoniae productores de ESBL y/o carbapenems durante 3 años (2017 - 2019) y posteriormente se secuenció el genoma completo utilizando tecnologías de segunda generación (Illumina) y tercera generación (Pacific Biosciences y Oxford Nanopore). Para proporcionar contexto y establecer una colección que nos permitiera dilucidar las relaciones entre los aislados de K. pneumoniae del SKPCV con los de hospitales españoles y a nivel mundial, incluimos aislados recogidos previamente en algunos hospitales del NLSAR, así como datos externos de tres bases de datos diferentes: RefSeq, GenBank y ENA. Utilizando estos datos, recogimos más de 13,000 genomas. Trabajar con conjuntos de datos grandes y garantizar la calidad de los datos puede ser un desafío, por lo tanto, creamos un filtro de control de calidad con pasos jerarquizados que evaluaron la asignación taxonómica y la contaminación interespecífica, la calidad del ensamblado, la contaminación intraespecífica y, finalmente, la similitud genómica de toda la colección. Utilizando este filtro de calidad, obtuvimos una gran colección con 1,604 genomas del SKPCV, 395 aislados retrospectivos recogidos en tres hospitales del NLSAR y más de 10,000 genomas globales disponibles en las bases de datos públicas. Finalmente, encontramos que las composiciones de linajes del SKPCV y NLSAR eran muy diversas, pero también similares a las de los genomas españoles depositados en las bases de datos. De hecho, la mayoría de los aislados de NLSAR estaban relacionados con aislados recogidos en otras regiones de España, lo que sugiere historias evolutivas similares. Nuestro análisis reveló que solo un linaje, ST307, fue responsable de la mayoría de las infecciones resistentes a 3GC y carbapenems, así como de las transmisiones interhospitalarias. También descubrimos que los determinantes de la resistencia a 3GC y carbapenems, junto con los linajes portadores correspondientes, se distribuían de manera distinta en los hospitales y que, excepto por ST307 que portaba blaCTX-M-15, la mayoría de los linajes y combinaciones de determinantes de AMR se limitaban mayormente a un solo hospital. De hecho, las poblaciones hospitalarias eran diferentes entre sí. Nuestros hallazgos sugieren que la carga de la RAM y K. pneumoniae en esta región fue el resultado de una diversidad de factores, que incluyen linajes únicos que probablemente se originaron en la comunidad o en la microbiota previa de los pacientes, así como una compleja interacción entre la transmisión de linajes entre hospitales y la proliferación local de clones problemáticos dentro de cada hospital. Nuestros hallazgos muestran que la aparición inicial de resistencia a carbapenems y la diseminación del grupo hospitalario universitario (HGUV) ocurrieron durante un corto período de un año y fue muy compleja. Encontramos seis linajes diferentes que comprendían la mayoría de la población de CRKp en el HGUV, diseminando diferentes mecanismos de resistencia (AmpC, OXA-48, NDM-1 y NDM-23) en diferentes variantes de plásmidos. Estos linajes experimentaron una expansión clonal local, con varios casos de posible transmisión directa dentro del hospital. Finalmente, utilizamos la epidemiología genómica para describir la aparición y diseminación en varios hospitales de un nuevo gen de resistencia a carbapenems, denominado blaNDM-23. Pudimos dilucidar el efecto fenotípico y el entorno genético del gen. El gen estaba contenido en un plásmido resistente a múltiples fármacos con 18 genes adicionales de resistencia a antibióticos, lo que produjo un fenotipo de resistencia a múltiples fármacos. El gen y el plásmido se encontraron en una cepa ST437. Descubrimos que el plásmido no era movilizable, por lo que la diseminación del blaNDM-23 se produjo a través de una expansión clonal. La diseminación de este linaje ST437 portador del blaNDM-23 afectó al menos a cuatro hospitales diferentes de la Comunitat Valenciana desde 2016 hasta al menos 2019, cuando concluyó nuestro muestreo

    Digital agriculture: research, development and innovation in production chains.

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    Digital transformation in the field towards sustainable and smart agriculture. Digital agriculture: definitions and technologies. Agroenvironmental modeling and the digital transformation of agriculture. Geotechnologies in digital agriculture. Scientific computing in agriculture. Computer vision applied to agriculture. Technologies developed in precision agriculture. Information engineering: contributions to digital agriculture. DIPN: a dictionary of the internal proteins nanoenvironments and their potential for transformation into agricultural assets. Applications of bioinformatics in agriculture. Genomics applied to climate change: biotechnology for digital agriculture. Innovation ecosystem in agriculture: Embrapa?s evolution and contributions. The law related to the digitization of agriculture. Innovating communication in the age of digital agriculture. Driving forces for Brazilian agriculture in the next decade: implications for digital agriculture. Challenges, trends and opportunities in digital agriculture in Brazil
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