Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-03-13T13:00:52Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Maria Paloma Silva de Barros_PPGG UFPE.pdf: 8103324 bytes, checksum: 8a69c263eb54cdd37eb3275985c8eb30 (MD5) / Made available in DSpace on 2015-03-13T13:00:52Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Maria Paloma Silva de Barros_PPGG UFPE.pdf: 8103324 bytes, checksum: 8a69c263eb54cdd37eb3275985c8eb30 (MD5) Previous issue date: 2012-09-14 / A peste é uma doença que permanece enraizada em inúmeros focos naturais por todo o mundo. Embora o Brasil passe por um período de silenciamento epidemiológico, anticorpos antipestosos são detectados nas atividades de vigilância, sugerindo que estes focos permanecem ativos. A Yersinia pestis, agente causador da peste, apresenta uma história evolutiva recente e é considerada uma espécie, geneticamente, muito homogênea. Diante da necessidade de estudos mais aprofundados sobre as características moleculares e evolutivas dos isolados de Y. pestis dos focos do Brasil, realizamos neste trabalho uma caracterização de cepas da coleção biológica (Fiocruz-CYP) de Yersinia, provenientes de cinco focos de peste do Brasil, com a finalidade de compreender a adaptação da bactéria no ambiente. Realizamos a padronização e a análise de macrorestrição (PFGE) em 22 cepas de Y. pestis, 17 de um surto ocorrido em 1986 e cinco isoladas da atividade de vigilância. O PFGE não separou os isolados da rotina e do surto, entretanto foi capaz de revelar diversidade genética entre as cepas. As técnicas MLVA e CRISPR também foram utilizadas na genotipagem das cepas de Y. pestis. Doze locos VNTR (MLVA) analisados em 37 cepas, pertencentes a dois eventos epidemiológicos distintos, permitiram observar a separação dos grupos por evento e estabelecer uma relação epidemiológica. Três locos CRISPR (YPa, YPb e YPc) foram analisados em 146 cepas, apenas dois (YPa e YPb) se mostraram polimórficos. A análise desta região permitiu realizar uma caracterização intraespecífica e microevolutiva dos isolados de peste dos focos brasileiros. O MLVA e o CRISPR demonstraram uma melhor relação entre os dados epidemiológicos e moleculares, enquanto o PFGE apenas diferenciou os isolados de Y. pestis. Os dados gerados pelas três técnicas estudadas permitiram confirmar que houve apenas a entrada de um clone de Y. pestis no Brasil e observar que alguns processos adaptativos foram necessários para sua interiorização e fixação nos focos do país.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T17:45:25Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-MariaPalomaBarros.pdf: 8071319 bytes, checksum: 0b3f7f14ac7d15cf16b3a5e185a6f89d (MD5) / Made available in DSpace on 2017-04-10T17:45:25Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-MariaPalomaBarros.pdf: 8071319 bytes, checksum: 0b3f7f14ac7d15cf16b3a5e185a6f89d (MD5) Previous issue date: 2012-09-14 / A peste é uma doença que permanece enraizada em inúmeros focos naturais por todo o mundo. Embora o Brasil passe por um período de silenciamento epidemiológico, anticorpos antipestosos são detectados nas atividades de vigilância, sugerindo que estes focos permanecem ativos. A Yersinia pestis, agente causador da peste, apresenta uma história evolutiva recente e é considerada uma espécie, geneticamente, muito homogênea. Diante da necessidade de estudos mais aprofundados sobre as características moleculares e evolutivas dos isolados de Y. pestis dos focos do Brasil, realizamos neste trabalho uma caracterização de cepas da coleção biológica (Fiocruz-CYP) de Yersinia, provenientes de cinco focos de peste do Brasil, com a finalidade de compreender a adaptação da bactéria no ambiente. Realizamos a padronização e a análise de macrorestrição (PFGE) em 22 cepas de Y. pestis, 17 de um surto ocorrido em 1986 e cinco isoladas da atividade de vigilância. O PFGE não separou os isolados da rotina e do surto, entretanto foi capaz de revelar diversidade genética entre as cepas. As técnicas MLVA e CRISPR também foram utilizadas na genotipagem das cepas de Y. pestis. Doze locos VNTR (MLVA) analisados em 37 cepas, pertencentes a dois eventos epidemiológicos distintos, permitiram observar a separação dos grupos por evento e estabelecer uma relação epidemiológica. Três locos CRISPR (YPa, YPb e YPc) foram analisados em 146 cepas, apenas dois (YPa e YPb) se mostraram polimórficos. A análise desta região permitiu realizar uma caracterização intraespecífica e microevolutiva dos isolados de peste dos focos brasileiros. O MLVA e o CRISPR demonstraram uma melhor relação entre os dados epidemiológicos e moleculares, enquanto o PFGE apenas diferenciou os isolados de Y. pestis. Os dados gerados pelas três técnicas estudadas permitiram confirmar que houve apenas a entrada de um clone de Y. pestis no Brasil e observar que alguns processos adaptativos foram necessários para sua interiorização e fixação nos focos do país. / Plague disease remains present in numerous natural foci worldwide. Although Brazil goes through a period of epidemiological silence antiplague antibodies are detected in the surveillance activities, suggesting that Brazilians foci remain active. Yersinia pestis, causative agent of plague, has a recent evolutionary history and is considered a very genetically homogeneous species. Given the need for further studies on the molecular and evolutionary characteristics of the isolates of Y. pestis of the Brazilian plague areas, we perform in this work a characterization of strains biological collection (Fiocruz-CYP) of Yersinia, from five outbreaks of plague in Brazil, with the aim of understanding the adaptation of bacteria to the environment. We carried out the standardization and macrorestriction analysis (PFGE) in 22 Y. pestis strains, 17 from an outbreak occurred in 1986 and five from surveillance activity. PFGE did not separate the isolates from surveillance and from outbreak, but it was able to reveal genetic diversity among strains. MLVA and CRISPR techniques were also used in genotyping Y. pestis strains. Analysis of 12 VNTR loci (MLVA) in 37 Y. pestisstrains, of two different epidemiological events, allowed observing the separation of groups establish an epidemiological link among the isolates. Three CRISPR loci (YPa, YPb and YPc) were analyzed in 146 Y. pestis strains, only two loci (YPa and YPb) were polymorphic. The analysis of this region allowed an intraspecific characterization and microevolutionary of the plague isolates in the Brazilian foci. The CRISPR and MLVA showed a better relationship between the epidemiological and molecular data, while PFGE differ only the Y. pestis strains. The data generated by the three techniques studied confirmed that there was only one entry clone Y. pestis in Brazil and observed that some adaptive processes were necessary for its internalization and fixation of these foci in our country.

PNPase is a gene implicated as a potential target for cancer therapy; human mutations also present with deafness, myopathies, and neuropathies. In this study, C. elegans was used to investigate the effect of knocking out PNPase in a whole animal. C. elegans knockdown studies have reported an extended lifespan via an increase in ROS production. Further noted are larger mitochondria and an increase in fzo-1 expression. Knockout animals previously constructed using CRISPR/Cas9 were used for this study. We aimed to confirm these findings validating previous studies. It was discovered that PNPase knockout animals demonstrated a similar lifespan extension that was resolved with the addition of antioxidants in the media. All subsequent findings contradicted those of the knockdown studies. Resequencing of knockout animals demonstrated no existing mutation and studies were discontinued. New mutants will advance future analyses and validate prior investigations.

Pluripotente Stammzellen haben das Potential, in unterschiedliche Zelltypen zu differenzieren und können genutzt werden, um organähnliche Mikrostrukturen zu generieren. Somit können molekulare Unterschiede verschiedenster künstlich differenzierter Gewebe, etwa zwischen Mensch und Schimpanse, anhand von pluripotenten Ausgangszellen untersucht werden. Da die Genome unserer nächsten ausgestorbenen Verwandten Neandertaler und Denisovaner aus konservierter DNA in alten Knochen sequenziert wurden, könnten ebenso Unterschiede zwischen Mensch und diesen Spezies oder dem letzten gemeinsamen Vorfahren untersucht werden. Dies erfordert jedoch die Generierung neandertalisierter Stammzellen durch künstliche Integration von Neandertalerallelen in humane Stammzellen, etwa durch die CRISPR Genomeditierungstechnik. Durch CRISPR kann ein DNA-Doppelstrangbruch an einer gewünschten Stelle im Genom eingefügt werden. Die zelluläre Reparatur des Doppelstrangbruchs ermöglicht dann die Editierung des Genoms. Basierend auf einer DNA-Matrize, die die gewünschte Modifikation trägt, kann das Genom an dieser Stelle präzise editiert werden. Die Effizienz präziser Editierung ist jedoch sehr niedrig im Vergleich zu unpräziser Reparatur. Um möglichst effizient neandertalisierte Stammzellen generieren zu können, wurden im Zuge dieser Doktorarbeit Methoden entwickelt, welche die präzise Genomeditierungseffizienz drastisch steigern. Zum einen wurde aus mehreren niedermolekularen Substanzen, welche mit Proteinen der DNA-Reparaturen interagieren, ein optimierter Mix entwickelt. Weiterhin konnte durch eine Mutation in einem zentralen Reparaturprotein die Effizienz für die Editierung eines einzelnen Gens auf 87% erhöht werden. Diese hohe Effizienz ermöglicht erstmals die präzise homozygote Editierung von vier Genen auf einmal in ein und derselben Zelle

Pre-mRNA splicing is the most critical process in gene expression regulation across eukaryotic species. This reaction is carried out by the spliceosome, a large, dynamic, and well-organized ribonucleoprotein complex. The spliceosome is composed of five major small nuclear RNAs and an excessive number of associated protein factors. Many protein splicing factors bind and release during splicing to assist the assembly and the modulation of many RNA structures and proteins within the spliceosome. CWC25 is a splicing protein factor that functions in modulating the conformational structure of the spliceosome at the first transesterification reaction. CWC25 binds with its N-terminus to the major groove of the catalytic spliceosome triggering the spliceosome activity. Here, we employed CRISPR/Cas9 genome engineering system for targeted mutagenesis to generate CWC25 functional knock-out mutants to understand its molecular function, contribution to splicing regulation and implication in fine-tuning responses to abiotic stress in rice. Our genotyping analysis of the OsCWC25 locus revealed the presence of two mono-allelic and 18 bi-allelic mutant lines. Phenotypic analysis of these mutants, including germination and root inhibition assays, showed that the cwc25 mutants are oversensitive to abiotic stresses such as ABA and salinity. Our data demonstrate that CWC25 plays an important role in regulating plant responses to abiotic stresses.

At the post-transcriptional level, the splicing of the pre-mRNA plays a vital role in cell fate determination and respond to biotic and abiotic stresses. Through alternative splicing, mRNAs variants can be produced from a single gene. SF3B is a heptameric protein complex that is essential for pre-mRNA splicing. It contains seven subunits: SF3b155, SF3b130, SF3b145, SF3b49, SF3b14b, P14/SF3b14a and SF3b10 and they play an important role in BS (branch point sequence) recognition. P14/SF3b14a interacts with the branch point Adenosine (BPA), directing the binding of U2 complex. Several studies performed on the mutations of SF3b complex as it is associated with many diseases. Further studies are needed to deeply analyze the molecular function of P14/SF3b14a in plant growth and development. CRISPR/Cas9 system employed in gene editing among eukaryotes. The capability of the system is not only limited to the scope of bioengineering but also for functional studies of genes. CRISPR/Cas9 system assists in revealing the function of genes and the genetic networks through establishing a functional knockout and can help in understanding the molecular basis behind these processes. Here, we report the successful targeted mutagenesis of SF3b14a/p14 gene in Oryza sativa and the recovery of homozygous and heterozygous mutants. Phenotypic analyses have shown that SF3b14a/p14 is hypersensitive to abiotic stresses compared to the wild type plants. Further physiological and molecular studies are needed to reveal the role of p14 during plant growth and development, and responses to abiotic stresses.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22707号 / 農博第2423号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5300(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 教授 吉村 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Human immunodeficiency virus still remains the leading cause of death globally including women of child-bearing age. The rate of AIDS-related death has significantly declined since the introduction of antiretroviral treatment and other non-medical interventions such as the distribution and use of condoms. The introduction of antiretroviral treatment has however led to insulin resistance amongst users. Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated nuclease 9 (Cas) has been used to knockout NFκB to understand the pathway at which antiretroviral treatment causes insulin resistance. Heteroduplex mobility assay has shown that CRISPR-Cas9 knock out the gene of interest. These results have played a foundation in understanding how CRISPR-Cas9 can be integrated and utilized in medical research. / Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2020. / National Research Foundation (NRF) / Chemical Pathology / MSc (Chemical Pathology) / Restricted

Zebrafish is a frequently used model organism with an array of transgenic lines that have been used indevelopmental and physiological studies. We aim to generate novel transgenic zebrafish reporter lines to study subpopulations of spinal neurons in vivo. The gene editing system called CRISPR/Cas9 system was used to knock in reporter genes such as green fluorescent protein (GFP) or Gal4 transcription factor, to generate transgenic fish lines. Zebrafish embryos were injected with gRNAs targeting gabrb1 or nr4a2a and GFP or Gal4 plasmid, respectively. F0 larvae were screened, positive fish were raised until sexual maturity, and founders characterized to verify germline insertion. Three founders were found for gabrb1 and the location and the direction of the insert verified. The GFP expression was studied during development and differential expression patterns were identified whereas all founders had expression in brain and spinal cord. In parallel, positive fish from the Gal4 injections were raised and will be screened. Immunohistochemistry was performed to check if nr4a2a is expressed in the same cells as known neuronal markers. However, no co-localization was detected. The three gabrb1 founders identified in this study highlight the challenges into creating stable transgenic lines recapitulating true expression of the gene of interest. Sequencing, in-situ hybridization and immunohistochemistry should be performed to verify the line. A possible reason for the varying expression may be that through the knock-in we may interfere with regions regulating gene. The nr4a2a-Gal4 line will be used to perform functional studies. Those experiments will be performed using reporter genes, such as opsins or GCaMP, controlled by Upstream Activation Sequence (UAS). These transgenic lines will provide important insights regarding neuronal subpopulations that express gabrb1 and nr4a2a to unravelhow the locomotor network is formed.

The development of molecular tools that enable precise manipulation and control of biological systems would allow for a broader understanding of cellular functions and applications in biotechnology, synthetic biology, and therapeutic research. The discovery of CRISPR/Cas systems and the understanding and repurposing of their mechanisms have revolutionized the field of molecular biology. Here, I identified and characterized novel CRISPR/Cas systems and applied them for different in vivo and in vitro applications. In this work, I interrogated various Cas13 effector proteins and identified the most efficient Cas13 effector (CasRx) for in planta applications. I adapted CasRx to engineer plant immunity against different plant RNA viruses. CasRx showed robust activity and specificity against RNA viruses, demonstrating its suitability for studying key questions relating to virus biology. To expand the Cas13 toolbox and enable new applications, I performed a homology search of Cas13 enzymes in prokaryotic genomes and metagenomes, and identified previously uncharacterized, novel CRISPR/Cas13 effector proteins. I first identified and functionally characterized a small size, miniature Cas13 effector (named here as mCas13) and combined it with isothermal amplification to develop a simple and sensitive CRISPR-based SARS-CoV-2 diagnostic platform. In addition, I discovered and biochemically characterized the first known thermostable Cas13 proteins and showed that these thermostable proteins are phylogenetically related. I harnessed the unique features of these thermostable enzymes to develop the first one-pot, RT-LAMP coupled Cas13-based nucleic acid detection assay, which was utilized for highly sensitive, specific, and easily programmable detection of SARS-CoV-2 and other viruses. Lastly, I utilized CRISPR/Cas12a to develop a detection assay of plant ssDNA geminiviruses with easy-to-interpret visual readouts, making it suitable for point-of-use applications. In addition, I leveraged the self vs. non-self-discrimination and pre-crRNA processing capabilities of CRISPR/Cas12a, with the allosteric transcription factors (aTFs)- regulated expression of CRISPR array to engineer a field-deployable small molecule detection platform. I demonstrated the ability of the developed platform to detect different tetracycline antibiotics with high sensitivity and specificity. In conclusion, my work demonstrates that the discovery and characterization of programmable nucleic acid targeting systems could enable their utility for biotechnological innovations, including technologies for inhibition of viral replication and diagnostics.