Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.157-158

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept 22, 2008). Includes bibliographical references (p. 42-45).

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 2009. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/07/02). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / "May 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

In this study, for the first time we used CRISPR/Cas9 gene editing technology in sturgeons i.e., sterlets (Acipenser ruthenus). The sequences of sgRNA and primers were designed based on published dnd1 sterlet sequence. Each pair of sgRNA oligos after ligation ready duplex DNA fragment was cloned into vector pX330-U6-Chimeric_BB-CBh-hSpCas9 backbone and thereafter the transformation to competent cells Escherichia coli DH5 was done. The plasmid carried sgRNA was extracted for downstream applications. We diluted extracted plasmid with 10% of 2 M KCl and injection into animal pole of fertilized eggs of sterlets at one to four-cell-stage, 4 hours post fertilization (hpf). At the same time, second microinjection with 2.5% FITC-biotin-dextrans was injected into vegetal pole for labelling PGCs. In the control groups, the eggs were only injected by 2.5% FITC into vegetal pole. PGCs of sterlet were visualized and photographed using a uorescent stereo microscope Leica M165 FC. To confirm the presence or deletion/insertion occurring in the target gene, we used MCE-202 MultiNA microchip electrophoresis system for DNA analysis, in which the targeted gene after amplifying by PCR was analyzed. Mutations in both treated and control embryos of sterlet were further assessed by Sanger sequencing of the PCR product. In present study, we successfully established basic protocols such as preparation of competent cells, construction of vector carrying sgRNA and its transformation into competent cells to carry out the CRISPR/Cas9 technology in sturgeons. Less number of PGCs was observed in embryos that were treated with CRISPR/Cas9; however, sequencing did not provide us a reliable evidence for mutation of the targeted gene probably due to an unspecific PCR. Therefore, more authentication of dnd1 knockout should be done in future by more specific PCR and repeated sequencing.

DEAF1 is a transcription factor linked to suicide and depression and is recurrently mutated in non-syndromic intellectual disorder (ID). In humans with major depressive disorder (MDD), DEAF1 is reported to have altered expression in the prefrontal cortex and the dorsal raphe nucleus of females but not males, and therefore may function in sex-specific depression. Standard (whole body) knockout of Deaf1 has been reported to alter 5-hydroxytryptamine (serotonin) receptor 1A (Htr1a) levels in the frontal cortex and dorsal raphe nucleus. We hypothesized that mice with targeted deletion of Deaf1 in brain would produce behavior phenotypes analogous to human MDD and ID, and that changes in Deaf1-target gene expression would be associated with behavior changes. To test this hypothesis, we produced a mouse line to allow conditional gene targeting of exons 2-5 (Deaf1-flox). Deaf1-flox mice were bred to congenic status onto a C57BL/6 background, and were then bred to mice transgenic for the Nestin-Cre gene to produce embryonic knockout of Deaf1 in neuronal precursor cells. Adult mice were tested for anxiety behavior using the Elevated Plus Maze and Open Field Exploration tests, and were tested for depression-like behavior using the Porsolt forced swim and sucrose preference tests. Relative to control mice, both male and female mice with homozygous deletion of Deaf1 in brain displayed increased anxiety measured by the anxiety tests, and no differences were measured in tests for depression-like behavior. Rotarod testing showed no deficits in motor skills of male mice. Learning and memory in mice were tested using Morris Water Maze (MWM) and fear conditioning. No change in long-term memory in male mice was observed in MWM, but both male and female mice lacking Deaf1 displayed severe deficits in fear conditioning tests. Eif4g3 and Tmem80 are target genes of Deaf1 and decreased mRNA levels were observed for Eif4g3 and Tmem80 throughout the brain of Deaf1 knockout mice, with no change in Htr1a mRNA levels. Our results demonstrate that conditional knockout of Deaf1 in neuronal precursors produces anxiety behavior and deficits in learning and memory in adult mice, potentially without changes in Htr1a mRNA levels, and that this mouse model may be useful in understanding the molecular mechanisms underlying MDD and ID in humans.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-01-13T10:59:16Z No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-01-20T14:53:34Z (GMT) No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) / Made available in DSpace on 2017-01-20T14:53:34Z (GMT). No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) Previous issue date: 2016-04-05 / Esse estudo objetiva avaliar se a express?o da enzima de reparo de DNA Neil3 ? importante para o desenvolvimento funcional dos neur?nios. Estudos previos tem demonstrado que Neil3 interfere tanto na neurog?nese adulta como na fazer embrionaria. Eu utilizei whole cell patch clamp para estudar propriedades sinapticas e de membrana das c?lulas granulares do giro denteado. O giro denteado ? uma das regi?es com maior express?o de Neil3 no c?rebro e estudos previos tem demonstrado que a neurog?nese reativa em camundongos adultos ? afetada pela ausencia da enzima de reparo Neil3. Eu encontrei que a maioria das propriedades de membrana nas c?lulas granulares de camundongos knockout para Neil3 s?o normais com exce??o ? resposta de membrana ?s correntes de hiperpolariza??o e p?s-hiperpolariza??o. Diferentemente de neur?nios imaturos, as c?lulas granulares do giro denteado de camundongos com aus?ncia de Neil3, na qual as correntes de hiperpolariza??o ativadas s?o geralmente as ultimas a aparecerem durante o desenvolvimento. Al?m disso, correntes sinapticas excitatorias foram similar em amplitude mas apresentaram um decaimento ligeiramente mais rapido em c?lulas de camundongos knockout de Neil3. Esses resultados podem indicar um balan?o diferente entre os receptores AMPA e NMDA em camundongos knockout. Analises morfologicas de neur?nios preenchidos com biotina e reconstru??o post hoc n?o apresentaram grandes diferen?as na morfologia dendritica entre animais controle e knockout. Esse estudo mostra que, em rela??o diferen?as entre animais controle, neur?nios do giro denteado de animais knockout de Neil3 n?o podem ser classificados como imaturos. Eu encontrei diferen?as pontuais na corrente de hiperpolariza??o e pequenas diferen?as em propriedades sinapticas. Ainda devem ser avaliadas se essas diferen?as podem ser respons?veis por altera??es comportamentais encontradas em camundongos Neil3-knockout. Al?m disso, estudos futuros utilizando marcadores de neur?nios rec?m-nascidos s?o necess?rios para analisar o efeito da elimina??o da enzima Neil3 no desenvolvimento de neur?nios. / This study aims to assess whether the expression of the DNA repair enzyme Neil3 is important for the functional development of neurons. Previous studies have demonstrated that Neil3 interferes with both adult and embryonic neurogenesis. I have used whole cell patch clamp to study membrane and synaptic properties of granule cells of the dentate gyrus. The dentate gyrus is one of the regions with the highest expression of Neil3 in the brain, and previous studies have shown that reactive neurogenesis in adult mice is affected by Neil3 deletion. I found that most membrane properties of granule cells in Neil3-knockout mice are normal except from the membrane response to hyperpolarization currents and afterhyperpolarization currents. Different from immature neurons, granule cells of the dentate gyrus from Neil3-knockout mice, in which hyperpolarizing activated currents, are generally the last to appear during development. In addition, excitatory synaptic currents were similar in amplitude but showed a slightly faster decay in cells from Neil3-knockout mice. These results could indicate a different balance between AMPA and NMDA receptors in Neil3-knockout mice cells. Morphological analysis of neurons filled with biocytin and reconstructed post hoc showed no gross difference in dendritic morphology between dentate gyrus neurons of control and Neil3-knockout mice. This study shows that, while different from those of control littermates, dentate gyrus neurons of Neil3-knockout mice cannot be classified as ?immature?. I found specific differences in hyperpolarizing activated currents and small differences in synaptic properties. Whether these differences may account for behavioral changes found in Neil3-knockout mice is yet to be assessed. In addition, future studies using markers of newly born neurons are necessary for analyzing the effect of Neil3 deletion in developing neurons.

Sepse é a primeira causa de morte em Unidades de Terapia Intensiva. A gravidade dessa doença é considerada conseqüência de um desequilíbrio da resposta inflamatória e, apesar dos avanços em diagnóstico e tratamento, os índices de mortalidade se mantêm inalterados. O papel dos receptores Fc de immunoglobulinas nesta situação é pouco esclarecido. Tais receptores deflagram respostas imunes opostas, que dependem do receptor envolvido e podem ser tanto ativatórias, quanto inibitórias. As respostas ativatórias são atribuídas a um motivo chamado ITAM, enquanto as inibitórias são relacionadas ao motivo ITIM. Camundongos apresentam dois receptores de IgG ativatórios (FcγRI e FcγRIII), que portam motivos ITAM, associados a uma sub-unidade conhecida como cadeia gamma, assim como um receptor de IgG que apresenta um motivo ITIM na sua porção intra-citoplasmática (FcγRII). Este trabalho teve como objetivo o estudo do papel destes receptores em bacteremias e sepse. Para isso, utilizamos um modelo de peritonite induzida por ligadura e punção cecal. Este projeto descreve pela primeira vez, um papel importante do FcRγII na indução de apoptose em linfócitos B, durante infecção bacteriana severa. Nossos resultados colocaram em evidência, ainda, o fato de que animais deficientes em cadeia gamma apresentam mortalidade diminuída, quando submetidos a esse modelo de peritonite, e que essa diminuição é associada a menores valores de TNFα no soro e nos fluidos peritoneais, menor recrutamento peritoneal de células inflamatórias, assim como a um surpreendente aumento na fagocitose de E. coli. Hemocultura e cultura do lavado peritoneal desses animais revelaram uma flora multimicrobiana, enquanto camundongos selvagens apresentaram uma forte predominância de E. coli e um número total bastante superior de bactérias. Esse papel inibitório da cadeia gamma pode estar relacionado a mecanismos de auto-tolerância. Lisado total de células peritoneais de camundongos deficientes em cadeia gamma apresentam fosforilação aumentada de diversas proteínas, quando comparados a lisados obtidos, a partir de camundongos selvagens. Estudos semelhantes realizados com camundongos transgênicos para o receptor de IgA (FcαRI), entretanto, não demonstraram um papel crucial desse receptor nesta doença. Este trabalho abre, portanto, novas perspectivas para o tratamento de doenças infecciosas, através de intervenção sobre a cadeia gamma e coloca em rediscussão os conceitos atuais de ITAM e ITIM. / Sepsis is the first cause of death in Critical Care Units and despite the development in its diagnosis and treatment, mortality remains unaffected. The role of immunoglobulin Fc receptors in sepsis is not clearly understood. These receptors initiate opposing responses, depending on their aggregation by the ligand and can induce activating or inhibitory responses. The activating responses are attributed to a motif known as ITAM, and the inhibitory responses to another one called ITIM. Mice express two activating IgG receptors (FcγRI et FcγRIII) which have ITAM motifs in the intracytoplasmic domain of an associated subunit, called the FcRγ chain, as well as an inhibitory IgG receptor which possesses an ITIM motif in its intracytoplasmic domain. The objective of this work is to study the importance of these receptors in bacteremia and in sepsis. To this aim, we have used a peritonitis model, induced by cecal ligation and puncture (CLP). This project describes for the first time, an important role of FcγRII in B lymphocytes apoptosis. Moreover, our results show that FcRγ chain knockout mice have a decreased mortality in this model, which is associated to diminished TNFα serum and peritoneal fluids levels, to a reduced recruitment of peritoneal inflammatory cells and to a surprising increase in E. coli phagocytosis. Blood and peritoneal fluid cultures have shown a polymicrobial flora 24 hours post-CLP for FcRγ-chain deficient mice, whereas wild-type mice present a strong predominance of E. coli in the same cultures and an increased bacteria total count. Lysates from FcRγ-chain deficient peritoneal cells revealed augmented phosphorylation of many proteins, as compared to wild-type cells. This FcRγ chain inhibitory role could be related to self-tolerance mechanisms. This work opens new perspectives for the treatment of bacterial diseases, proposing FcRγ chain targeting and the reexamination of the actual concepts of ITAM and ITIM.