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Macrophage Activation and Differentiation with Cholesterol CrystalsBurrowes, Hannah Mahony January 2012 (has links)
Cholesterol crystals have been linked to activation of the NLRP3
inflammasome and the formation of foreign body giant cells (FBGCs). It
has been hypothesized that FBGCs have a role in advanced atherosclerotic
plaque formation. This thesis examined the feasibility of producing
stable cultures of FBGCs starting with human monocytes with the
goal to examine pterin production by these cells in comparison to
human monocyte derived macrophages (HMDMs). The study also
investigated the effect of cholesterol crystals on 7,8-dihydroneopterin
(7,8-NP) production and modulation of IL-1β levels in macrophages.
7,8-Dihydroneopterin is a potent antioxidant generated by macrophages
which also down regulates the expression of macrophage scavenger
receptor CD36. The use of alpha-tocopherol and IL-4 as FBGC fusion
mediators was explored. Using these mediators, large numbers of
FBGC were successfully cultured. The rates of fusion achieved in the
cultures were low, and the cells had poor adhesion, which prevented
pterin measurement. FBGC, which are thought to remove crystallized
cholesterol from the plaque, cleared 21% of cholesterol crystal compared
to 50% cleared by HMDM cells. Due to this result, the effect of
cholesterol crystals on pterin production in monocytes and macrophages
was explored. Cholesterol crystals cause inflammation through the
activation of the NLRP3 inflammasome, however, it was unknown
whether they could modulate 7,8-NP production. Cholesterol crystals
caused an intracellular dose-dependent loss of 7,8-NP to its oxidized form,
neopterin, in HMDM cells. Cholesterol crystals induced intracellular
synthesis of 7,8-NP in HMDMs. 7,8-NP was released into the supernatant
and oxidized to neopterin in media. Monocytes treated with cholesterol
crystals released up to 100 nM of neopterin and 120 nM of 7,8-NP in
the media after 48 hours. The combination of IFN- and cholesterol
crystals appeared to inhibit the release of 7,8-NP into the media for the
first 48 hours, after this time 7,8-NP release rapidly increased. The
addition of exogenous 200 μM 7,8-NP showed that in the presence of
monocytes, cholesterol crystals did not cause the oxidation of 7,8-NP to
neopterin, as seen in HMDMs but possibly to 7,8-dihydroxanthopterin
or xanthopterin. The presence of 7,8-NP increased IL-1β expression in
the presence of cholesterol crystals after 24 hours incubation. FBGCs
and the removal of cholesterol crystals may be a key process in the
resolution of atherosclerotic plaques. It appears that cholesterol crystals
are able to modulate inflammatory processes including activation of
the inflammasome and balance of 7,8-dihydroneopterin to the oxidized
neopterin. The infiltrating monocytes may provide antioxidant protection
against the inflammation induced by cholesterol crystals and the activity
of the infammasome.
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Giant quartz vein zones of the Great Bear magmatic zone, Northwest Territories, CanadaByron, Suzanne 11 1900 (has links)
The Great Bear magmatic zone, Northwest Territories, hosts numerous giant quartz veins and stockwork zones. These zones can be up to 100m wide and up to 10km long, with two or more generations of quartz. A few of the giant quartz vein zones host base-metal uranium mineralization, and some are proximal to mineralization, although most are barren. Cathodoluminescence imaging shows the quartz veins have complex growth zones and a trace element study suggests that these zones are the result of Al and Li substitution in the quartz lattice. Oxygen isotope (18Oqtz) values of quartz generally fall between +8 to +14.6 (VSMOW). Fluid inclusion homogenization temperatures range from 100 to 375C, and the fluids have variable salinities. The fluids that created the giant quartz veins are epithermal in nature with a meteoric water brine signature, and formed as a result of multiple fluid pulses and re-fracturing events.
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Characterising Crim1 in Vertebrate DevelopmentGenevieve Kinna Unknown Date (has links)
This thesis investigates the role of Crim1, a transmembrane protein that is expressed in a number of areas in the vertebrate embryo including the developing kidney, eye, testis and spinal cord, which we believe may be a regulator of vertebrate tissue development. To dissect the function of Crim1 in normal mammalian development, two vertebrate models were used, zebrafish and mice. The results show that in zebrafish, crim1 is expressed early in development from the 16-cell stage through to 30 hours post fertilisation (Chapter 3). At 24 hours post fertilisation crim1 is expressed in the intermediate cell mass (icm), the site of haemangioblast development. Haemangioblasts are precursor cells that contribute to the formation of the blood and endothelial cell lineages. Injection of crim1 antisense oligonucleotides into zebrafish embryos (crim1 morphants) lead to an expansion of the icm and defects in the trunk, tail, somites and vasculature. The injection of crim1 antisense oligonucleotides into transgenic fli:GFP zebrafish revealed defects in the intersegmental, dorsal longitudinal anastomotic and parachordal vessels. Although crim1 is expressed during haemagiogensis the primary defect in the crim1 morphant zebrafish appears to be vascular. Further experiments used a ‘knock-in’ mouse, Crim1KST264, in which a loss of functional Crim1 leads to defects in limb (syndactyly), skeleton, eye, vascular, kidney and placental development. Analysis of the kidney phenotype in the embryonic Crim1KST264 homozygotes showed that a loss of Crim1 affects ERK1/2 and phosphorylated-Smad1/5/8 protein expression, although has no direct effect on BMP or TGFβ protein expression (Chapter 4). Analysis of the adult Crim1 outbred kidneys revealed they have albuminuria and leaky vasculature. The complex phenotype presented by the Crim1KST264 homozygote kidneys suggests Crim1 may be regulating multiple growth factor pathways. As Crim1 was shown to be expressed in the placenta, we characterised the role of Crim1 in placental development using the Crim1KST264 mouse (Chapter 5). Crim1KST264 homozygote placentas and embryos are smaller than their wild-type littermates. Our investigations revealed that Crim1 is expressed in trophoblast giant cells and in spongiotrophoblasts. A loss of Crim1 causes a developmental defect in that the junctional zone (region of the placenta containing spongiotrophoblasts and glycogen cells) is expanded, although this phenotype does not appear to be due to a defect in proliferation or apoptosis. Further analysis of E15.5 Crim1KST264 homozygote placentas revealed there was a reduction in the number of labyrinth trophoblast gaint cells. Thus, by using zebrafish and mouse as two model organisms of vertebrate development, this thesis has showed that Crim1 is clearly important for normal embryonic development. To dissect the complex phenotype presented by the Crim1KST264 mouse, further studies of Crim1 and its interaction with other growth factor pathways is needed to elucidate how and to what extent they interact with Crim1 to determine its biological effect on vertebrate tissue.
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Engineering biomaterial interfaces to control foreign body response : reducing giant cell formation and understanding host response to porous materials /Tsai, Annabel T. January 2007 (has links)
Thesis (118-130)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 118-130).
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The giant dipole resonance in highly excited nulei : does the width saturate? /Kelly, Michael P. January 1999 (has links)
Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (p. [141]-152).
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Étude de partenaires protéiques d’une protéine associée aux microtubules, MAP65-3, indispensable à la formation des cellules géantes induites par le nématode à galles Meloidogyne incognita : caractérisation du complexe de surveillance de la mitose chez Arabidopsis / Non disponiblePaganelli, Laëtitia 11 June 2013 (has links)
Les nématodes à galles du genre Meloidogyne sont des parasites obligatoires des plantes. Lors de l’interaction compatible, ils induisent la formation de cellules nourricières hypertrophiées et plurinucléées leur permettant d’assurer croissance et reproduction. L'étude des mécanismes moléculaires impliqués dans la formation de ces cellules géantes a permis d’identifier une protéine associée aux microtubules, MAP65-3, essentielle à la formation de ces cellules géantes et au développement du nématode. Un des partenaires protéiques de MAP65-3 est un homologue de BUB3, membre du « Mitotic Checkpoint Complex » (MCC). Le MCC est un point de contrôle de la mitose assurant la fidélité de la ségrégation des chromosomes. Au cours de ma thèse, j'ai caractérisé chez la plante modèle Arabidopsis thaliana les homologues du MCC: BUB3.1, MAD2 et la famille multigénique composée de BUBR1, BRK1 et BUB1.2. J’ai démontré les interactions in planta entre les membres du complexe, certaines interactions ayant lieu au niveau des noyaux, voire au niveau des centromères. J’ai réalisé l’analyse fonctionnelle de ces gènes et montré qu’ils étaient exprimés dans les tissus enrichis en cellules en division comme MAP65-3. L’étude de la localisation subcellulaire des protéines a révélé une localisation cytoplasmique pour BUB3.1, BUB1.2 et MAD2, nucléaire pour BUBR1 et centromérique pour BRK1. Nous avons pu également montrer que lorsque des défauts d’attachement des microtubules du fuseau mitotique sont provoqués, BUB3.1, BUBR1 et MAD2 se relocalisent au niveau des kinétochores. L’étude de la famille BUB1/BUBR1 a révélé que l’inactivation des gènes correspondants induisait une sensibilité accrue à un traitement chimique déstabilisant les réseaux de microtubules. L’étude de la mitose chez ces mutants a révélé que BUBR1 est essentielle à la réalisation d’une mitose sans erreur chez Arabidopsis. Ce travail a ainsi permis de caractériser pour la première fois le MCC chez A. thaliana. / Root-knot nematodes from the genus Meloidogyne are obligate biotrophic plant parasites. During a compatible interaction, they induce the redifferentiation of root cells into multinucleated and hypertrophied feeding cells to ensure their growth and reproduction. The study of molecular and cellular mechanisms underlying giant cell ontogenesis has led to the identification of a Microtubule-Associated Protein, MAP65-3, essential for giant cell ontogenesis and nematode development. One of the MAP65-3 interacting partners is a BUB3 homologue, member of the Mitotic Checkpoint Complex (MCC). The MCC is a surveillance mechanism ensuring that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the mitotic spindle. During my thesis, I have characterized the Arabidopsis thaliana orthologs of the MCC, BUB3.1, MAD2 and the multigenic family composed of BUBR1, BRK1 et BUB1.2. I have demonstrated that MAP65-3 and all the MCC members interact together in planta, some interactions taking place within the nuclei or at the centromeres. As MAP65-3, all these genes are expressed in dividing cells. The study of the subcellular localization of the protein showed a cytoplasmic localization for BUB3.1, BUB1.2 and MAD2, nuclear for BUBR1 and centromeric for BRK1. Thus, the MCC proteins did not relocalize to the kinetochore during a normal mitosis in planta. BUB3.1, BUBR1 and MAD2 localize to the unattached kinetochores following defects in spindle assembly as observed in cells treated with microtubule poisons. The functional analysis of BUB1/BUBR1 multigenic family showed that the knock-out mutants were more sensitive to microtubule-destabilizing drugs. Furthermore, analysis of mitosis revealed that BUBR1 is essential for an error-free mitosis in Arabidopsis. This work represents the first characterization of the MCC in A. thaliana.
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Análise da expressão gênica no tumor ósseo de células gigantes /Babeto, Erica. January 2011 (has links)
Orientador: Paula Rahal / Banca: Aparecida Maria Fontes / Banca: Débora Aparecida Pires de Campos Zuccari / Banca: Ana Elizabete Silva / Banca: Jane Lopes Bonilha / Resumo: O tumor ósseo de células gigantes (TCG) é um tumor benigno, que causa destruição osteolítica, com comportamento biológico incerto e com características particulares como um elevado número de células gigantes multinucleadas e comportamento agressivo. A recorrência local do TCG é freqüentemente observada em 20 a 50% dos casos. Mais agravante que a recorrência, é o fato de que após a recidiva, o paciente muitas vezes também apresenta metástases em outros órgãos, principalmente no pulmão. Dessa forma, o objetivo do trabalho foi á investigação da expressão gênica para identificar genes diferencialmente expressos no TCG, que podem estar envolvidos na biologia molecular e desenvolvimento da doença. A hibridização subtrativa rápida (RaSH) foi utilizada para identificar novos genes diferencialmente expressos, como KTN1, NEB, ROCK1 e ZAK, que foram validados por PCR quantitativo em tempo real (qPCR) e a imuno-histoquímica em amostras de TCG comparadas ao tecido ósseo normal. A anotação funcional indica que estes genes estão envolvidos em processos celulares relacionadas ao fenótipo tumoral. A expressão dos genes KTN1 e ROCK1 encontra-se aumentada e o gene ZAK tive sua expressão reduzida nas amostras de TCG analisadas. Pela presença de ilhas CpG na região promotora e baixa expressão no tecido tumoral, o padrão de metilação do gene ZAK foi analisado por MSP-PCR. Os genes identificados KTN1, ROCK1 e ZAK pode ser responsável pelas perda de homeostase celular no TCG uma vez que são responsáveis pela várias funções relacionadas com a tumorigênese, como a migração celular, organização do citoesqueleto, apoptose, controle do ciclo celular e, portanto esses resultados poderão contribuir para a compreensão das bases moleculares do TCG, assim ajudando a melhorar o diagnóstico, prognóstico e tratamento dos pacientes / Abstract: Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction, with a number of particular characteristics. These tumors can have uncertain biological behavior, often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1 and ZAK, using qRT-PCR and immunohistochemical in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1 and ZAK was independently confirmed by qRT-PCR and immunohistochemical. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemical and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified, KTN1, ROCK1 and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB / Doutor
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Isolamento e caracterização de um novo vírus gigante de amebas : Golden mussel marseillevirus / Isolation and characterization of a new giant virus in amoebae : Golden mussel marseillevirusSantos, Raíssa Nunes dos January 2016 (has links)
Marseilleviridae é uma família de vírus gigantes cujos membros infectam amebas de vida livre. Esses vírus têm sido encontrados em amostras ambientais de água, larvas de inseto, torres de resfriamento e mais recentemente em amostras humanas. Eles possuem capsídeo icosaédrico medindo entre 190-250 nm e genoma de DNA dupla fita circular ou linear. Sua replicação ocorre no citoplasma amebiano onde observam-se fábricas virais. Este trabalho tem como objetivo investigar a presença de vírus gigantes em mexilhões-dourados (Limnoperna fortunei) que habitam o Lago Guaíba, Porto Alegre, Brasil. Quarenta indivíduos foram coletados e agrupados em pools de 5 amostras (água interna e corpo, totalizando dezesseis pools). Os pools foram homogeneizados em tampão fosfato, centrifugados e o sobrenadante filtrado em membrana de 0,45μM. Foram cultivadas amebas da espécie Acanthamoeba polyphaga em meio PYG em microplacas de 24 poços, inoculadas com os sobrenadantes, incubadas a 30ºC e examinadas diariamente em busca de efeito citopático (ECP) até 72 horas após a inoculação. Quando CPE era evidente, os sobrenadantes foram coletados e ultracentrifugados através de um gradiente de sacarose de 25%. Um dos dezesseis pools induzindo CPE claro foi submetido à extração de DNA e sequenciamento do genoma viral completo um sequenciador de nova geração (Illumina MiSeq). O genoma do vírus chamado Golden mussel marseillevirus consiste de uma única molécula de DNA de 360610 pb, com um teor de G+C de 43,1%. A análise da sequência de nucleotídeos traduzida revelou a presença de proteínas virais que apresentam homologia com proteínas de outros membros da família Marseilleviridae, como Lausanevirus e vírus Insectomime, porém grande parte do seu genoma não apresenta identidade com proteínas depositadas no banco de dados. A análise filogenética do gene D6/D11 Helicase sugere que este vírus faça parte de uma nova linhagem de marseillevirus. Este é o primeiro estudo que demonstra o isolamento de um vírus gigante a partir de tecidos de mexilhão-dourado e infere que estes vírus estão distribuídos amplamente no meio ambiente. / Marseilleviridae is a family of giant viruses whose members infect free living amoebae. These viruses have been found in environmental samples of water, insect larvae, cooling towers and, more recently, in human samples. They have icosahedral capsids measuring between 190-250 nm and their genome is double-stranded circular or linear DNA. Replication occurs in the host cell cytoplasm, inside the viral factories. This study aims to investigate the presence of giant viruses in tissues of golden mussels (Limnoperna fortunei) that inhabit the Guaiba Lake, Porto Alegre, Brazil. Forty specimens were pooled in groups of 5 specimens (internal water and body, totalizing sixteen pools). The pools were homogenized with phosphate buffer, centrifuged and the supernatant was filtered in 0,45μM. Monolayers of Acanthamoeba polyphaga were cultivated with PYG medium in 24-well microplates, inoculated with the pooled samples, incubated at 30 ºC and examined daily in search for cytopathic effect (CPE) up to 72 hours after inoculation. When CPE was evident, the supernatants were collected, clarified and ultra centrifuged through a 25% sucrose cushion. One out of the sixteen CPE positive pools was submitted to DNA extraction and complete sequencing of the viral genome in a NGS apparatus (Illumina MiSeq). The genome of the virus named Golden mussel marseillevirus consists of a single DNA molecule of 360,610 bp, with a G+C content of 43.1%. The analysis of the translated nucleotide sequence reveals the presence of proteins which are homologs to proteins predicted in other members of the family Marseilleviridae like, e.g. Lausannevirus and Isectomime virus. However, part of the viral genome has no identity with the nucleotide sequences available at the database. The phylogenetic analysis of the D6/D11 Helicase gene suggests that this virus is part of a new lineage of marseillevirus. This is the first study where the isolation of a giant virus from golden mussel tissues is achieved, suggesting that these viruses are widely distributed in the environment.
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Underground measurement of hydrogen-burning reactions on 17;18O at energies of astrophysical interestBruno, Carlo Giulio January 2017 (has links)
The 17;18O(p,α)14;15N nuclear reactions play an important role in several astrophysical scenarios, and in Asymptotic Giant Branch (AGB) stars in particular. These stars are the site of several mixing and recirculating processes that transport matter from their hot cores to their cooler surfaces, and vice versa. Some of these mixing processes are still not well understood. Constraining them would improve our knowledge of stars that are in, or will enter, the AGB phase, including our own Sun. An ideal way to trace these poorly understood mixing processes are provided by the rare, stable 17;18O isotopes. Their abundances are strongly sensitive to the 17;18O(p,α)14;15N reactions. At temperatures of astrophysical interest, the 17O(p,α)14N reaction is dominated by a narrow, isolated resonance at Eproton=70 keV. This resonance has been studied several times in the past, using both direct and indirect methods. However, the picture painted in the literature is still not completely satisfying. The situation is more complex for the 18O(p,α)15N, for which an interference pattern between at least three resonances dominates the reaction rate at the temperatures of interest. This thesis work concerns an experimental campaign aimed at measuring both reactions at energies of astrophysical interest. These challenging measurements were performed by exploiting the low radiation background at the underground LUNA accelerator in Gran Sasso Laboratories, Italy. The two reactions were investigated in direct kinematics. A proton beam was accelerated onto solid Ta2O5 targets and the alpha particles produced were detected at backward angles using an array of silicon detectors mounted in a purpose-built scattering chamber. Our results indicate that the 17O(p,α)14N reaction rate at temperatures of astrophysical interest is approximately a factor of two higher than previously reported, solving a long standing puzzle on the origin of some pre-solar grains. For the 18O(p,α)15N reaction, we find a reaction rate largely in agreement with previous investigations, but with a significantly reduced uncertainty which could help improve the accuracy of stellar models of a number of stellar sites.
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Magnetická orientace rypoše obřího a rypoše stříbřitého / Magnetic orientation in the giant mole-rat and the silvery mole-ratOLIVERIUSOVÁ, Ludmila January 2008 (has links)
The magnetic orientation was study in several species of rodents. Aim of this work was verify magnetic sense in two species of subterranean rodents: the giant mole-rat and the silvery mole-rat. A spontaneous directional preference in the magnetic field was tested in circular arena.
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