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Stefanou, Eunice-Georgia G. "Chromosome painting using microdissection techniques." Thesis, University of Glasgow, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364084.
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Vergier, Béatrice. "Intérêts de la microdissection unicellulaire dans l'étude des lymphomes." Bordeaux 2, 2001. http://www.theses.fr/2001BOR28871.
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Les lymphomes se caractérisent par une très grande hétérogénéité cellulaire conduisant à utiliser des approches unicellulaires. Notre travail de thèse a été de mettre au point la technique de microdissection unicellulaire et d'optimiser les méthodes d'amplification de l'ADN pour analyse à l'échelon d'une cellule de plusieurs événements moléculaires. Nous avons adapté une méthode de pré-amplification génomique (PEP-PCR) permettant, à partir d'un lymphocyte, l'étude combinée du réarrangement des gènes du TCRγ (sensibilité 28 %), des IgH (sensibilité 40 % ), et des translocations t(14 ; 18) ou t(11 ; 14). Nous avons appliqué cette méthode à différentes problématiques. Tout d'abord à l'étude des lymphomes bigénotypiques : l'incidence d'un double réarrangement majoritaire des gènes IgH et TCRγ était parmi les 398 lymphomes analysés de 13 % pour les lymphomes B et de 10 % pour les lymphomes T. L'analyse combinée de 4 lymphomes représentatifs a montré dans 2 cas (1 syndrome de Sézary et 1 lymphome du manteau) qu'il s'agissait de "vrais" lymphomes bigénotypiques (la même cellule portant les 2 réarrangements) et dans 2 cas (un lymphome B diffus à grandes cellules et un lymphome T angio-immunoblastique) que chaque réarrangement était porté par 2 populations différentes. La deuxième application a permis le suivi évolutif moléculaire d'un patient porteur successivement d'un lymphome du MALT (Epstein Barr Virus, EBV négatif), d'une maladie de Hodgkin (EBV +) et d'un lymphome B diffus à grandes cellules (EBV+). Nous avons prouvé l'homogénéité clonale de la population lymphomateuse qui était morphologiquement et phénotypiquement hétérogène. Enfin la dernière application a porté sur l'étude de la t(14 ; 18) dans les lymphomes B folliculaires cutanés. Au total, l'analyse combinée de plusieurs gènes après microdissection unicellulaire permet de corréler à l'échelon d'une cellule sa morphologie, sa localisation, son phénotype, son génotype et ses anomalies moléculairesLymphomas consist of heterogenous cells making necessary the use of unicellular analysis. So, we have developed single cell microdissection and adapted PCR analysis to study at single cell level several molecular events. After a whole genome amplification step, we have designed a single cell combined TCR γ (sensibility, 28 %), IGH (sensibility, 40 %) gene analysis and t(14 ; 18) detection. We applied this method to analyse different problems. Firstly the bigenotypic lymphomas : we have observed a dual genotype in 13 % of B-cell lymphomas among the 398 lymphoma cases. This single cell combined PCR approach allowed to identify, among 4 cases studied, 2 true bigenotypic lymphomas (one Sézary Syndrome and one mantle cell lumphoma) as both IgH and TCR γ monoclonal rearrangements were detected in the same cells. Conversely, in the 2 other cases (one diffuse large B-cell lymphoma and one angio-immunoblastic T-cell lymphoma), large CD22 + single cells exhibited only the monoclonal IgH rearrangement but not the TCR γ gene that was detected in CD3+ single cells. Secondly this approach was found useful for the molecular follow-up of different lymphoproliferations arising in same patient. We studied a patient who have presented first MALT Lymphoma (EBV -) then mediastinal Hodgkin disease (EBV +) and at last large B-cell lymphoma of the colon (EBV+). Our method, proved the common clonal origin of large cells despite the fact that they were morphologically and phenotypically (CD30 + or-, CD22 + or -, EBV+ or -) different. Lastly, we studied the t(14 ; 18) in follicular B-cells lymphoma by comparing 2 techniques (real-time PCR, Taqman vs "classic" PCR). Finally, this single cell/multiple gene analysis makes it possible to attribute specific genetic abnormalities, such as translocations and/or oncogenic alterations, to lymphoid cells defined both by their location, morphology, phenotype and their antigen receptor gene rearrangement
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Fang, Yu-Yan. "Microdissection and molecular cloning of extra small ring chromosomes of human /." Title page, contents and summary only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phf211.pdf.
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Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics, 1998.Copies of author's previously published articles inserted. Errata pasted onto front end-paper. Includes bibliographical references (leaves 111-139).
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Matsuo, Hidemasa. "Purification of leukemic blast cells from blood smears using laser microdissection." Kyoto University, 2018. http://hdl.handle.net/2433/232315.
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Stuart, Charles A., William L. Stone, Mary E. A. Howell, Marianne F. Brannon, H. Kenton Hall, Andrew L. Gibson, and Michael H. Stone. "Myosin Content of Individual Human Muscle Fibers Isolated by Laser Capture Microdissection." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/4642.
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Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle.
The technical challenges of human skeletal muscle fiber type identification have evolved over the past three decades (8). The typical normal adult has roughly equal amounts of slow- and fast-twitch fibers, designated type I and II fibers. In addition, a variable portion of the type II fibers is mixed, containing both fast- and slow-twitch fiber markers, called type IIa fibers, whereas type II fibers that contain only the fast-twitch phenotype are designated type IIx in humans. Exercise training can cause modest shifts in fiber composition from one of these types to a contiguous type, with the relationship being type I to IIa to IIx or type IIx to IIa to I. The tail end of each myosin heavy chain is attached to the tail of another myosin heavy chain, and each of these forms a complex with two myosin light chains. Many heavy and light chain complexes are intertwined to form the thick filaments of each sarcomere. Thin filaments are composed of actin, troponin, and tropomyosin. The myosin heavy chains contain ATPase, which is essential for shortening of the contractile apparatus in the sarcomere, resulting in muscle-generated movement of a body part. The pH optimum of the ATPase has been classically the histochemical technique for identifying fast, slow, and mixed fibers. However, for more than a decade, monoclonal antibodies that correlated with the ATPase designation of fast, slow, and mixed fibers by bright-field or immunohistochemical methods have been used (2). The widely used fast and slow myosin monoclonal antibodies were obtained from mice immunized with only partially purified human skeletal muscle myosin antigens. More recently, antibodies that were raised against specific individual myosin heavy and light chain proteins became commercially available.
The 15 human genes that code myosin heavy chains are designated MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH7B, MYH8, MYH9, MYH10, MYH11, MYH12, MYH13, MYH14, MYH15, and MYH16 (17). MYH9, MYH10, and MYH11 are expressed primarily in smooth muscle. At least eight separate genes that code myosin light chains, MYL1, MYL2, MYL3, MYL4, MYL5, MYL6, MYL6B, and MYLPF, have been identified, and at least three of these have a second isoform (3).
Our initial investigation of the expression of myosin heavy and light chains using laser capture microdissection (LCM) to obtain specific fiber type samples from human vastus lateralis biopsies yielded some unexpected results. These observations led us to question which isoforms of myosin heavy and light chains are actually characteristic of “fast” or “slow” fibers in human skeletal muscle. We used immunoblots, mass spectroscopic (MS) proteomics, and next-generation sequencing of muscle homogenates and of LCM-generated samples of individual fiber types from normal control subjects and subjects with extremely different muscle fiber composition to approach this question by evaluating muscle specimens from subjects with diverse and extremely different fiber compositions. The hypothesis that drove these studies was that fibers of each type would have consistent myosin heavy and light chains that are characteristic of the fiber type. This is the first report that the abundance of different myosin heavy and light chains corresponds to different muscle fiber types.
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Mohamed, Allie. "Colorado microdissection needle versus cold steel scalpel for incisions in third molar surgery." Thesis, University of the Western Cape, 2014. http://hdl.handle.net/11394/4089.
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Magister Chirurgiae Dentium - MChDThis study compares the CMN to the steel scalpel by assessing incision time, incisional blood loss, postoperative pain, wound healing, and the incidence of lingual and long buccal nerve injury. Twenty standardised cases were included in an analytical prospective case series. Each case had one side cut with CMN and the other side with steel scalpel. Third molar surgery is the most commonly performed procedure by maxillo-facial and oral surgeons, and is associated with expected but transient sequelae such as pain, swelling and trismus. Modalities to reduce the severity of these sequelae are desirable. Several studies report that the use of conventional electrosurgical instruments and the Colorado Microdissection Needle (CMN) resulted in significant reductions in cutting time, incisional blood loss, postoperative pain, with no evidence of increased incidence of wound complications such as dehiscence and infection.
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Majithia, Haritika. "Determining cell-specific gene expression in two soybean mutants using laser capture microdissection." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119666.
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Soybean, Glycine max, (L.) Merr., is usually covered in trichomes and has three leaflets per compound leaf. Two mutant soybean cultivars, one without trichomes, cv. Glabrous, and the other with five leaflets per compound leaf, cv. 5-LF, are compared with a wild type cultivar to detect gene expression differences. Trichomes develop and differentiate from the epidermis and the fate of leaves, whether they are compound or simple, is decided in the meristem. Cell-specific gene expression of the epidermis as compared to the meristem is investigated in the three cultivars using Laser Capture Microdissection and high-throughput RNA sequencing. The results indicate about 200 differentially expressed genes in the two tissues (meristem and epidermis) of each of the three cultivars. The meristem had higher expression of genes containing sequence-specific DNA binding domains whereas the epidermis had higher expression of genes related to plant defense.Soya, Glycine max, (L.) Merr., est généralement couvert de trichomes et possède trois folioles par feuille composée. Deux cultivars de soya mutant, un sans trichomes, cv. Glabrous, et un avec cinq folioles par feuille composée, cv. 5-LF, ont été comparés avec un cultivar sauvage pour étudier la différence dans l'expression des gènes. Comme les trichomes se développent et se différencient depuis l'épiderme et comme le sort des feuilles (qu'elle devienne composée ou simple) se décide au niveau du méristème, l'expression des gènes des cellules spécifiques de l'épidermes a été comparée au méristème dans les trois cultivars via un instrument de microdissection au laser ainsi qu'à l'aide de séquençage d'ARN à haute capacité. Les résultats indiquent qu'environ 200 gènes distincts dans les deux tissues (méristème et épiderme) ont été exprimés différemment dans chacun des trois cultivars. Le méristème avait une expression plus élevée de domaines de liaison à l'ADN spécifiques de séquence alors que l'épiderme avait une plus forte expression de gènes liés à la défense des plantes.
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Vlachouli, Christina. "Microarray analysis of GFP-expressing mouse Dopamine neurons isolated by laser capture microdissection." Doctoral thesis, SISSA, 2009. http://hdl.handle.net/20.500.11767/4762.
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The Central Nervous System (CNS) contains an enormous variety of cell types which organize in complex networks. The lack of adequate markers to discern unequivocally among this cellular heterogeneity make the task of dissecting out such neural networks and the cells that comprise them very challenging. The present study represents a “bottom-up” approach that entails a description of A9 and A10 nuclei, which are components of the mesencephalic dopaminergic system, and the identification of their molecular make-up through microarray analysis of their gene expression profiles. These mesencephalic dopaminergic nuclei give rise to the mesocortical and mesostriatal projections and are well known for their roles in initiation of movement, reward behaviour and neurobiology of addiction. Moreover, in post mortem brains of Parkinson Disease patients a specific topographic pattern of degeneration of these neurons, also recapitulated in experimental animal models, is noted, with A9 neurons presenting with a higher vulnerability to degeneration with respect to A10 cells among which, neuron loss is almost negligible. Molecular differences may be at the basis of this different susceptibility. In this study we have optimized a protocol for laser-assisted microdissection of fluorescent-expressing cells and have taken advantage of a line of transgenic mice TH-GFP/21-31, which express GFP under the TH promoter in all CA cells, to guide laser capture microdissection of A9 and A10 mDA neurons for differential informative cDNA microarray profiling. Results show that our optimized method retains the GFP-fluorescence of DA cells and achieves good tissue morphology visualization. Moreover, RNA of high quality and good reproducibility of hybridizations support the validity of the protocol. Many of the genes that resulted differentially expressed from this analysis were found to be genes previously known to specifically define the different identities of the two DA neuronal nuclei. Transcripts were verified for expression, in DA neurons, using the collection of in situ hybridization in the Allen Brain Atlas. We have identified 592 differentially expressed transcripts (less than 8%) of which 242 showing higher expression in A9 and 350 showing higher expression in A10. Categorical analysis showed that transcripts associated with mitochondria and energy production were enriched in A9, while transcripts involved in redox homeostasis and stress response resulted enriched in A10. Of all the differentially expressed genes, eight transcripts (Mif, Hnt, Ndufa10, Aurka, Cs, enriched in A9 neurons and Pdia5, Whrn, and Gpx3 enriched in A10 neurons), verified with the Allen Brain Atlas and not noted or confirmed as differentially expressed before, emerged from this analysis. These and other selected genes are discussed.
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Griffiths, T. R. Leyshon. "The pathophysiological and clinical significance of TP53 in bladder cancer." Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299358.
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Asplund, Anna. "Molecular Analysis of Normal Human Skin and Basal Cell Carcinoma Using Microdissection Based Methods." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5795.
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Palmier, Mathilde. "Evolution des réseaux ostéocytaire et vasculaire lors de la maturation, du vieillissement physiologique et dans un contexte physiopathologique de réparation osseuse." Electronic Thesis or Diss., Bordeaux, 2023. http://www.theses.fr/2023BORD0500.
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Le vieillissement de la population fait naître des problématiques de santé publique telles que l’augmentation du nombre de fractures dues à la fragilisation des os et la nécessité d’adapter les traitements. Aujourd’hui, un certain nombre de stratégies sont adoptées pour prévenir ou ralentir la perte de masse osseuse et pour traiter les fractures. Elles présentent toutes des limitations qui poussent la recherche vers l’identification de nouvelles cibles de traitements. Les ostéocytes représentent 95 % des cellules de l’os et vivent plusieurs dizaines d’années à l’intérieur de la matrice minéralisée. Ils ont une forme caractéristique avec des prolongements partant du corps cellulaire vers les autres ostéocytes, les cellules à la surface de l’os et les vaisseaux sanguins. Pendant longtemps, ils ont été considérés comme passifs parce qu’emmurés au cœur de la matrice. Cependant, le développement d’outils in vitro et in vivo a permis d’identifier leur rôle central dans le maintien de la masse osseuse. En effet, les ostéocytes sont les cellules de l’os les plus sensibles aux variations de sollicitations mécaniques provoquées par l’exercice physique ou l’immobilisation. En réponse, ils envoient des signaux aux ostéoblastes et ostéoclastes pour renforcer la matrice ou l’éliminer. Le vieillissement provoque des changements métaboliques et hormonaux systémiques qui affectent le réseau ostéocytaire. Cependant, comme il est aujourd’hui encore difficile d’étudier les ostéocytes dans leur environnement d’origine, beaucoup reste à explorer. Notamment, leur rapport au réseau vasculaire qui subit également des changements lors du vieillissement ainsi que l’impact de ce dernier sur leur métabolisme énergétique. De plus, ils ont été très peu considérés en tant qu’acteurs de la régénération osseuse pouvant potentiellement avoir un impact sur la qualité de la réparation. Les fractures problématiques, qui ne se réparent pas spontanément sont appelées défauts critiques. Pour les réparer, des solutions sous forme de substituts osseux sont depuis des années en développement. Parmi ceux-ci, les biocéramiques bénéficient d’un intérêt particulier car elles sont capables de libérer des ions Ca2+ et PO43- dans leur environnement. Leur effet sur les ostéocytes a été très peu étudié bien qu’ils régulent le métabolisme du calcium et du phosphate. Pour aborder ces différents aspects, le projet de thèse a d’abord consisté en l’optimisation d’une méthode de microdissection laser pour spécifiquement collecter les ostéocytes dans leur environnement. Ensuite, cette méthode a été utilisée pour analyser leur expression génique dans différents contextes : d'une part la maturation et le vieillissement physiologique, d'autre part la réparation d’un défaut critique avec ou sans biocéramique chez la souris mâle. Lors de la première phase du projet, en supplément des données d’expression génique pour les ostéocytes, l’évolution morphologique des réseaux ostéocytaire et vasculaire a été décrite lors de la maturation et du vieillissement grâce à des techniques d’imagerie en fluorescence. Les changements opposés observés sur la morphologie de l’os étaient accompagnés de changements spécifiques à chaque réseau, sans lien apparent. L’objectif de la deuxième phase (effectuée dans un laboratoire aux Etats-Unis) était d’établir des techniques d’analyse du métabolisme énergétique des ostéocytes en s’intéressant aux acides gras à longue chaîne comme source d’énergie. Cela a mené à l’établissement de protocoles de mesure bioénergétique in vitro et d’imagerie ex vivo. Finalement, l’étude de l’expression génique des ostéocytes dans les phases précoces du processus de réparation a suggéré une contribution de leur part et un effet des biocéramiques via les gènes Il6 et Dmp1. Les outils mis au point et les résultats produits serviront de base pour initier des études visant à mieux comprendre le fonctionnement des ostéocytes dans des contextes encore peu explorésPopulations live longer raising public health concerns related to aging, such as the increase in fracture number due to bone frailty and the necessity to adapt treatments. Nowadays, multiple strategies are followed to prevent or slow down the loss of bone mass, and to treat fractures. They all present limitations forcing researchers to look for new treatment targets. Osteocytes represent 95 % of the cells in bone and live decades embedded inside their mineralized matrix. They have a specific shape with dendrites extending from their body towards other osteocytes, cells at the bone surface, and towards blood vessels. For a long time, they have been considered passive because of their location. However, the development of in vitro and in vivo tools enabled to identify their central role in bone mass maintenance. This is due to the fact that osteocytes are the most mechanosensitive cells in bone, meaning that they react to variations in mechanical loading coming from exercise or disuse. They are able to send signals to osteoblasts and osteoclasts to form and resorb the matrix where it is needed. Aging causes systemic hormonal and metabolic changes affecting the osteocyte network. However, a lot remains to be explored because it is still difficult to study them in their environment. In particular, the nature of their interactions with the vascular network and the changes in energy metabolism with aging need to be investigated. Moreover, very few studies considered osteocytes as having a role in the bone healing process, or an impact on the quality of the repair. Difficult fractures do not repair spontaneously and are called critical. To repair them, bone substitutes have been under development for years. Among them, bioceramics benefit from a specific interest because they are able to release Ca2+ et PO43- in their environment. Their impact on osteocytes has not been well studied, although these cells regulate calcium and phosphate metabolism. To address these different aspects, the first task of the Ph.D. work was to optimize a laser-assisted microdissection protocol to specifically collect osteocytes in their environment. Then, this method was applied to the analysis of osteocyte gene expression during maturation, aging, and during the repair of a critical-size defect in male mice. For the first part of the project, in addition to the osteocyte gene expression analysis, the evolution of the osteocyte and blood vessel network morphologies was described during maturation and aging, with the help of fluorescent imaging techniques. The opposite changes in bone morphology observed during maturation and aging were characterized by distinct, network-specific changes. The second part of the project was elaborated within a lab in the USA, the goal was to establish different techniques to analyze osteocyte energy metabolism using long-chain fatty acids as a fuel source. This led to the optimization and use of in vitro bioenergetics assays and ex vivo imaging. In the last part of the project, the osteocyte gene expression during the early phases of bone repair was analyzed. Among the genes tested, a contribution of osteocytes was identified through the genes Il6 and Dmp1, as well as an impact of the presence of the bioceramics. The different tools and techniques optimized, and the results produced during this PhD project will enable the initiation of new research studies to better understand osteocyte function in contexts still underexplored
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Telu, Kalyani. "Differential localization of mRNA using laser microdissection in the polarized hyphal tip of Fusarium oxysporum." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file,147 p, 2009. http://proquest.umi.com/pqdweb?did=1885754551&sid=1&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Rocha, Lívia Sartoratto Rodrigues Teixeira 1985. "Estudo citogenético de espécies de Dendropsophus (Anura: Hylidae)." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317681.
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Orientador: Luciana Bolsoni Lourenço MorandiniDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O gênero Dendropsophus (Anura: Hylidae) é composto por 94 espécies neotropicais, das quais somente 31 foram estudadas citogeneticamente. Embora as espécies desse gênero apresentem o mesmo número cromossômico (2n=30), diferem quanto ao número fundamental e em relação à localização de NORs. Entretanto, a escassez de marcadores citogenéticos e o alto número de espécies com cariótipos ainda não descritos impedem inferências acerca dos possíveis eventos envolvidos na diferenciação cariotípica nesse gênero. Com o intuito de fornecer novos dados para a análise evolutiva cariotípica em Dendropsophus, no presente trabalho foram descritos os cariótipos de duas espécies do grupo de D. marmoratus, D. seniculus e D. soaresi, ampliada a caracterização dos cariótipos de D. decipiens, D. meridianus e descrito o de D. werneri, três espécies do grupo de D. microcephalus. Para a localização cromossômica do gene ribossomal 5S foram utilizadas sequências isoladas de D. soaresi. Também foram utilizadas sondas para verificar a localização cromossômica de sequências teloméricas em todas as espécies estudadas. Por fim, na tentativa de gerar sondas cromossômicas para futuros estudos dos cromossomos telocêntricos encontrados em Dendropsophus, experimentos de pintura cromossômica foram conduzidos com D. nanus e D. walfordi. Dendropsophus seniculus e D. soaresi apresentaram cariótipos muito semelhantes, com a NOR localizada no braço longo dos cromossomos do par 9. Tais cariótipos se assemelham também àqueles das outras três espécies já cariotipadas do grupo de D. marmoratus (D. marmoratus, D. melanargyreus e D. nahdereri), embora o cariótipo de D. nahdereri difira dos demais por apresentar a NOR no braço curto dos cromossomos do par 1. Dois tipos de sequências de DNAr 5S foram isolados de D. soaresi. A sequência identificada como do tipo I é composta por 512 pb, dos quais 390 pb pertencem à região não-transcritora (NTS); já a sequência do tipo II apresenta NTS com apenas 249 pb. A sequência nucleotídica do DNAr 5S do tipo I é idêntica a uma sequência encontrada no hilídeo Gastrotheca riobambae. Quando utilizado como sonda, esse segmento de DNAr 5S foi mapeado no braço longo dos cromossomos do par 2 de D. seniculus e D. soaresi. Sondas compostas por sequências teloméricas detectaram exclusivamente as regiões cromossômicas terminais de D. seniculus, assim como previamente observado em D. marmoratus e D. melanargyreus. Já em D. soaresi, as sondas teloméricas também resultaram em marcações centroméricas e pericentroméricas, exceto nos pares telocêntricos identificados como 5 e 6. Dentre as três espécies do grupo D. microcephalus aqui viii analisadas, D. decipiens, D. meridianus e D. werneri, algumas diferenças puderam ser notadas. Enquanto D. meridianus e D. werneri apresentaram apenas um par de cromossomos telocêntricos de tamanho mediano, no cariótipo de D. decipiens três pares de cromossomos telocêntricos de tamanho mediano puderam ser observados. D. berthalutzae, espécie considerada do mesmo clado de D. decipiens, também apresenta cariótipo com três pares de telocêntricos de tamanho mediano. Em relação à localização da NOR, os cariótipos de D. decipiens, D. meridianus e D. werneri se assemelham, já que em todos a NOR foi localizada na região terminal do braço longo de cromossomos telocêntricos/subtelocêntricos identificados como 12. A partir de cromossomos telocêntricos microdissecados de D. nanus foi construída uma sonda capaz de identificar o par de cromossomos 15 dessa espécie. A sonda obtida, quando hibridada em cariótipos de outras linhagens de D. nanus e no cariótipo de D. walfordi, também detectou cromossomos telocêntricos de tamanho pequeno, sugerindo a homeologia entre eles. Este é o primeiro estudo que utiliza a técnica de pintura cromossômica para analisar os cromossomos telocêntricos de Dendropsophus e os resultados preliminares aqui apresentados sugerem que essa pode vir a ser uma interessante ferramenta na investigação da evolução cromossômica no gênero
Abstract: The genus Dendropsophus (Anura: Hylidae) comprises 94 neotropical species, from which only 31 were studied cytogenetically. The species of this genus have the same chromosome number (2n=30) but differ on fundamental number and NORs location. Cytogenetic markers, however, are scarce for this genus and a large number of species has not been karyotyped yet. Therefore, it is still not possible to infer about the possible events involved in the karyological divergence in this genus. In order to provide new data for evolutionary karyotype analysis in Dendropsophus, we described cytogenetically two species of D. marmoratus group, D. seniculus and D. soaresi, and one species of D. microcephalus group, D. werneri; we also detailed the karyotypes of two other species of D. microcephalus group, D. decipiens and D. meridianus. Sequences isolated from D. soaresi genome were used for chromosomal localization of the 5S rDNA gene. Probes were also used to observe the chromosomal localization of telomeric sequences in all the species in study. Finally, in an attempt to generate chromosomal probes for future studies of the telocentric chromosomes found in Dendropsophus, chromosome painting experiments were conducted with D. nanus and D. walfordi. Dendropsophus seniculus and D. soaresi had very similar karyotypes, with the NOR located on the long arm of chromosome pair 9. These karyotypes are similar to those of the other three species of D. marmoratus group already karyotyped (D. marmoratus, D. nahdereri and D. melanargyreus), although the karyotype of D. nahdereri differs from the others by presenting the NOR in the short arm of chromosome pair 1. Two types of 5S rDNA sequences were found in D. soaresi. While type I 5S rDNA sequence consists of 512 bp and includes a NTS composed of 390 bp, the NTS of type II 5S rDNA has only 249 bp. Type I 5S rDNA nucleotide sequence is identical to the 5S RNA gene found in Gastrotheca riobambae. When used as a probe, the type I 5S rDNA of D. soaresi was mapped in the long arm of chromosome pair 2 of D. seniculus and D. soaresi. Telomeric sequences used as probes detected chromosomal ends in D. seniculus, as previously observed in D. marmoratus and D. melanargyreus. However, the telomeric probes also resulted in centromeric and pericentromeric signals in the chromosomes of D. soaresi, except the telocentric pairs identified as 5 and 6. Among the three species of the D. microcephalus group analyzed herein, D. decipiens, D. meridianus and D. werneri, some differences could be noted. While D. meridianus and D. werneri had only one pair of telocentric chromosomes of medium size, D. decipiens karyotype had three telocentric pairs. The x species D. berthalutzae, which is included in the same clade of D. decipiens, has also three telocentric pairs of a medium size. The karyotypes of D. decipiens, D. meridianus and D. werneri are also similar with regards to the NOR, which was located terminally in the long arm of a telocentric/subtelocentric chromosome identified as 12. The smallest telocentric chromosomes of D. nanus were microdissected and used to construct a probe, which was able to detect chromosome pair 15 of this species. When this probe was hybridized to the karyotype of different lineages of D. nanus and D. walfordi, a small telocentric chromosome pair was also detected, which suggests their homeology. This is the first work that includes chromosome painting to study the telocentric chromosomes in Dendropsophus and preliminary results presented here suggest that this may be an interesting tool to assess the chromosomal evolution in this genus
Mestrado
Biologia Celular
Mestra em Biologia Celular e Estrutural
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Dos, Santos Alexandre. "Identification de régulateurs clés de la carcinogenèse hépatique humaine : Études clinico-pathologiques, moléculaires et fonctionnelles." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS382.
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Le carcinome hépatocellulaire (CHC) est la forme la plus fréquente de cancer du foie et l’une des principales causes de mortalité par cancer dans le monde. Il s’agit d’une maladie de mauvais pronostic, aux ressources thérapeutiques limitées, hétérogène sur le plan immunophénotypique et génomique, qui se développe très souvent sur un foie remanié cirrhotique. Les études moléculaires ont révélé plusieurs sous-classes de CHC caractérisés par des signatures génomiques et protéomiques distinctes. Au cours de mon travail de thèse, nous avons contribué à améliorer notre compréhension de la biologie des CHC et des classifications moléculaires en cartographiant le génome non-codant de tumeurs de CHC induites par des virus hépatotropes (VHB, VHC) et en étudiant la sous-classe moléculaire de CHC la plus agressive KRT19-positif. Nous avons établi la première carte de transcriptome à ARN non codants du CHC et révélé une forte activation intra-tumorale des rétrotransposons à LTR, qui sont principalement inhibés dans les cellules hépatiques normales, dans des CHC induits par les VHB et VHC. Certains des transcrits dérivés de LTR se sont révélés être des régulateurs clés de l’expression génique et donc activer la croissance cellulaire. Dans la deuxième étude, nous identifions une nouvelle voie de régulation des CHC KRT19-positif affectant le métabolisme énergétique de ces tumeurs. Les CHC KRT19-positif sont des tumeurs fortement glycolytiques liée à une activation de la réponse à l’hypoxie. L’excès de production par les CHC KRT19-positif de l’oncométabolite 2-hydroxyglutarate en absence de mutation des gènes IDH1 et IDH2 était associé à un profil aberrant hyperméthylé sur la lysine 9 de l’histone H3 (H3K9me3) suggérant une répression de la transcription notamment des gènes impliqués dans la différenciation cellulaireHepatocellular carcinoma (HCC) is the main primary liver cancer and one of the most leading cause of cancer-related death worldwide. This heterogeneous disease with a worse prognosis has been subjected of numerous studies aimed to establish global phenotypic profiles. During my thesis, I dedicated my work to improve these classifications by identifying signatures on the non-coding genome and working on a very aggressive form of HCC expressing progenitors markers. With help of a Japanese team, we demonstrated that LTR-derived ncRNAs were active in HCC and that correlation correlates with expression of common cancer markers (GPC3) ans TP53 mutations. This signature can also be used to discriminate HCCs at high risk of recurrence. Finally, we have showed that these LTRs are detectable on prenoplastic stages in the Mdr2 KO mouse model. In parallel, I worked on HCC that expresses progenitor markers such as cytokeratin 19. Using proteomic and transcriptionnal approaches and in silico analyses, we propose that the occurrence of this type of cancer id due to an hypoxic event likely related to trans-arterial chemoembolization. These tumors have a highly glycolytic phenotype with production of an oncometabolite (2-hydroxyglutarate) that has been generally foubd in IDH1/2 mutated cholangiocarcinomas. Finally we suggest the use of metformin, type 2 diabetes drug, to reverse metabolic reprogramming and restore sensitivity to chemotherapy
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Guan, Xin-Yuan. "Cytogenetic and molecular analysis of complex chromosome rearrangements in human cancer: Application of chromosome microdissection." Diss., The University of Arizona, 1993. http://hdl.handle.net/10150/186228.
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Cytogenetic analysis was performed on 50 short-term culture of human ovarian carcinoma from 48 patients. All 50 cases evidenced clonal karyotypic abnormalities with 32/50 displaying numeric changes, and 49/50 displaying structural alterations. The most notable numeric abnormalities were loss of the X chromosome and gain of chromosome 7. Structural alterations appeared to be nonrandom. Chromosomes most frequently involved in structural alterations included chromo-somes 1>7>11>12>3>6. When the chromosomal breakpoints were analyzed they were shown to cluster to several chromosomal band regions including: 1p31-p36, 1p13-q1, 6q22-q25, 7p15-p22, 7q31-34, 11p15, and 12p11-pl13. In addition, a novel procedure for chromosome microdissection and in vitro amplification of dissected DNA was developed and applied to several areas. Microdissection and in vitro amplification of dissected chromosomal fragments were performed, followed by labeling for fluorescent in situ hybridization (FISH) to normal metaphase chromosomes (Micro-FISH). Micro-FISH probes have been used successfully to determine the derivation of chromosome segments unidentifiable by conventional cytogenetic analysis. Micro-FISH probes were also generated following microdissection of unidentifiable portions of nonreciprocal translocations, allowing determination of the derivation of these unknown chromosome segments. Another application of the microdissection procedure has been to generate whole chromosome "painting" probes by microdissection of an entire chromosome. Finally, a chromosome microdissection combining microcloning technique has been developed and used to generate chromosomal band-specific DNA libraries for physical mapping. A genomic library of >20,000 clones, which is highly enriched for sequences encompassing 6q21, was constructed. Clones from this library have been characterized and localized to the target region by hybridization to a chromosome-6 mapping panel.
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Signour, Thomas. "Extraction de signatures de bactéries par microspectroscopie Raman et chimiométrie : application à l’étude de la composition biologique des aérosols dans l’environnement." Thesis, Lille 1, 2017. http://www.theses.fr/2017LIL10152/document.
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Depuis plusieurs années, l’étude et le contrôle de la qualité de l’air sont au cœur de toutes les préoccupations. En 2012, la DGA (Direction Générale de l’Armement) met en place le programme ASTRID (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense) accompagnant les travaux de recherche duale civile et militaire. Cette thèse s’inscrit dans cette démarche et propose d’étudier la faisabilité du concept de détection et d’identification rapides des microorganismes présents dans un échantillon d’air par microspectroscopie Raman, avec une résolution au niveau de l’espèce. Pour cela, nous construisons un modèle chimiométrique de classification des microorganismes représentatifs de la biodiversité naturelle en acquérant, sans a priori, d’une part les spectres Raman de ces microorganismes après biocollecte et étalement sur la lame d’un microspectromètre Raman, et d’autre part les séquences génomiques codant les ARN 16S de ces mêmes microorganismes.Les travaux de recherche présentés dans cette thèse présentent donc les différentes études mises en œuvre lors du développement d’un nouveau protocole permettant l’analyse des bactéries issues d’aérosols naturels environnementaux. Nous démontrons la nécessité d’optimiser l’acquisition des spectres Raman sur les bactéries et le traitement statistique des données spectrales permettant le développement de modèles de classification présentant des taux de reconnaissance élevésFor several years, the study and the control of the quality of the air are at the heart of all the concerns. In 2012, the DGA (Direction Générale de l’Armement) employs the ASTRID program (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense), to accompany the dual civil and military research work. This thesis is part of this approach and proposes the feasibility study, by Raman microspectroscopy, of the concept of rapid detection and identification of microorganisms present in an air sample, with a resolution at the species level. For this, we construct a chemometric model for the classification of micro-organisms representative of the natural biodiversity. Such a model is built by acquiring, without a priori i) the Raman spectra of these microorganisms after biocollection; and ii) the genomic sequences encoding the 16S RNAs of these same microorganisms. The research presented in this thesis therefore presents the different studies carried out during the development of a new protocol allowing the analysis of bacteria from natural environmental aerosols. We demonstrate the need to optimize the acquisition of Raman spectra on bacteria and the statistical processing of spectral data that allows the development of classification models with high recognition rates
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Sanders, Christine. "THE USE AND DEVELOPMENT OF LASER MICRODISSECTION TO SEPARATE SPERMATOZOA FROM EPITHELIAL CELLS FOR STR ANALYSIS." Master's thesis, University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3869.
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Short Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser microdissection (LMD) for the separation of pure populations of spermatozoa from two-donor cell mixtures. In this study, cell separation was demonstrated by microscopic identification of histologically stained spermatozoa and female buccal cell mixtures, and STR analysis of DNA obtained from the separated sperm cells. Clear profiles of the male donor were obtained with the absence of any additional alleles from the female donor. Five histological stains were evaluated for use with LMD and DNA analysis: hematoxylin/eosin, nuclear fast red/picroindigocarmine, methyl green, Wright's stain, and acridine orange. Hematoxylin/eosin out-performed all other stains however nuclear fast red/picroindigocarmine could be used satisfactorily with STR analysis. In addition, three DNA isolation methods were evaluated for LMD collected cells: QIAamp (Qiagen), microLYSIS (Microzone Ltd.) and Lyse-N-Go (Pierce Chemical Co.). MicroLYSIS performed poorly, yielding low levels of PCR product. Lyse-N-Go extraction was effective for the recovery of DNA from LMD collected sperm cells while QIAamp isolation performed best for the recovery of DNA from LMD collected epithelial cells. LMD is shown to be an effective, low-manipulation separation method that enables the recovery of sperm while excluding epithelial cell DNA.M.S.
Department of Chemistry
Arts and Sciences
Industrial Chemistry
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Beasley, Brooke, Aubrey Sciara, Tiffani Carrasco, Gregory Dr Ordway, and Michelle Dr Chandley. "Laser Capture Microdissection Analysis of Inflammatory-Related Alterations in Postmortem Brain Tissue of Autism Spectrum Disorder." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/asrf/2019/schedule/34.
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Autism spectrum disorder (ASD) is a social, sensory and developmental condition that affects one in 59 children and specifically one in 42 boys. Despite the 15% increase in prevalence in the last two years, there is no specific etiology, objective diagnostic criteria, or drug treatment. However, up-regulation of inflammation in ASD patients has been demonstrated in blood samples. Increased peripheral inflammation could have devastating effects on the developing brain. Peripheral inflammation in the blood could cross the blood-brain-barrier to stimulate microglia in the brain to produce aberrant levels of cytokines that regulate neuroinflammation such as insulin-like growth factor one (IGF1) that could alter neuronal cell-surface expression and neurotransmission. Additionally, arginase serves as a marker of inflammation, produced and expressed during cellular remodeling during brain injury. A balance of neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), is critical to facilitate inter-regional signaling in the brain. Alterations of inflammatory molecules and the effects on glutamatergic neurons ability to uptake GABA in certain brain areas is currently unknown in ASD. Pathological changes in brain areas associated with social behaviors have been identified in postmortem tissue from ASD donors when compared to typically developing (TD) age and gender matched control tissue, as well as, in imaging scans of living individuals with ASD. We hypothesize that expression of inflammatory related molecules are increased in the identified brain areas related to symptoms of ASD and can be associated with altered gene expression changes in neurons as shown by gamma-aminobutyric acid type A receptor alpha 1 subunit (GABRA1). Dysfunction of GABRA1 on glutamatergic neurons could disrupt the typical neuronal balance of glutamate and GABA signaling. Inflammatory markers, IGF1 and insulin-like growth factor one receptor (IGF1R), were evaluated using quantitative polymerase chain reaction (QPCR). Additionally, IGF1 and arginase were evaluated using immunohistochemistry in both white and gray matter from the anterior cingulate cortex (ACC). Laser capture microdissection (LCM) was used to obtain single cell captures of glutamatergic neurons. IGF1R and GABRA1 gene expression was measured using end point PCR. A significant increase in IGF1 expression was obtained in the white matter punch in comparison to typically developed age-matched subjects using QPCR during initial statistical significance, however, was ultimately not significant. Additionally, IGF1R expression was significantly increased in ASD neurons in comparison to TD subjects utilizing the LCM method. However, a decrease expression in GABRA1 trended significance indicating a possible alteration in the neuron’s ability to facilitate proper signaling. These findings are the foundation of future investigations of signaling pathways in ASD that may uncover cell-specific etiologies and drug therapies for a condition that is only projected to increase in prevalence.
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Tan, Jing [Verfasser], and Thomas [Akademischer Betreuer] Klopstock. "Laser capture microdissection of single muscle fibers for mitochondrial proteomic investigations / Jing Tan ; Betreuer: Thomas Klopstock." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1205664874/34.
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Brugière, Charlotte. "L'invasion péri-nerveuse des carcinomes épidermoïdes cutanés humains." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC193.
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Le carcinome épidermoïde cutané (CEC) représente un enjeu important par sa fréquence et sa gravité potentielle.L’agressivité de ce cancer est liée à l’invasion péri-nerveuse (IPN), mode d’envahissement tumoral reconnu comme un facteur de mauvais pronostic.L’objectif de ce travail est de s’intéresser aux mécanismes favorisant l’IPN, en comparant 2 groupes appariés de CEC humains, avec et sans IPN.Pour cela nous avons réalisé une étude de facteurs et récepteurs neurotrophiques, de marqueurs de la transition épithélio-mésenchymateuse (TEM), et de la molécule NCAM1, par analyse immunohistochimique à partir de pièces chirurgicales de CEC et par analyse moléculaire en droplet digital PCR sur des cellules tumorales microdisséquées.L’analyse immunohistochimique a trouvé une forte expression de BDNF, TrkB, p75NGFR, Snail 1 et NCMA1 dans les cellules tumorales péri-nerveuses, contrastant avec une faible expression de ces marqueurs dans les cellules tumorales à distance du nerf. L’E-cadhérine était diminuée dans les cellules tumorales péri-nerveuses.L’analyse moléculaire en ddPCR montrait une diminution d’expression de l’E-cadhérine et une surexpression de BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 et NCAM1 dans les cellules tumorales péri-nerveuses par rapport aux cellules tumorales distantes du nerf.Nous avons démontré dans ce travail que l’invasion péri-nerveuse dans les CEC humains est liée aux neurotrophines, à la TEM et implique NCAM1Cutaneous squamous cell carcinoma (SCC) is an important issue because of its frequency and potential severity.The aggressiveness of this cancer is related to perineural invasion (PNI), a mode of tumor dissemination recognized as a poor prognosis factor.The aim of this work is to study the mechanisms of PNI, comparing 2 matched- groups of human SCC with and without PNI.For this, we studied neurotrophins, epithelial-mesenchymal transition (EMT) markers, and the NCAM1 molecule, by immunohistochemistry analysis on surgical pieces of SCC and by molecular analysis with digital-droplet PCR on laser-microdissected tumor cells.Immunohistochemistry analysis found strong expression of BDNF, TrkB, p75NGFR, Snail 1 and NCMA1 in perineural tumor cells, contrasting with weak expression of these markers in tumor cells distant from the nerves. E-cadherin was decreased in perineural tumor cells.Molecular analysis in ddPCR showed decreased expression for E-cadherin and overexpression of BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 and NCAM1 in perineural tumor cells compared to tumor cells distant from the nerves.We have demonstrated in this work that PNI in human SCC is linked to neurotrophins and EMT, and involves NCAM1
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Quanico, Jusal. "Development of On-Tissue Mass Spectrometric Strategies for Protein Identification, Quantification and Mapping." Thèse, Université de Sherbrooke, 2014. http://hdl.handle.net/11143/5867.
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Résumé : L’imagerie par spectrométrie de masse est une technique sans marquage permettant la détection et la localisation de protéines à partir de coupes de tissus. Afin de répondre à des problématiques biologiques, le nombre de protéines identifiées doit être amélioré. Une stratégie consiste à réaliser une micro-jonction liquide sur des régions particulières des coupes de tissus afin d’extraire les peptides issus de la digestion in situ des protéines. Plus de 1500 protéines ont identifié sur une zone de 650µm, correspondant à environ 1900 cellules. Une corrélation entre ces données avec celles générées par MSI a augmenté le nombre de protéines localisées. Afin d’obtenir dans le même temps, la localisation et l’identification de protéines, une méthode consiste à réaliser la microdissection de l’ensemble de la coupe après l’avoir déposée sur une lame recouverte de prafilm. Parafilm-Assisted Microdissection (PAM) a également été appliquée à l’étude de l'expression différentielle de protéines dans des tumeurs de prostate. Les résultats identifiés glutamate oxaloacétate transférase 2 (GOT2) en tant que biomarqueur de protéine candidate impliquée dans le métabolisme du glucose, en plus de celles qui ont déjà été indiqué précédemment. Réunis ensemble, ces méthodes MS d'analyses directes fournissent un moyen robuste d’étude de protéines dans leur état natif afin de fournir des indications sur leur rôle dans des systèmes biologiques. // Abstract : Mass spectrometry-based methods for direct tissue analysis, such as MS imaging, are label-free techniques that permit the detection and localization of proteins on tissue sections. There is a need to improve the number of protein identifications in these techniques for them to comprehensively address biological questions. One strategy to obtain high protein IDs is to realize liquid microjunction on localized regions of tissue sections to extract peptides from the in situ digestion of proteins. More than 1500 proteins were identified in a 650μm spot, corresponding to about 1900 cells. Matching these IDs with those from MSI increased the number of localized proteins. In order to achieve simultaneous identification and localization of proteins, a method consisting of microdissecting entire tissue sections mounted on parafilmcovered slides was developed. Spectral counting was then used to quantify identified proteins, and the values were used to generate images. Parafilm-Assisted Microdissection (PAM) was also used to examine the differential expression of proteins on prostate tumors. Results identified glutamate oxaloacetate transferase 2 (GOT2) as a candidate protein biomarker involved in glucose metabolism, in addition to those that have already been reported previously. Taken together, these direct MS analysis methods provide a robust means of analyzing proteins in their native state and are expected to provide insights to their role in biological systems.
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Ordway, Gregory A., Attila Szebeni, Michelle M. Duffourc, and Katalin Szebeni. "Laser Capture Microdissection and RT- PCR Analyses of Specific Cell Types in Locus Coeruleus From Postmortem Human Brain." Digital Commons @ East Tennessee State University, 2007. https://dc.etsu.edu/etsu-works/8624.
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Morphological studies have shown pathology of neurons and glia in many brain disorders, including psychiatric disorders such as major depression. However, most biochemical characterizations of postmortem human brain tissue have not made a distinction between neurons and glia. Laser capture microdissection (LCM) to isolate specific cell types has the potential to advance our understanding of human brain pathologies. Here, RT-PCR was used to evaluate the utility of LCM in the capture of noradrenergic neurons, astrocytes and oligodendrocytes from the locus coeruleus (LC) of postmortem human brain. The 3 LC cell types were individually identified using modifications of established histological and morphological methods. LCM settings were optimized for each cell type and captured cell bodies were those having no nearby cell body of a different phenotype. LC neurons (200), astrocytes (500), and oligodendrocytes (500) were captured within the LC from 3 postmortem brains. RNA was isolated, reversed transcribed, and markers for neurons (tyrosine hydroxylase [TH], dopamine beta-hydroxylase [DBH]), astrocytes (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (myelin oligodendrocyte glycoprotein [MOG]), along with 3 references (actin, GAPDH, ubiquitin C) were PCR amplified and quantified by standardized end-point PCR. RNA quality as assessed by RIN was not altered by LCM as compared to RNA isolated from homogenized tissue. TH gene expression was found only in neurons in 2 of the 3 brains. DBH gene expression was ~5-fold greater in neurons than in astrocytes and oligodendrocytes. GFAP gene expression in astrocytes was 7- and 5-fold greater than that in neurons and oligodendrocytes, respectively. MOG gene expression was only detected in oligodendrocytes. Different expression ratios of marker genes between neurons and glia suggest that simple cross contamination of mRNA is unlikely. Glial cells may contain DBH mRNA. Alternatively, DBH, but not TH, mRNA may occur in neuronal dendrites or axons in close association with glial cells that become captured with glia during LCM. GFAP may be expressed in low levels in neurons and oligodendrocytes, or alternatively, GFAP mRNA may be located in astrocytic processes in close association with neuronal and oligodendrocyte cell bodies. Use of a single marker to identify a cell type may be insufficient; other cell types for comparison or additional markers may be required. Multiple well-characterized markers can be used to evaluate clarity of cell capture for each sample. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of specific cell types in postmortem human brain.
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Guillier-Gencik, Zuzana. "Des galliformes à l'homme : approche cytogénomique." Paris 6, 2004. http://www.theses.fr/2004PA066146.
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Ordway, Gregory A., Attila Szebeni, Craig A. Stockmeier, Michelle M. Duffourc, and Katalin Szebeni. "Glial Deficits in the Noradrenergic Locus Coeruleus in Major Depression Revealed by Laser Capture Microdissection and Quantitative PCR." Digital Commons @ East Tennessee State University, 2008. https://dc.etsu.edu/etsu-works/8625.
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BENSAADA, MUSTAPHA. "Nouvelles approches pour l'analyse cytogenetique des chromosomes humains par l'etude de leur degre de methylation et leur microdissection." Paris 7, 1999. http://www.theses.fr/1999PA077025.
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Ce travail a pour objectif la mise au point de nouveaux outils destines a la cytogenetique. Nous nous sommes efforces : - d'etablir un nouveau caryotype sur la base de l'etude qualitative et quantitative de la distribution des methylcytosines (mc) du genome humain, - d'etablir un protocole pour la microdissection des chromosomes par le laser, capable de fournir des sondes genomiques avec un delai et un cout raisonnables. Dans le processus tumoral une relation etroite entre la variation du nombre de mc et le caractere malin d'une cellule a deja ete etabli. La possibilite d'analyser cette variation le long des chromosomes metaphasiques de cellules normales ou de lignees leucemiques au moyen d'anticorps, anti-mc (amc) nous a permis de degager les donnees suivantes : - les mc sont reparties le long des chromosomes de cellules normales d'une maniere heterogene produisant un profil de type bande r. Nous avons pu definir avec les amc deux types de bandes r : hautement (rhf) et faiblement (rff) fluorescentes. - sur les chromosomes des cellules provenant de lignees leucemiques, nous avons defini deux autres types de bandes : les rhf + plus fluorescentes que les rhf et les rff 0 qui le sont moins. - il y a une chute du niveau global des mc dans les lignees leucemiques par rapport aux cellules normales. L'analyse fine de plusieurs mc au voisinage des genes mdr1, -globine, l'oncogene c-myc et le gene suppresseur de tumeur p53, montre que ces genes sont differemment methyles dans les cellules normales et issues de lignees leucemiques. Nos conditions experimentales apportent une methode complementaire au banding traditionnel, empruntant une approche structurelle et fonctionnelle. La microdissection des chromosomes pour la preparation de sondes pour l'his est peu employee en raison des infrastructures qu'elle necessite et des difficultes de mise en uvre. Nous avons mis au point un protocole, suffisamment simple qui permet d'obtenir en moins de 24 h. Des fragments d'adn a partir d'une seule copie, de taille variee (300 pb a 4 kb).
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Bartel, Jan [Verfasser]. "Laser Capture Microdissection in Paraffin eingebetteter Gewebe als Werkzeug zur Bestimmung des Sialylierungsstatus von ausgewählten Zellpopulationen / Jan Bartel." Gießen : Universitätsbibliothek, 2017. http://d-nb.info/1141574675/34.
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Jumper, Natalie. "Application of a site-specific in situ approach to keloid disease research." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/application-of-a-sitespecific-in-situ-approach-to-keloid-disease-research(f0a9bcae-93f0-4335-8839-afa5747f40d6).html.
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Keloid disease (KD) is a cutaneous fibroproliferative tumour characterised by heterogeneity, locally aggressive invasion and therapeutic resistance. Clinical, histological and molecular differences between the keloid scar centre and margin as well as recent evidence of the importance of epithelial-mesenchymal interactions (EMI) in KD pathobiology contribute to the complexity and diversity of KD, which coupled with the lack of a validated animal model have hindered research and effective management. Despite significant progress in the field of KD research, reliance on conventional monolayer cell culture and whole tissue analysis methods have failed to fully reflect the natural architecture, pathology and complexity of KD in vivo. In order to address these challenges, a site-specific in situ approach was therefore employed here for the first time in KD research. The first aim of this work was to compare the value of this contemporary approach with traditional methods of tissue dissection. The second aim was to compare the genomic expression between well-defined, distinct keloid sites and normal skin (NS). The third aim was to develop and explore hypotheses arising from this site-specific gene expression profiling approach, so as to enhance understanding of KD pathobiology as a basis for improved diagnostic and therapeutic strategies in future KD management. The fourth aim was to probe these hypotheses with relevant functional in vitro studies. The current site-specific in situ approach was achieved through a combination of laser capture microdissection and whole genome microarray, allowing separation of epidermis from dermis for keloid centre, margin and extralesional sites compared with NS. This in situ approach yielded selective, accurate and sensitive data, exposing genes that were overlooked with alternative methods of dissection. Identification of significant upregulation of the aldo-keto reductase enzyme AKR1B10 in all three sites of the keloid epidermis (KE) in situ, implicated dysregulation of the retinoic acid (RA) pathway in KD pathogenesis. This hypothesis was supported by showing that induced AKR1B10 overexpression in NS keratinocytes reproduced the keloid RA pathway expression pattern. Moreover, co-transfection with a luciferase reporter plasmid revealed reduced RA response element activity. Paracrine signals released by AKR1B10-overexpressing keratinocytes into conditioned medium resulted in TGFβ1 and collagen upregulation in keloid fibroblasts, suggesting the disturbed RA metabolism exerts a pro-fibrotic effect through pathological EMI, thus further supporting the hypothesis of RA deficiency in KE. Gene expression profiling further revealed an upregulation of NRG1 and ErbB2 in keloid margin dermis. Exogenous NRG1 led to enhanced keloid fibroblast migration with increased Src and PTK2 expression, which were attenuated with ErbB2 siRNA studies. Together with the observed failure to recover this expression with NRG1 treatment, suggested the novel KD pathobiology hypothesis that NRG1/ErbB2/Src/PTK2 signaling plays a role in migration at the keloid margin. In addition to these hypotheses, LCM methodology with comprehensive analysis of the data permitted the development of additional novel working hypotheses that will inform future KD research, including inflammatory gene dysregulation and cancer-like stem cells that may contribute to the therapeutic resistance characteristic of KD.
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Ordway, Gregory A., Attila Szebeni, Michelle J. Chandley, Craig A. Stockmeier, Michelle M. Duffourc, and Katalin Szebeni. "Abnormal Gene Expression in Noradrenergic Neurons and Surrounding Glia in Brains of Depressed Suicide Victims Revealed by Laser Capture Microdissection and qPCR." Digital Commons @ East Tennessee State University, 2009. https://dc.etsu.edu/etsu-works/8628.
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Bertheau, Philippe. "Caractérisation moléculaire et étude des mécanismes de la réponse à la chimiothérapie des cancers du sein inflammatoires ou localement avancés." Paris 7, 2002. http://www.theses.fr/2002PA077203.
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Edlund, Karolina. "Microdissection of well defined cell populations for RNA isolation in the analysis of normal human skin and basal cell carcinoma." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6148.
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The human skin provides us with an excellent protective barrier and possesses a remarkable ability of constant renewal. Basal cell carcinoma is the most common type of skin cancer. The aim of this project was to verify results from an earlier study investigating the molecular differences between basal cell carcinoma (BCC) and basal cells of normal human epidermis. In that study microdissection of cell populations from BCC and basal cells of normal epidermis respectively was performed in five cases of confirmed BCC. Following RNA extraction and amplification, a gene expression analysis was performed using a 46 k human cDNA microarray. Comparison of expression profiles showed a differential expression of approximately 300 genes in BCC. An upregulation of signaling pathways previously known to be of importance in BCC development could be observed, as well as a downregulation of differentiation markers, MHC class II molecules, and proteins active in scavenging of oxygen radicals. We wanted to confirm these findings for a number of selected genes, using real time PCR. The focal point of this project was microdissection of cells from BCC and subsequent isolation of RNA. Microdissection based methods offer a possibility of selecting well defined cell populations for further analysis by using a focused laser beam. Initially tests in order to optimize the method were also performed, concerning the dehydration process and choice of slides used in microdissection. Isolation of RNA may, as we experienced, be associated with problems due to destruction of RNA by degrading enzymes.
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Ordway, Gregory A., Attila Szebeni, Michelle M. Duffourc, Sophie Dessus-Babus, and Katalin Szebeni. "Gene Expression Analyses of Neurons, Astrocytes, and Oligodendrocytes Isolated by Laser Capture Microdissection From Human Brain: Detrimental Effects of Laboratory Humidity." Digital Commons @ East Tennessee State University, 2009. https://dc.etsu.edu/etsu-works/8606.
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Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.
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Moncada, Benavides Camilo Andres. "EFFECT OF NICOTINE ON LUNG S-ADENOSYLMETHIONINE AND PNEUMOCYSTIS PNEUMONIA DEVELOPMENT." Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/206623.
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BiochemistryPh.D.
Infection with "Pneumocystis" causes a ≥ 99% depletion of plasma S-adenosylmethionine (AdoMet) levels in both "Pneumocystis" pneumonia (PcP) animal models and patients. AdoMet is a critical cellular metabolic intermediate, with a pivotal role as methyl donor in a myriad of biochemical processes and necessary for the synthesis of the essential polyamines spermidine and spermine. In the target tissue of "Pneumocystis", the lung, levels of AdoMet were previously shown to be depleted experimentally using nicotine. Here we show that chronic administration of nicotine in an animal model of PcP resulted in decreased lung AdoMet content. Since "Pneumocystis" is dependent on this metabolite, PcP burden was also relived. We hypothesized that the underlying mechanism behind nicotine-induced AdoMet depletion was an increased consumption of AdoMet through the polyamine pathway where the increased activity of N-1-spermidine/spermine acetyl transferase raises the catabolic / anabolic cycling of polyamines, a process that utilizes AdoMet. In a critical test of our hypothesis, we found that blockage of polyamine metabolism via inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) hinders the effect of nicotine on lung AdoMet levels. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of AdoMet toward polyamine synthesis and away from methylation reactions; these shifts are also reversed by inhibition of ODC. Because the nicotine effect on "Pneumocystis" is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection (LCM), we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ODC activity in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on "Pneumocystis" pneumonia. Our studies demonstrate the feasibility of pharmacological manipulation of the polyamine pathway in order to reduce AdoMet levels in the lung and prompted the assessment of compounds alternative to nicotine with the potential to achieve a comparable effect. In vitro evaluation of the polyamine analog DENSPM along with putrescine in type II alveolar cell lines, indicates that although such a combination has the potential to induce polyamine flux, an apparent competition for the same polyamine transport system impairs simultaneous uptake of both compounds at effective concentrations. In conclusion, we showed that chronic nicotine administration causes reduction of AdoMet levels in rat lung following 21 days of treatment, by a mechanism involving the induction of polyamine flux, which is responsible of increased AdoMet utilization for polyamine biosynthesis. According to LCM-based analysis, this effect seems to be confined to the alveolar regions of the lung.
Temple University--Theses
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Thwe, Le Myo. "Biomarker Analysis in Paediatric Tumours Diagnosed within A Single Institution." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/16297.
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The incidence of childhood cancers in Australia represents approximately 0.6% of all cancers diagnosed. It can therefore be challenging to undertake investigations of rare childhood tumours, especially those diagnosed within a single institution. As biological molecules in tissue samples degrade over time, biomarker expression may not be validly compared in samples stored for different lengths of time. Sample storage time could therefore significantly affect protein detection in childhood cancer samples of different ages, and therefore affect the results of comparative analyses. Informative neuroblastoma (NB) patient cohorts were identified for 1985-2005, an overall cohort of 174, and an analysis sub-cohort of 56 NB and ganglioneuroblastomas (GNB). Immunohistochemical (IHC) analyses were done on tissue microarrays using five different biomarkers with digital and visual IHC scoring. Significant inverse associations were identified between sample storage time and digital or visual IHC scores for NB84 in the archival TMA, for NSE and NB84 in the analysis TMA, and for TPD52 in the archival TMA. A significant difference in digital TPD52 IHC staining was measured in two NB differentiation groups, and significant differences between digital ALK and MGMT IHC scores were measured in stage 4 and non-stage 4 NB. A significant difference between serum NSE levels was also identified between stage 4 and non-stage 4 patients. Significant differences between digital and visual NSE or NB84 IHC staining were also identified in amplified and non-amplified MYCN gene status. Patients with high digital IHC scores for MGMT, low visual IHC scores for NSE and high visual scores for TPD52 showed significantly poor overall survival. Differential IHC staining of NSE, NB84, ALK, MGMT and TPD52 was visually identified in duplicate tissue cores for 15/46 NB cases. Genomic DNA was extracted from laser captured 6 NB cases and normal kidney tissue, followed by PCR amplification using TPD52 and ALK primers. No significant associations were measured between total yields of extracted DNA and LCM cutting areas or the age of FFPE specimens. The TPD52 product of was successfully PCR amplified from both positive control, NB and normal samples but the ALK products was only successful on a positive control.
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Szathmari, Alexandru. "Étude du transcriptome dans les tumeurs périventriculaires du système nerveux central : recherche des marqueurs diagnostiques et pronostiques." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10037.
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Les services de Neurochirurgie du Groupement Hospitalier Est de Lyon ont une expérience reconnue pour l’exérèse des tumeurs des régions périventriculaires du système nerveux central notamment au niveau de la région pinéale. Dans ce contexte neurochirurgical favorable et avec l’opportunité d’utiliser des techniques de biologie moléculaire, notre objectif a été l’identification de marqueurs diagnostiques pour chaque type tumoral par une étude transcriptomique en microarray, la caractérisation d’un sous-type de tumeur du parenchyme pinéal (TPP) pléïomorphe, l’évaluation de la synthèse de mélatonine par les TPP et l’étude du transcriptome de certains organes circumventriculaires (OCV) microdisséqués chez le rat. L’analyse du transcriptome des tumeurs périventriculaires a permis de regrouper les tumeurs par leur signature moléculaire et d’identifier des marqueurs diagnostiques pour chaque type tumoral. De nouveaux marqueurs pronostiques potentiels (HoxD13, Prame, CD24 et Pou4f2 et gènes de la voie Aurora kinase B) sont proposés en vue d’améliorer la classification des TPP. Pour ces dernières, une étude multicentrique a permis de caractériser un sous-type tumoral, les tumeurs pléïomorphes, souvent surgradées. L’étude des TPP ex vivo et in vivo montre leur capacité de synthèse de mélatonine. Toutefois, nous n’avons pu obtenir une lignée de cellules tumorales stable. La microdissection des OCV du rat, parfois vestigiaux chez l’homme et qui pourraient être à l’origine de tumeurs périventriculaires, a permis d’étudier leur transcriptome et de mettre en évidence des marqueurs nouveaux ou déjà associés dans la littérature à ces structuresNeurosurgery of periventricular tumors, especially of pineal region tumors, is well developed at the Neurosurgical Hospital in Lyon. Taking opportunity of this background, our objective was identification of new diagnostic markers for each of these tumors using microarray transcriptome analysis, characterisation of a pleomorphic pineal parenchyma tumor (PPT) subtype, evaluation of melatonin synthesis in PPTs and the microarray analysis of molecular signature of some of circumventricular organs (CVO) after their laser microdissection in rat. The microarray analysis of periventricular tumors allowed molecular classification of the tumours and revealed different diagnostic markers for each type of tumors. Potentially new prognostic candidate genes (HoxD13, Prame, CD24 and Pou4f2 and Aurora kinase B pathway genes) are proposed for improvement of PPT classification. A PPT multicenter study allowed the characterisation of a pleomorphic subtype frequently managed as a higher grade tumour in the literature. The study of PPT ex vivo and in vivo showed their preserved capacity for melatonin production. However a stable PPT cell line culture could not be obtained. The laser microdissection of OCV in rat, sometimes vestigial in humans and potentially at the origin of the periventricular tumors, associated with a microarray study highlighted some potentially new or already described specific markers of these structures
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Ordway, Gregory A. "Abnormal Gene Expression in Noradrenergic Neurons and Surrounding Glia in Brains of Depressed Suicide Victims Revealed by Laser Capture Microdissection and qPCR." Digital Commons @ East Tennessee State University, 2009. https://dc.etsu.edu/etsu-works/8666.
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Fischer, Anthony John. "Augmenting antiviral host defense in the respiratory epithelium." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/500.
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The airway epithelium has many roles in innate immunity including detection of pathogens and transmitting danger signals to other cell types. However, its role as a primary defender against infection is not well recognized. We have investigated methods of augmenting antiviral immunity by application of agents that stimulate viral killing, either in the extracellular space or within the cytoplasm. A recently described property of airway epithelial cells is direct oxidative killing of bacteria through the coordination of Duox and lactoperoxidase enzymes. We have exploited this property by supplementing airway cells with the lactoperoxidase substrate iodide to prevent viral infection. A second method for enhancing antiviral defenses is to supply small interfering RNAs
(siRNAs) targeting essential viral genes. We have optimized antiviral siRNAs targeting respiratory syncytial virus by designing them to specifically target positive sense viral RNAs. Finally, we have initiated a project to discover host defense genes that are expressed in either the submucosal glands surface epithelium of human airway. This information will enable a better characterization of the roles for these structures in host defense pathways, and may identify other targets for augmentation of antiviral immunity.
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Busto, Germain. "Bases cellulaires et moléculaires de l’apprentissage et de la mémorisation dans le bulbe olfactif de souris." Thesis, Lyon 1, 2009. http://www.theses.fr/2009LYO10086.
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Durant ma thèse, j’ai étudié dans le bulbe olfactif (BO) de souris adulte, les mécanismes cellulaires et moléculaires impliqués dans l’apprentissage et de la mémorisation olfactive. Le BO est le premier relai central de l’information olfactive. A ce niveau, des phénomènes de plasticité locaux interviendraient dans la conservation d’une trace mnésique de l’apprentissage. J’ai tout d’abord évalué, dans la couche granulaire, les conséquences d’un apprentissage olfactif associatif sur l’expression de l’IEG Zif268 induite par une stimulation odorante. Les souris ayant une expérience préalable avec l’odorant ne présentent pas d’augmentation de l’expression de Zif268. Cependant, le patron d’expression cellulaire de Zif268 est modifié par l’apprentissage. J’ai ensuite isolé par microdissection laser, à partir des patrons d’expression de Zif268, les populations de cellules de la couche granulaire impliquées dans le traitement de l’odorant suite à l’apprentissage. Dans ces régions, l’étude de l’expression des gènes à large échelle m’a permis de mettre en évidence que la voie des neurotrophines était modulée dans la phase précoce de l’apprentissage alors que les acteurs de la LTP étaient modulés lors de la phase tardive. Enfin, j’ai montré que des souris inactivées pour zif268 présentaient des déficits d’acquisition et de consolidation de l’apprentissage olfactif ainsi que de discrimination d’odorants perceptivement proches. Ces résultats indiquent que l’acquisition par l’odorant d’une signification lors d’un apprentissage olfactif modifie son traitement dans le BO. D’autre part, des acteurs moléculaires potentiellement impliqués dans ces modifications cellulaires ont été identifiésMy research was about cellular and molecular mechanisms implicated in olfactory learning and memory in the adult mouse olfactory bulb (OB). The OB is the first relay of olfactory information in the central nervous system. At this level, phenomenon of local plasticity could be involved in the conservation of a memory trace associated with learning process. First, I evaluated in the granule cell layer, the consequences of an olfactory associative learning on the IEG Zif268 odour-induced expression. Mice with a prior behavioural experience with the odour do not show increase in Zif268 expression. However, the specific odour-induced Zif268 expression pattern is modified by learning. Then, I isolated using laser capture microdissection activated cell populations of the granule cell layer, based on Zif268 expression patterns, after an olfactory associative learning. In those regions, I studied gene expression at a large scale. I found that neurotrophine pathway was modulated during the early phase of learning process whereas molecular actors of LTP are modulated during the consolidation phase. Finally, I showed that Zif268 knock-out mice exhibit associative learning and memory deficits. Those mice also present deficits to discriminate between closely related odorants. Those results indicate that acquisition by odorant of a behavioural meaning during olfactory learning modify odorant processing at the level of OB. Moreover we identified candidate genes that could be implicated in the cellular modifications
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Owens, Misty. "BDNF-Related Gene Expression of Laser Capture Microdissected Glutamate Neurons from the Anterior Cingulate Cortex in Mouse Models of Autism Spectrum Disorder." Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/etd/3805.
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Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting social behaviors. ASD affects 1 in 59 children with males affected more frequently. ASD is postulated to result from excitatory and inhibitory neurotransmission imbalances. Brain-derived neurotrophic factor (BDNF) signaling affects ASD by influencing synaptogenesis, plasticity, and survival. Studying early in-utero neuropathological changes within ASD requires the use of animal models. Expression of BDNF-associated genes were analyzed within laser capture microdissected pyramidal neurons from the anterior cingulate cortex of male and female BTBR and valproic acid mouse models. No expression differences were found in any gene comparing the three groups. Gender comparisons did identify differences in NTRK2 and EFNB2. Significant correlations of gene expression were identified for male NTRK2 with EFNB2 and GRIN1 and EFNB2 with GRIN1 and female BDNF with GRIN1 expressions (p
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Owens, Misty. "BDNF-Related Gene Expression of Laser Capture Microdissected Glutamate Neurons from the Anterior Cingulate Cortex in Mouse Models of Autism Spectrum Disorder." Digital Commons @ East Tennessee State University, 2008. https://dc.etsu.edu/etd/3805.
Full textAbstract:
Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting social behaviors. ASD affects 1 in 59 children with males affected more frequently. ASD is postulated to result from excitatory and inhibitory neurotransmission imbalances. Brain-derived neurotrophic factor (BDNF) signaling affects ASD by influencing synaptogenesis, plasticity, and survival. Studying early in-utero neuropathological changes within ASD requires the use of animal models. Expression of BDNF-associated genes were analyzed within laser capture microdissected pyramidal neurons from the anterior cingulate cortex of male and female BTBR and valproic acid mouse models. No expression differences were found in any gene comparing the three groups. Gender comparisons did identify differences in NTRK2 and EFNB2. Significant correlations of gene expression were identified for male NTRK2 with EFNB2 and GRIN1 and EFNB2 with GRIN1 and female BDNF with GRIN1 expressions (p
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Nikolaidou, Theodora. "Mammalian atrioventricular junction anatomy, electrophysiology and ion channel remodelling in health and disease." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/mammalian-atrioventricular-junction-anatomy-electrophysiology-and-ion-channel-remodelling-in-health-and-disease(ed62c1fd-89d7-4db1-a6be-9b002ceff503).html.
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The atrioventricular junction (AVJ) is a complex anatomical structure. It has an important role in maintaining synchronised atrioventricular conduction and protects from ventricular tachycardia, as well as bradycardia. Its embryological development and function is under tight transcription factor control. Heart failure is a chronic systemic condition, affecting one million people in the UK alone. Slowing of atrioventricular conduction in heart failure is associated with increased morbidity and mortality. The molecular and anatomical basis of abnormal atrioventricular conduction was studied in a rabbit model of heart failure due to aortic insufficiency and abdominal aortic constriction. The PR interval was significantly prolonged in heart failure animals. Using laser-assisted microdissection, the tiny tissues of the AVJ were collected for RT-PCR analysis. HCN1, Cav1.3, Cx40 and Cx43 transcripts were significantly downregulated by heart failure, with a compensatory increase in CLCN2, Nav1.1, Navβ1, SUR2A and PAK1. Immunolabelling for Cx43 showed reduction in protein level and longitudinal dissociation not only in the inferior nodal extension but also in the His bundle in heart failure animals. Anatomical studies of the AVJ have previously been limited by its small size and inaccessible location. Contrast-enhanced micro-CT scanning allowed non-destructive imaging of the AVJ anatomy. AVJ length and volume were increased in the rabbit model of heart failure, which is expected to contribute to atrioventricular conduction abnormalities. Micro-CT additionally resolved the anatomy of the canine AVJ and atria, including fibre orientation in the pulmonary vein sleeves and Bachmann’s bundle. The physiological effects of loss of T-box transcription factor 5 (Tbx5) in the AVJ were studied in a transgenic inducible Tbx5 knockout mouse model using optical mapping. Tbx5-deficient mice had a prolonged PR interval in vivo and a higher incidence of atrioventricular block and ventricular conduction abnormalities in Langendorff-perfused hearts.
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Behrens, Fauke. "Analysis of the human centromere : an investigation into the use of a centromeric microdissection library for isolating and mapping of centromeric DNA sequences." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264957.
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Consentino, Laurent. "Mécanismes d'acquisition du fer de l'hôte chez Bacillus cereus : rôle du couple bacillibactine-FeuA et expression des gènes impliqués dans l'homéostasie du fer in vivo durant l’infection intestinale chez l’insecte." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLA018/document.
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L’apport de fer est essentiel pour la plupart des organismes vivants, incluant la majorité des bactéries pathogènes. Cependant, le fer libre est toxique : il est lié à des protéines de stockage et de transport (e.g. ferritine, hémoprotéines…) et voit son homéostasie finement régulée. Afin d’extraire le fer de ces protéines, les bactéries utilisent divers systèmes tels que des protéines de surface ou encore des sidérophores. Bacillus cereus est une bactérie Gram-positive sporulante, pathogène opportuniste chez l’homme, 2ème cause en France de toxi-infection alimentaire collective. Chez B. cereus, la protéine de surface IlsA et le sidérophore bacillibactine (BB) sont impliqués dans l’acquisition du fer de la ferritine exogène et elles sont importantes pour l’infection de l’insecte modèle Galleria mellonella. Mes travaux présentaient deux parties : tout d’abord, l’étude de l’import du complexe BB-Fe3+ dans la cellule par FeuA, protéine de liaison de ce complexe à la surface de la bactérie, souligne le rôle central du couple BB-FeuA. La délétion des gènes codants pour ces deux molécules limite l’acquisition par B. cereus du fer de la ferritine, de l’hème, de l’hémoglobine et du fer inorganique in vitro. En revanche, elle présente un phénotype de virulence in vivo comparable à la souche de référence dans le cas d’injection intra-hémocœlique de larves de G. mellonella. Ce résultat surprenant suggère un probable rétrocontrôle sur l’expression de facteurs de virulence lorsque B. cereus ne produit ni BB ni FeuA, et se trouve par conséquent fortement carencé en fer. Le second volet de mes travaux s’intéresse à l’expression des gènes liés à l’homéostasie du fer in vivo, au cours de l’infection de l’intestin de larves de G. mellonella axéniques. Nous avons choisi une approche de type microgénomique, en prélevant les échantillons par microdissection laser, sur de façon à prélever de petits échantillons dans une zone définie, puis en analysant l’expression de quelques gènes ciblés par RT-qPCR et ddPCR à 3h et 16h post ingestion. Nos résultats montrent que : i) la colonisation intestinale de G. mellonella est impactée lorsque B. cereus est dépourvu du couple BB-FeuA ; ii) ilsA est exprimé lors de l’infection intestinale ; iii) les gènes ciblés impliqués dans l’homéostasie du fer sont activés dès le début de l’infection, suggérant un rôle dans l’adaptation et la pathogénicité ; iv) une faible modulation de l’expression est observée entre les deux temps. Ces travaux ouvrent de nouvelles connaissances fondamentales sur l’homéostasie du fer et des perspectives quant à l’utilisation de nouvelles techniques pour l’étude in situ des interactions hôte-pathogèneIron acquisition is essential for most living organisms, including many pathogenic bacteria. However, free iron is toxic: it is bound into storage or transport proteins (e.g. ferritin, hemoproteins…) and iron homeostasis is tightly regulated. To scavenge iron from these sources, bacteria possess several systems to acquire the bound iron, by surface proteins or siderophores. Bacillus cereus is a sporeforming Gram-positive bacterium, opportunistic human pathogen, 2nd cause of food-borne disease in France. It has been demonstrated that the B. cereus surface protein IlsA and the siderophore bacillibactin (BB) are involved in iron acquisition from ferritin and that these two molecules are important for infection of the insect model G. mellonella. My thesis project focused on two parts: first the study of the BB-Fe3+ complex import into the cell by the siderophore binding protein FeuA highlights the central role of both BB and FeuA. The deletion of the genes encoding for these two molecules limits iron acquisition by B. cereus from ferritin, heme, hemoglobin and inorganic iron in vitro. On the other hand, the virulence phenotype during intra-haemocelic infection of G. mellonella is similar to the Wild-type strain. These results suggest a possible feedback on the expression of virulence factor genes when B. cereus is unable to synthetize both BB and FeuA, and therefore are under high stress. The second part of my work focused on the expression of genes involved in iron homeostasis in vivo, during gut infection of germ-free larvae of G. mellonella. We chose to perform a microgenomic approach, using laser-capture microdissection to get small samples in targeted areas, and then analysing the expression of chosen genes by RT-qPCR and ddPCR at two time points post ingestion The results show that : i) the colonisation of G. mellonella gut is impacted when B. cereus is deprived of both BB and FeuA ; ii) ilsA is expressed during gut infection ; iii) iron homeostasis is involved in adaptation and pathogenicity from the early step of infection of the insect gut ; iv) only weak gene expression modulation occured between the two timepoints This work gives new fundamental knowledge about B. cereus iron homeostasis, and highlights the use of new techniques regarding the in situ study of host-pathogen interactions
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Stuart, Charles A., and Michael H. Stone. "Reply to "Letter to the Editor: Comments on Stuart Et Al. (2016): 'myosin Content of Individual Human Muscle Fibers Isolated by Laser Capture Microdissection'"." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/4675.
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Parillaud, Romain. "Définir le rôle de chimiokines comme médiateurs pathologiques de la neuroinflammation dans le modèle MPTP de la Maladie de Parkinson." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066627.
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La maladie de Parkinson (MP) est marquée par la présence d'une inflammation, pouvant être bénéfique ou délétère à la neurodégénérescence dopaminergique (DAgique). Nous avons adressé la nature des interactions pathologiques entre neurones DAgiques, cellules gliales et leucocytes infiltrant, nécessaire à la mise en place de cette inflammation. Dans un modèle MPTP murin de la MP, les objectifs de ma thèse ont été 1) d'identifier des signaux inflammatoires neuronaux et gliaux, par une approche transcriptomique associée à de la microdissection laser et 2) de déterminer leurs rôles dans la neuroinflammation ainsi que leurs effets sur la perte DAgique. Nous avons retenu parmi les candidats identifiés: les axes CXCL16-CXCR6 et CCL2-CCR2. Nous reportons dans le modèle MPTP, une expression microgliale de CXCL16 ainsi qu'une infiltration de population lymphocytaire CXCR6. Bien que la déplétion de CXCR6 permette de réduire cette infiltration, aucun effet n'est observé sur la perte DAgique. Nous décrivons une infiltration de monocyte CCR2 en concomitance avec une expression astrocytaire précoce de CCL2 dans le modèle MPTP murin, ainsi qu'une expression plus prolongée de CCL2 chez le primate non-humain MPTP, suggérant une relevance de l'axe CCL2-CCR2 dans la MP. En effet nous montrons que des souris surexprimant CCL2, intoxiquées au MPTP, ont non seulement une augmentation accrue de l'infiltrat monocytaire CCR2, mais également de la lésion DAgique. De manière inattendue, nous montrons que la neurotoxicité accrue observée chez des souris CX3CR1-/- MPTP passe indirectement par la voie CCL2-CCR2. Ainsi, nos données supportent l'hypothèse d'une neurotoxicité des monocytes CCR2 dans la MPParkinson's disease (PD) presents signs of neuroinflammation, which can be beneficial or deleterious for dopaminergic (DA) neurodegeneration. We have analyzed the characteristics of such pathological interactions between DA neurons, glial cells and infiltrating immune cells. Using the neurotoxic MPTP mouse model of PD and focusing on chemokines, my thesis objectives were: 1) to identify using laser-microdissection and RNA profiling, the neuronal and glial inflammatory signals in the affected Substantia Nigra (SN) and 2) to assess the role of promising identified chemokine candidates during DA neurodegeneration. We have focused on the lymphocytic CXCL16-CXCR6 and the monocytic CCL2-CCR2 axes. We have found early microglial CXCL16 induction and parallel SN infiltration of CXCR6 lymphocyte subpopulations. CXCR6-deletion reduced infiltration of specific lymphocyte subpopulations, but did not affect the known deleterious infiltration of CD4 T-lymphocytes. For the CCL2-CCR2 axis, we found evidence for limited SN infiltration of CCR2 monocytes, which was preceded by transient astrocytic CCL2 induction in MPTP mice, but a prolonged CCL2 induction in MPTP monkeys, suggesting a potential relevance for human PD. While CCR2-gene deletion did not affect loss of DA neurons, astrocytic CCL2 overexpression increased MPTP induced DA neural loss, revealing the principally neurotoxic nature of infiltrating CCR2 monocytes in a PD-like environment. Unexpectedly, we also found that the known increased DA loss in CX3CR1-KO mice was mediated indirectly via over-induction of the CCL2-CCR2 axis. Combined, our results suggest a potential deleterious role of the CCL2-CCR2 axis in actual human PD
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Millar, Jenna. "Anatomical and transcriptomic characterization of the canola (Brassica napus) maternal seed subregions during ovule and seed development." Plant Science, 2015. http://hdl.handle.net/1993/31776.
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Canola (Brassica napus) contributes $19.3 billion dollars to the Canadian economy each year as a result of its oil- and protein-rich seeds. These economically important seed products are produced in highest concentration in the embryo. Embryo development is supported nutritionally and structurally by the maternal subregions, which include the inner (ISC) and outer distal seed coat (OSC), the chalazal seed coat (CZSC), and the chalazal proliferating tissue (CPT). Research on the maternal seed subregions is limited to the SC as a result of its accessibility; the embedded CZSC and CPT subregions have yet to be characterized in canola. Using light and transmission electron microscopy, I found the CZSC and CPT to be anatomically distinct and experience profound changes throughout seed development. To understand these changes at the RNA level, laser microdissection and RNA sequencing were used to profile these subregions spatially and temporally from the ovule to mature green stage of seed development. Employing vigorous bioinformatics analyses, I found that the maternal subregions are transcriptomically distinct and possess unique RNA populations. From here I began to elucidate the biological processes operating within the maternal subregions. As a whole, the maternal subregions appear to have a critical role in transporting nutrients to the filial subregions as well as in coping with oxidative stress produced during these energy-rich processes. Additionally, using CanEnrich, I was able to generate predictive transcriptional circuits regulating the biological processes occurring within the maternal seed. This research has produced the most comprehensive dataset on the canola seed to date and will provide a valuable resource for research on seed development as well as seed improvement.October 2016
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Gatto, Kaleb Pretto 1987. "Análise dos cromossomos sexuais de Pseudis tocantins (Anura, Hylidae)." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317686.
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Orientadores: Luciana Bolsoni Lourenço, Carmen Silvia BusinDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Mestrado
Biologia Celular
Mestre em Biologia Celular e Estrutural
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Devouassoux, Shisheboran Mojgan. "Analyse génétique des tumeurs germinales : recherche d'instabilité génétique et caractéristiques génotypiques." Lyon 1, 2001. http://www.theses.fr/2001LYO1T132.
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Clatot, Florian. "Expression et valeur pronostique du couple CXCL 12/CXCR 4 dans les carcinomes épidermoïdes de la sphère ORL chez l'homme." Rouen, 2014. http://www.theses.fr/2014ROUES041.
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La chémokine CXCL 12 et ses récepteurs CXCR 4 et CXCR 7 sont impliqués dans de nombreux modèles de dissémination métastatique en cancérologie. Nous avons évalué l’expression et la valeur pronostique de l’axe CXCL 12/CXCR 4 à partir d’une cohorte de patients traités à visée curative pour un carcinome épidermoïde ORL. Sur une série de 71 patients, nous avons pu établir que le taux d’expression intratumoral de CXCL 12 évalué par RT-PCR quantitative était un facteur pronostique majeur et indépendant. L’analyse par qRT-PCR haut débit puis clustering non supervisé de l’expression de 42 gènes (impliqués dans l’angiogénèse, l’inflammation, le métabolisme, l’hypoxie et les intéractions à la matrice) pour 61 échantillons, nous a permis d’identifier 2 groupes de gènes présentant une probabilité d’évolution métastatique très différente. Puis, un score basé sur l’expression de 9 gènes et capable de prédire à 90% à quel groupe un échantillon donné appartient a été développé. Au niveau tissulaire, l’analyse après microdissection de 23 échantillons a confirmé que le stroma péri-tumoral produit la majorité de CXCL 12, et a permis de montrer de façon originale que CXCR 4 est aussi exprimé au niveau du stroma et non de la tumeur. CXCR 7 ne présente pas d’expression différentielle entre tumeur et stroma. Enfin, l’hyperméthylation du promoteur de CXCL 12, évaluée par pyroséquençage, n’expliquait l’hypoexpression de CXCL 12 que pour 20% des tumeurs. Dans leur ensemble, ces résultats suggèrent une fonction de gène suppresseur de tumeur du gène CXCL 12 dans les carcijnomes épidermoïdes de la sphère ORL et justifient une évaluation prospective de sa potentielle utilisation à visée pronostiqueThe CXCL 12 chemokine and its receptors CXCR 4 and CXCR 7 are involved in many models of metastatic spread in cancer. Based on a cohort of patients treated for a curative intent for a head and neck cancer, we assessed the expression and the prognostic value of the CXCL 12/CXCR 4 axis in this setting. In our cohort of 71 patients we showed that the intratumoral level of expression of CXCL 12 assessed by quantitative PCR was a strong and independent prognostic factor. Using a high-throughput qRT-PCR, we performed an unsupervised clustering analysis based on the expression of 42 genes (involved in hypoxia, metabolism, matrix interactions, inflammation and angiogenesis) for 61 patients. We identified 2 groups of genes that were related to metastatic evolution. Next, we established a 9-gene model that was capable of classifying the samples into the 2 clusters with 90% accuracy. At the tissue level, the analysis of 23 samples after microdissection confirmed that the stroma is the main provider of CXCL 12. It also showed in a pioneering way that CXCR 4 was mainly produced by the stroma instead of the tumor. CXCR 7 expression did not differ between the 2 compartments. Finally, the methylation of the CXCL 12 promoter assessed by pyrosequencing could only explain the low intra-tumoral expression of this gene in 20% of cases. Taken together, these results suggest that CXCL 12 may be a tumor suppressor gene in head and neck cancers. A prospective evaluation of the CXCL 12 prognostic value is needed
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Crawford, Jessica D. "Cellular-based Brain Pathology in the Anterior Cingulate Cortex of Males with Autism Spectrum Disorder." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etd/2443.
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Autism spectrum disorder (ASD) now affects 1 in 68 children in the United States. Disorders within this spectrum share hallmark deficits in verbal and nonverbal communication, repetitive behavior, and social interaction. The cause of ASD is still unknown. Even though hundreds of genetic abnormalities have been identified in ASD, these markers account for less than 1% of all ASD cases. Researchers continue to search for pathological markers common to all or most cases of ASD. The research presented in this dissertation used a novel combination of state-of-the-art methods to investigate brain pathology in ASD. Postmortem anterior cingulate cortex (ACC) from ASD and typically developing brain donors was obtained from 2 national brain banks. The ACC was chosen for study because of its documented role in influencing behaviors characteristically disrupted in ASD. An initial study revealed elevated glial fibrillary acidic protein (GFAP) in ACC white matter from ASD brain donors compared to typically developing control donors. Laser capture microdissection was then employed to isolate specific cell populations from the ACC from ASD and control brain donors. Captured cells were used to interrogate potential gene expression abnormalities that may underlie biological mechanisms that contribute behavioral abnormalities of ASD. The expression of 4 genes associated with synaptic function, NTRK2, GRM8, SLC1A1, and GRIP1, were found to be significantly lower in ACC pyramidal neurons from ASD donors when compared to control donors. These expression abnormalities were not observed in ACC glia. Given robust evidence of neuronal and glial pathology in the ACC in ASD, a novel method for whole transcriptome analysis in single cell populations was developed to permit an unbiased analysis of brain cellular pathology in ASD. A list of genes that were differentially expressed, comparing ASD to control donors, was produced for both white matter and pyramidal neuron samples. By examining the ASD brain at the level of its most basic component, the cell, methods were developed that should allow future research to identify common cellular-based pathology of the ASD brain. Such research will increase the likelihood of future development of novel pharmacotherapy for ASD patients.
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Signour, Thomas. "Extraction de signatures de bactéries par microspectroscopie Raman et chimiométrie : application à l’étude de la composition biologique des aérosols dans l’environnement." Electronic Thesis or Diss., Lille 1, 2017. http://www.theses.fr/2017LIL10152.
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Depuis plusieurs années, l’étude et le contrôle de la qualité de l’air sont au cœur de toutes les préoccupations. En 2012, la DGA (Direction Générale de l’Armement) met en place le programme ASTRID (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense) accompagnant les travaux de recherche duale civile et militaire. Cette thèse s’inscrit dans cette démarche et propose d’étudier la faisabilité du concept de détection et d’identification rapides des microorganismes présents dans un échantillon d’air par microspectroscopie Raman, avec une résolution au niveau de l’espèce. Pour cela, nous construisons un modèle chimiométrique de classification des microorganismes représentatifs de la biodiversité naturelle en acquérant, sans a priori, d’une part les spectres Raman de ces microorganismes après biocollecte et étalement sur la lame d’un microspectromètre Raman, et d’autre part les séquences génomiques codant les ARN 16S de ces mêmes microorganismes.Les travaux de recherche présentés dans cette thèse présentent donc les différentes études mises en œuvre lors du développement d’un nouveau protocole permettant l’analyse des bactéries issues d’aérosols naturels environnementaux. Nous démontrons la nécessité d’optimiser l’acquisition des spectres Raman sur les bactéries et le traitement statistique des données spectrales permettant le développement de modèles de classification présentant des taux de reconnaissance élevésFor several years, the study and the control of the quality of the air are at the heart of all the concerns. In 2012, the DGA (Direction Générale de l’Armement) employs the ASTRID program (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense), to accompany the dual civil and military research work. This thesis is part of this approach and proposes the feasibility study, by Raman microspectroscopy, of the concept of rapid detection and identification of microorganisms present in an air sample, with a resolution at the species level. For this, we construct a chemometric model for the classification of micro-organisms representative of the natural biodiversity. Such a model is built by acquiring, without a priori i) the Raman spectra of these microorganisms after biocollection; and ii) the genomic sequences encoding the 16S RNAs of these same microorganisms. The research presented in this thesis therefore presents the different studies carried out during the development of a new protocol allowing the analysis of bacteria from natural environmental aerosols. We demonstrate the need to optimize the acquisition of Raman spectra on bacteria and the statistical processing of spectral data that allows the development of classification models with high recognition rates