Dissertations / Theses: 'Laser capture microdissection'

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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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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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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 élevés
For 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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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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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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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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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 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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Biochemistry
Ph.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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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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Molina, Mariana. "Abordagem para análise proteômica de neurônios contendo neuromelanina na substância negra, isolados por microdissecção a laser." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-22012016-104704/.

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Atualmente observa-se que a proporção de pessoas com 60 anos ou mais está crescendo mais rápido do que a de outras faixas etárias. Um dos resultados desta transição epidemiológica é o aumento das doenças cujo fator de risco é o envelhecimento, entre elas, a doença de Parkinson. Embora muitas regiões exibam os sinais neuropatológicos da doença de Parkinson, a degeneração dos neurônios, contendo neuromelanina, da substância negra é considerada como sendo uma característica importante, representando o critério cardinal para o diagnóstico. No entanto, ainda não está claro por que certas regiões do cérebro, como a substância negra, são vulneráveis em algumas doenças neurodegenerativas e alguns neurônios vizinhos, às vezes morfologicamente indistinguíveis, permanecem preservados. Análises moleculares de populações de neurônios podem conduzir a uma melhor compreensão sobre a fisiologia dos mesmos, bem como os mecanismos envolvidos nos processos de doença. Na era pós genômica, realizar análises proteômicas são de grande interesse científico, pois permitem avanços no conhecimento dos processos biológicos. A técnica de microdissecção e captura a laser tem sido uma ferramenta importante e cada vez mais utilizada para aquisição de populações puras de células a partir de secções histológicas, evitando que áreas não pertencentes ao tecido alvo sejam dissecadas. A união destes métodos pode contribuir de maneira relevante para o entendimento fisiopatológico dos neurônios contendo neuromelanina da substância negra. No entanto, para que a microdissecção e captura a laser e as análises proteômicas sejam eficazes, é imprescindível a aplicação de um protocolo bem estruturado. Dentro desse contexto, o presente trabalho tem como objetivo criar um protocolo de microdissecção a laser de neurônios contendo neuromelanina em indivíduos cognitivamente normais, para subsequente análise proteômica. Os casos utilizados neste estudo são provenientes do Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral. Para o desenvolvimento da nossa proposta, contamos com a colaboração do Centro de Proteômica Médica da Universidade de Bochum, Alemanha. O protocolo foi desenvolvido baseado em outros previamente descritos na literatura e otimizado de acordo com objetivos pretendidos. Analisamos o plano anatômico de amostragem do tecido, o método de congelamento, a espessura do corte para a microdissecção, a solução utilizada para a coleta do tecido durante a microdissecção e o método de digestão proteolítica para posteriores análises proteômicas. Através de ensaios comparativos, alcançamos os resultados desejados e os mesmos foram validados através de análises por espectrometria de massas. Consequentemente, também fomos capazes de reconhecer fatores técnicos que possivelmente impossibilitariam um efetivo estudo do proteoma
Currently the worldwide proportion of people aged 60 years and over is growing faster than any other age group. This strikingly epidemiological transition results in an increase of aging related diseases, including Parkinson\'s disease (PD). Although many brain areas exhibit the neuropathological signs of Parkinson\'s disease, the degeneration of neuromelanin containing cells in the substantia nigra is considered a hallmark feature, representing cardinal diagnostic criteria for PD. However, why certain brain regions -- such as the substantia nigra -- are vulnerable in some neurodegenerative diseases, while some neighboring morphologically indistinguishable neurons remain preserved, is still unclear. Molecular analysis of specific neuronal populations can lead us to a better understanding about the physiological role played by these neurons and mechanisms involved in disease\'s processes. In a post-genomic era, proteomic analyses are of great scientific interest since they allow a better understanding of the biological processes. The laser capture microdissection technique has also became an important tool in biological research, being increasingly used for acquisition of pure populations of cells from histological sections, preventing the dissection of areas outside the target tissue. The combination of these methods can significantly contribute to understand the pathophysiological role of the containing neuromelanin neurons of the substantia nigra. However, for an effective application of both techniques, laser capture microdissection and proteomic analysis, it is essential the application of an efficient protocol. In this context, this study aims to establish a protocol for laser microdissection of containing neuromelanin neurons in cognitively normal individuals for subsequent proteomic analyses. We selected cases from the Brain Bank of the Brazilian Aging Brain Study. A collaboration with the Medical Proteome Center, University of Bochum, Germany took part during the development of our proposal. Our protocol was developed based on previous published protocols and optimized according the intended aims. We analyzed anatomical planes for neuronal collection, freezing methods, thickness of tissue for microdissection sections, solution for tissue collection during laser microdissection and the proteolytic digestion methods. Through our comparative tests, we have achieved the desired results and validated them by mass spectrometry analyses. Consequently, we were also able to exclude technical factors that could possibly preclude one effective proteome analysis

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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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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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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.

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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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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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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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Macedo, Carolina Carneiro Soares 1989. "Análise da expressão diferencial de proteínas em ilhas neoplásicas grandes e pequenas por espectrometria de massas baseada em proteômica e sua relação com o prognóstico." [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/289257.

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Orientadores: Adriana Franco Paes Leme, Ricardo Della Coletta
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-28T02:02:25Z (GMT). No. of bitstreams: 1 Macedo_CarolinaCarneiroSoares_M.pdf: 3754068 bytes, checksum: 36ddb6796cb6690a37ce3e4e9447009c (MD5) Previous issue date: 2015
Resumo: O carcinoma espinocelular oral (CEC) é o tipo de neoplasia maligna mais comum em cabeça e pescoço, com alta prevalência e morbidade. O tratamento é baseado em sistemas de classificação pouco precisos e o prognóstico é ruim em muitos casos. Diferentes padrões histológicos já foram descritos na tentativa de melhor compreender o curso da doença, predizer prognóstico e auxiliar no tratamento. As diferentes áreas do tumor apresentam características morfológicas e moleculares distintas resultando em comportamentos clínicos específicos, e estudos recentes apontam a região de invasão tumoral (do inglês invasive front tumor) como área de interesse para análises de perfil molecular e identificação de possíveis marcadores de prognóstico. O padrão de invasão neoplásico tem relação com a agressividade tumoral e a presença de ilhas no fronte invasivo já foi descrita como pior padrão. Assim, o objetivo desta dissertação foi comparar a composição diferencial de proteínas totais de ilhas neoplásicas grandes e pequenas do fronte e do interior do tumor, e correlacionar essas proteínas com o prognóstico. A proteômica foi associada à microdissecção a laser (ML), consideradas juntas como ferramentas de alta robustez para identificação de proteínas em tecidos neoplásicos em regiões específicas. Vinte peças cirúrgicas de CEC oral de língua fixadas em parafina foram submetidas a ML para obtenção das amostras compostas pelas seguintes regiões do tecido: 1) ilhas neoplásicas grandes da região frontal, 2) ilhas neoplásicas pequenas da região frontal, 3) ilhas neoplásicas grandes do interior do tumor e 4) ilhas neoplásicas pequenas do interior do tumor, seguida pela extração de proteínas e análise por cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS). A anotação funcional das proteínas e a correlação aos dados clínico-patológicos dos pacientes foram realizadas. Foram identificadas 1906 proteínas totais, sendo 1480 proteínas comuns entre as quatros regiões estudadas. Duas proteínas foram exclusivas nas ilhas grandes do fronte e sete nas ilhas grandes do interior. Oitenta e cinco proteínas foram diferencialmente expressas entre a região do fronte e o interior tumoral, e destas, 57 foram relacionadas a dados clínico-patológicos importantes para o prognóstico. Os processos biológicos, como desenvolvimento da epiderme, adesão celular, apoptose, ciclo celular, degradação da matriz extracelular e expressão gênica, evidenciados entre as proteínas diferencialmente expressas confirmam as mudanças moleculares associadas à progressão neoplásica. A combinação de ML, MS e bioinformática foi capaz de identificar um painel de proteínas que podem auxiliar a desvendar o curso do CEC oral, predizendo agressividade e prognóstico. Em acréscimo, essa abordagem pode ajudar ainda na compreensão das diferenças e dos mecanismos de sinalização entre diferentes áreas do tecido
Abstract: Oral squamous cell carcinoma (SCC) is the most common type of malignant tumor in head and neck, with high prevalence and morbidity. Treatment is based on inaccurate classification systems and the prognosis is poor in many cases. Different histological patterns have been described in an attempt to better understand the course of the disease, predict prognosis and assist in treatment. Different areas of the tumor have different morphological and molecular characteristics resulting in specific clinical behaviors, and recent studies point to the region of tumor invasion as an area of interest for molecular profile analysis and identification of possible prognostic markers. The pattern of neoplastic invasion is related to tumor aggressiveness and the presence of islands in the invasive front has been described as worst invasion pattern. The objective of this work was to compare the protein differential expression of large and small islands neoplastic from the front and the inner tumor, and to correlate these proteins with prognosis. Proteomics was associated with laser microdissection (ML), and together they are considered robustness tools to identify proteins in neoplastic tissues in specific regions of interest. Twenty surgical specimens of oral tongue SCC fixed in paraffin were subjected to ML to obtain sample composed by the following regions of tissue: 1) large neoplastic frontal islands, 2) small neoplastic islands of the frontal region, 3) large neoplastic islands inside the tumor and 4) small islands within the neoplastic tumor, followed by extraction and analysis of proteins by liquid chromatography coupled to mass spectrometry (LC-MS/MS). The functional annotation of proteins and correlation with clinicopathological data from patients were performed. A total of 1906 proteins were identified, with 1480 common proteins between the four regions studied. Two proteins were exclusives in the large islands of the forehead and seven in large islands in the interior. Eighty-five proteins were differentially expressed between the front region and inner tumor, and of these, 57 were related to clinical and pathological data. The biological processes such as development of the epidermis, cell adhesion, apoptosis, cell cycle, disassembly of the extracellular matrix and gene expression, evidenced among the differentially expressed proteins confirmed the molecular changes associated with neoplastic progression. The combination of ML, MS, and bioinformatics was able to identify a panel of proteins that may help to unravel the course of oral SCC, predicting aggressiveness and prognosis. In addition, this approach may also help in understanding the differences and signaling mechanisms between different areas of the tumor tissue
Mestrado
Patologia
Mestra em Estomatopatologia

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Li, Su-Chen. "Small Intestinal Neuroendocrine Tumor Analyses : Somatostatin Analog Effects and MicroRNA Profiling." Doctoral thesis, Uppsala universitet, Endokrin Onkologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-233207.

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Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.

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Kollbeck, Sophie L. G. [Verfasser], Jens [Akademischer Betreuer] Habermann, and Frank [Gutachter] Köster. "Vergleichende Proteomanalyse zwischen Colitis Ulcerosa assoziierten und sporadischen kolorektalen Karzinomen nach Laser Capture Mikrodissektion : Comparative proteomic analysis of Ulcerative Colitis associated and Sporadic Colorectal Cancer after Laser Capture Microdissection / Sophie L. G. Kollbeck ; Gutachter: Frank Köster ; Akademischer Betreuer: Jens Habermann." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2020. http://d-nb.info/1207574155/34.

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24

Figueiredo, Joana Maria Serra de Oliveira Duarte. "The role of microRNAs in amyotrophic lateral sclerosis." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/7991.

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Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
MicroRNAs (miRNAs) are emerging as a primary mediator of gene regulation in many different cell types. There is increasing evidence that specific subsets of miRNA play a prominent role in the nervous system, both in development and in specific neurodegenerative diseases. This study aims to elucidate the role of microRNA in selective motor neuron death that is the hallmark of amyotrophic Lateral sclerosis (ALS). Pre-symptomatic time-point was chosen since the levels of miRNAs are highly likely to be altered as a secondary consequence of cell injury and death in ALS. Laser capture microdissection (LCM) was used to study miRNA profiles in motor neurons of spinal cord tissue from SOD1G93A mice, the best characterized model of ALS. In preliminary work, using miRNA specific chips we have identified 2 miRNAs which are dramatically upregulated before disease onset. In this study, high RNA quality was achieved from laser captured cells, which consist in a major advance towards obtaining meaningful results of these miRNAs expression in downstream applications. Despite LCM technology has become increasingly sophisticated; rapidly obtaining enough amount of starting material for downstream applications is still extremely challenging. The combination of this optimized technique with microarrays, followed by RT-qPCR may provide insights into potential contribution of microRNAs to progression of neurodegeneration of motor neurons in ALS.

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Majer, Anna. "Temporal deregulation of genes and microRNAs in neurons during prion-induced neurodegeneration." Virology Journal, 2010. http://hdl.handle.net/1993/30718.

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Prion diseases are fatal and incurable neurodegenerative diseases that share many pathological similarities to other neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. One of the earliest pathological signs commonly detected in all of these diseases is the dysfunction followed by loss of neuronal synapses, spines and eventually dendrites that collectively contribute to disruption of normal brain function. These pathologies tend to progressively accumulate within the brain tissue such that extensive damage typically precedes clinical symptom manifestation and ultimate death of neurons. Clearly, understanding the molecular processes responsible for these pathologies could uncover critical pathway(s) that are responsible for propagating this brain damage and could therefore be exploited for therapy development. However, molecular mechanisms implicated in this early pathology remain unidentified. To address this gap in knowledge, this thesis describes a transcriptional approach coupled with specific isolation of neuronal-enriched tissue which was used to help delineate cellular pathways involved in prion-induced neurodegeneration. Profiling cell bodies of CA1 hippocampal neurons known to be affected during early prion disease revealed temporal alteration in both gene and microRNA (gene regulators) expression throughout disease. On a gene expression level, changes in transcript expression during preclinical disease were reminiscent of an activity-dependent neuroprotective gene signature previously described in the literature. These neuroprotective genes were induced during preclinical disease, diminished as disease progressed and were abolished at clinical disease. In support of this process, upregulation of the phosphorylated form of the neuroprotective transcription factor CREB was detected during preclinical disease in these neurons. Furthermore, several genes known to be induced by CREB were also upregulated at preclinical disease in prion-infected mice. Interestingly, expression of numerous deregulated microRNAs paralleled the neuroprotective gene signature of which several are known to remodel neuronal spines and dendrites. To determine whether other preclinically induced microRNAs were also capable of remodeling neuronal structures, gain-of-function studies were performed in primary mouse hippocampal neurons for the uncharacterized miR-26a-5p. Neurons over-expressing miR-26a-5p had enhanced spine density and dendrite arborization, similar to other preclinically-induced microRNAs. Together, these data suggests that CA1 hippocampal neurons induce a neuroprotective transcriptional signature that is evident early in the course of disease within CA1 hippocampal neurons and is abolished by clinical disease. Reestablishment of key molecules that can induce this neuroprotective signature at a time when these genes begin to dissipate could prolong prion disease onset and delay clinical symptom manifestation.
October 2015

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Almeida, Marcia Rodrigues de. "Bases moleuculares da recalcitrância ao enraizamento adventício em Eucalyptus globulus Labill." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2015. http://hdl.handle.net/10183/131932.

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O eucalipto é uma das espécies arbóreas mais plantadas no mundo atualmente, principalmente devido ao seu uso como matéria prima para as indústrias de celulose, papel e madeireira. Eucalyptus globulus e seus híbridos possuem baixos teores de lignina e despertam grande interesse da indústria, já que essa característica facilita e diminui o custo do processo de extração da celulose. Entretanto, essa espécie é recalcitrante ao enraizamento adventício, o que dificulta a propagação vegetativa de suas mudas. Com o objetivo de melhor entender os mecanismos moleculares envolvidos na recalcitrância ao enraizamento em E. globulus, o presente estudo envolveu análises de parâmetros morfológicos, anatômicos e moleculares durante a rizogênese adventícia nesta espécie. A exposição à auxina exógena reverteu o fenótipo recalcitrante em E. globulus, aumentando significativamente a porcentagem de enraizamento. O câmbio vascular foi identificado como uma região de acúmulo de auxina e local de origem das raízes adventícias. Através de análises da expressão gênica em células do câmbio, observou-se que TOPLESS e IAA12, repressores da sinalização de auxina, e ARR1, envolvido na rota de sinalização de citocininas, parecem atuar como reguladores negativos do enraizamento adventício. A alta expressão destes genes em plantas controle foi significativamente diminuída com aplicação de auxina exógena. Comparativamente, em espécie de fácil enraizamento, E. grandis, a expressão destes genes se manteve em níveis mais baixos em ambas as condições de tratamento, e a concentração de ácido indol-3-acético endógeno em plantas controle mostrou-se mais elevada. Análises do padrão proteico durante o enraizamento em plantas de E. globulus tratadas ou não com auxina exógena identificaram proteínas envolvidas em diversos processos biológicos, principalmente estresse oxidativo e metabolismo energético. Diferenças interessantes foram identificadas ao comparar as diferentes condições e fases do enraizamento. Várias proteínas foram claramente relacionadas com o respectivo fenótipo apresentado pela planta em cada situação, principalmente considerando plantas controle. Os resultados aqui apresentados representam avanços relevantes no conhecimento sobre a rizogênese adventícia em plantas lenhosas, podendo ser utilizados como ferramentas no desenho de estratégias visando melhorar o enraizamento em genótipos recalcitrantes de valor para a indústria.
Eucalyptus is one of the most planted tree species in the world today, mainly due to its use as raw material for paper, cellulose and wood industries. Eucalyptus globulus and its hybrids have low lignin contents and are of great interest to industry, as this feature facilitates and reduces costs of the cellulose extraction process. However, this species is recalcitrant to adventitious rooting, making vegetative propagation by cuttings difficult. Aiming at a better understanding of the molecular mechanisms involved in rooting recalcitrance in E. globulus, this study analyzed changes in morphological, anatomical and molecular patterns during adventitious rooting in this species. Exogenous auxin exposure reversed the recalcitrant phenotype in E. globulus, significantly increasing rooting percentage. The vascular cambium was identified as a region of auxin accumulation and also the site from where adventitious roots originated. Gene expression analysis in cambium cells indicated that TOPLESS and IAA12, auxin signaling repressors, and ARR1, involved in cytokinin signaling pathway, appear to act as negative regulators of adventitious rooting. The high expression of those genes in control plants was significantly decreased by exogenous auxin treatment. Comparatively, in an easy-to-root species, E. grandis, the expression of these genes was significantlylower in both treatment conditions, and the concentration of endogenous indole- 3-acetic acid in control plants was higher. Analysis of the protein pattern during rooting in E. globulus plants treated or not with exogenous auxin allowed the identification of proteins involved in diverse biological processes, mainly oxidative stress and energy metabolism. Interesting differences were identified when comparing different rooting conditions or phases. Several proteins were clearly associated with the respective plant phenotype in each situation, particularly considering control plants. These results represent relevant advances in the knowledge about adventitious rooting in woody plants and can be used as tools in the design of strategies aiming at improving adventitious rooting in recalcitrant genotypes of industrial value.

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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-pathogène
Iron 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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Solimena, Michele, Anke M. Schulte, Lorella Marselli, Florian Ehehalt, Daniela Richter, Manuela Kleeberg, Hassan Mziaut, et al. "Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes." Springer, 2017. https://tud.qucosa.de/id/qucosa%3A33350.

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Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP).

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Dreja, Tanja S. "Microarray-basierte Expressionsanalysen des weißen Fettgewebes der NZO-Maus sowie der Langerhansschen Inseln der NZL-Maus : zwei Modelle für das metabolische Syndrom." Phd thesis, Universität Potsdam, 2009. http://opus.kobv.de/ubp/volltexte/2009/3237/.

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Übergewicht und Adipositas führen zu Insulinresistenz und erhöhen deutlich das Risiko für die Entwicklung von Typ-2-Diabetes und kardiovaskulären Erkrankungen. Sowohl Adipositas als auch die Suszeptibilität gegenüber Diabetes sind zu einem erheblichen Teil genetisch determiniert. Die relevanten Risikogene, deren Interaktion mit der Umwelt, insbesondere mit Bestandteilen der Nahrung, und die Pathomechanismen, die zur Insulinresistenz und Diabetes führen, sind nicht vollständig aufgeklärt. In der vorliegenden Arbeit sollte durch Genexpressionsanalysen des weißen Fettgewebes (WAT) und der Langerhansschen Inseln die Entstehung und Progression von Adipositas und Typ-2-Diabetes untersucht werden, um relevante Pathomechanismen und neue Kandidatengene zu identifizieren. Zu diesem Zweck wurden Diät-Interventionsstudien mit NZO- und verwandten NZL-Mäusen, zwei polygenen Mausmodellen für das humane metabolische Syndrom, durchgeführt. Eine kohlenhydrathaltige Hochfett-Diät (HF: 14,6 % Fettanteil) führte in beiden Mausmodellen zu früher Adipositas, Insulinresistenz und Typ 2 Diabetes. Eine fettreduzierte Standarddiät (SD: 3,3 % Fettanteil), welche die Entstehung von Adipositas und Diabetes stark verzögert, sowie eine diabetesprotektive kohlenhydratfreie Hochfett-Diät (CHF: 30,2 % Fettanteil) dienten als Kontrolldiäten. Mit Hilfe der Microarray-Technologie wurden genomweite Expressionsprofile des WAT erstellt. Pankreatische Inseln wurden durch laserbasierte Mikropräparation (Laser Capture Microdissection; LCM) isoliert und ebenfalls hinsichtlich ihres Expressionsprofils analysiert. Differenziell exprimierte Gene wurden durch Real-Time-PCR validiert. Im WAT der NZO-Maus bewirkte die HF-Diät eine reduzierte Expression nukleärer Gene der oxidativen Phosphorylierung und von lipogenen Enzymen. Dies deutet auf eine inadäquate Fettspeicherung und -verwertung in diesen Tieren hin. Die Reduktion in der Fettspeicherung und -oxidation ist spezifisch für das adipöse NZO-Modell und konnte bei der schlanken SJL Maus nicht beobachtet werden, was auf eine mögliche Beteiligung an der Entstehung der Insulinresistenz hinweist. Zusätzlich wurde bestätigt, dass die Expansion des Fettgewebes bei der adipösen NZO-Maus eine zeitlich verzögerte Infiltration von Makrophagen in das WAT und dort eine lokale Immunantwort auslöst. Darüber hinaus wurde die Methode der LCM etabliert und zur Gewinnung hochangereicherter RNA aus den Langerhansschen Inseln eingesetzt. In erstmalig durchgeführten genomweiten Expressionsanalysen wurde zu einem frühen Zeitpunkt in der Diabetesentwicklung der Einfluss einer diabetogenen HF-Diät und einer diabetesprotektiven CHF-Diät auf das Expressionsprofil von pankreatischen Inselzellen verglichen. Im Gegensatz zum WAT bewirkt die diabetogene HF-Diät in Inselzellen einerseits, eine erhöhte Expression von nukleären Genen für die oxidative Phosphorylierung und andererseits von Genen, die mit Zellproliferation assoziiert sind. Zudem wurden 37 bereits annotierte Gene identifiziert, deren differenzielle Expression mit der Diabetesentwicklung korreliert. Das Peptidhormon Cholecystokinin (Cck, 11,8-fach erhöht durch die HF) stellt eines der am stärksten herauf regulierten Gene dar. Die hohe Anreicherung der Cck-mRNA in Inselzellen deutet auf eine bisher unbekannte Funktion des Hormons in der Regulation der Inselzellproliferation hin. Der Transkriptionsfaktor Mlxipl (ChREBP; 3,8-fach erniedrigt durch die HF) stellt in Langerhansschen Inseln eines der am stärksten herunter regulierten Gene dar. Ferner wurde ChREBP, dessen Funktion als glucoseregulierter Transkriptionsfaktor für lipogene Enzyme bislang in der Leber, aber nicht in Inselzellen nachgewiesen werden konnte, erstmals immunhistochemisch in Inselzellen detektiert. Dies deutet auf eine neue, bisher unbekannte regulatorische Funktion von ChREBP im Glucosesensor-Mechanismus der Inselzellen hin. Eine durchgeführte Korrelation der mit der Diabetesentwicklung assoziierten, differenziell exprimierten Inselzellgene mit Genvarianten aus humanen genomweiten Assoziationsstudien für Typ-2-Diabetes (WTCCC, Broad-DGI-T2D-Studie) ermöglichte die Identifizierung von 24 neuartigen Diabetes-Kandidatengenen. Die Ergebnisse der erstmals am polygenen NZO-Mausmodell durchgeführten genomweiten Expressionsuntersuchungen bestätigen bisherige Befunde aus Mausmodellen für Adipositas und Diabetes (z.B. ob/ob- und db/db-Mäuse), zeigen in einigen Fällen aber auch Unterschiede auf. Insbesondere in der oxidativen Phosphorylierung könnten die Ergebnisse relevant sein für das Verständnis der Pathogenese des polygen-bedingten humanen metabolischen Syndroms.
Overweight and obesity cause insulin resistance and increase the risk of developing type 2 diabetes and cardiovascular diseases. Both, obesity and susceptibility to diabetes, are to a major part genetically predisposed. The relevant genes, their interaction with the environment – especially with food components – and the pathomechanisms causing insulin resistance and diabetes are not fully known yet. In the present study the development and progression of obesity and type 2 diabetes should be investigated by the means of gene expression analyses of the white adipose tissue (WAT) and the islets of Langerhans to identify underlying pathomechanisms and new causative candidate genes. For this purpose diet intervention studies on NZO- and related NZL-mice – two polygenic mouse models for the human metabolic syndrome – were performed. A carbohydrate containing high fat-diet (HF: 14.6 % fat) caused early obesity, insulin resistance and type 2 diabetes in both mouse models. A fat reduced standard chow (SD: 3.3 % fat) which strongly delayed the onset of obesity and diabetes, and a diabetes protective carbohydrate free high fat-diet (CHF: 30.2 % fat) served as control diets. Using microarray technology genome wide expression profiles of the WAT were generated. Pancreatic islets were isolated by the means of laser capture microdissection (LCM) and expression profiles of them were created, too. Differentially expressed genes were validated by quantitative real time PCR. The HF-diet reduced the expression of nuclear genes of the oxidative phosphorylation and lipogenic enzymes in the WAT of the NZO-mouse. This suggests an inadequate storage and utilization of fat in these animals. This is specific for the obese NZO-model and wasn’t observed for the lean SJL-mouse, indicating a role in the development of insulin resistance. Additionally, there was proof that the enlargement of the WAT triggers a retarded infiltration of macrophages into the WAT and there a local immune response. Moreover, the LCM technique was established and used for the isolation of highly enriched RNA from islets of Langerhans. For the first time the influence of carbohydrates in a high fat-diet on the expression profile of pancreatic islets was investigated by the use of genome wide expression analyses at an early time point at the onset of diabetes. Contrary to the WAT the diabetogenic HF-diet in islets cells increased the expression of both nuclear genes coding for the oxidative phosphorylation and genes associated with cell proliferation. Furthermore 37 already annotated genes correlated with diabetes progression were identified. The peptide hormone cholecystokinin (Cck: 11.8-fold enriched by the HF-diet) is one of the most up-regulated genes. The strong enrichment of Cck-mRNA in islets suggests a previously unknown function of the hormone in the regulation of the islet cell proliferation. The transcription factor ChREBP (Mlxipl: 3.8-fold reduced by the HF-diet) is one of the most down-regulated genes in the islets of Langerhans. Moreover, ChREBP, which has been already identified as a glucose regulated transcription factor for lipogenic enzymes in the liver but not in islets of Langerhans, was detected for the first time in islet cells, using immunohistochemistry. This points to an until now unknown regulatory function of ChREBP in the glucosesensor mechanism of the islet cells. Correlation of the differentially expressed genes associated with diabetes progression with gene variants from human genome wide association studies for type 2 diabetes (WTCCC, Broad-DGI-T2D-study) made the identification of 24 new diabetes candidate genes possible. The results of the genome wide expression analyses, which were done for the first time on a polygenic mouse-model, corroborated previous results for monogenic mouse-models for obesity and diabetes (e.g. ob/ob- and db/db-mice), however also demonstrated differences in some instances. Especially the results concerning the oxidative phosphorylation could be relevant for the comprehension of the pathogenesis of the polygenic human metabolic syndrome.

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Maniero, Carmela. "A functional study on novel genes involved in regulating aldosterone secretion in normal human zona glomerulosa and in aldosterone-producing adenomas." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/270313.

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Primary aldosteronism is the most common secondary cause of hypertension with a prevalence of about 10%. About half of PA cases are caused by aldosterone-producing adenomas (APA). Two APA subtypes, ZG-like and ZF-like APAs, have been described, according to the histological resemblance to normal zona glomerulosa (ZG) and zona fasciculata (ZF), underlying somatic mutations (KCNJ5 commonly found in ZF-like, CACN1AD, ATP1A1, ATP2B3, CTNNB1 in ZG-like APAs), and transcriptome profile. It is unknown if the process of tumorigenesis differs between ZG- and ZF-like APAs. In order to define ZG specific genes, we have compared the transcriptome of APAs and their adjacent adrenal glands by microarray assay. RNA was isolated by laser capture microdissection (LCM) from adjacent ZG, ZF and APAs from 14 patients with Conn’s and 7 patients with phaeocromocytoma. Two top hit genes from the comparison of ZG vs ZF were functionally studied, ANO4 and NEFM. NEFM, encoding neurofilament medium, was the fourth most up-regulated gene in ZG vs ZF, showing 14.8-fold-fold higher expression levels (p=9.16-12) in ZG than ZF. NEFM was also one of the most down-regulated genes in ZF-like vs ZG-like APAs. Immunohistochemistry (IHC) confirmed selective high expression of NEFM in ZG and ZG-like APAs. Silencing NEFM in H295R cells increased aldosterone secretion and cell proliferation. In addition, it increased stimulation and inhibition, respectively, of aldosterone secretion from H295R cells by the dopamine receptor D1R agonist fenoldopam and antagonist SCH23390. IHC showed predominantly intracellular staining for D1R in NEFM-rich ZG-like APAs, but membranous staining in NEFM-poor ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZG-like APAs was lower than in cells from ZF-like APAs. NEFM expression levels directly correlate with KCNJ5 phenotype: KCNJ5 mutations down-regulate NEFM mRNA and protein levels in H295R cells and in primary cells from ZG-like APAs. ANO4,encoding a Ca2+-activated chloride channel family member, was the third most upregulated gene, showing 19.9-fold higher expression levels (p=6.6x10-24) in ZG than ZF. IHC confirmed ZG selectivity of ANO4 protein in the adrenal cortex. The staining was mainly cytoplasmic. Unlike NEFM, there was no difference in expression of ANO4 between ZG- and ZF-like APAs, the levels being mid-way between those of ZF and ZG. Overexpression of ANO4 in H295R cells caused an increase in CYP11B2 and NR4A2 gene expression levels but basal aldosterone secretion was unchanged. In the presence of calcium agonists, ANO4 reduced aldosterone secretion. ANO4 subcellular localisation was confirmed as cytoplasmic by immunofluorescence microscopy of transfected cells. When exposed to calcium ionophores, ANO4 generated small chloride currents as detected by YFP assay. In summary, the comparison of transcriptome of ZG with paired ZF found unexpected up-regulated genes. Most of the highly up regulated genes in human ZG, including NEFM and ANO4, inhibit either basal or stimulated aldosterone secretion, and this may reflect an adaptive response to high salt intake. No clear-cut correspondence was found between transcriptome of APAs and their resembling zone of adrenal cortex. The down-regulation of NEFM following transfection of mutant KCNJ5 suggests that ZF-like properties may be a consequence of mutation, rather than tissue of origin.

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Scheel, Tobias. "Die B-Zell-Antwort im Synovialgewebe von Patienten mit Rheumatoider Arthritis." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/16026.

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Obwohl B-Zellen in der Pathogenese der Rheumatoide Arthritis (RA) eine wichtige Rolle spielen, ist über ihre Aktivierung und Differenzierung im Synovialgewebe (SG) nicht viel bekannt. Ein Merkmal von RA ist das Auftreten von Autoantikörpern (auto-AK). Trotz dessen sind bisher kaum Daten über den Einfluss des SG auf die auto-AK-Produktion und die Frequenz autoreaktiver synovialer B-Zellen bekannt. Diese Arbeit beschäftigt sich mit der Charakterisierung der synovialen B-Zell-Antwort und der Spezifität synovialer B-Lymphozyten. Dazu wurden B- und Plasmazellen (PC) aus dem Synovialgewebe von RA-Patienten mittels Mikrodissektion und Durchflusszytometrie isoliert und ihr Immunglobulin(Ig)-Repertoire bestimmt. Die Analyse der VH-Gene zeigte, dass sowohl naive als auch Gedächtnis-B-Zellen in das SG einwandern können. Ein Vergleich der VDJ-Rearrangements aus B-Zellen und PC belegte, dass hauptsächlich Gedächtnis-B-Zellen Antigen-abhängig aktiviert werden, klonal expandieren und zu PC differenzieren. Dabei können aktivierte B-Zellen ihre Ig-Klasse wechseln. Im Gegensatz dazu wurden nur rudimentäre Anzeichen somatischer Hypermutation nachgewiesen. Um die Spezifität synovialer B-Lymphozyten zu ermitteln, wurden rekombinante AK aus synovialen B-Zellen und PC generiert. Der Polyreaktivitätstest zeigte, dass naive B-Zellen aus dem SG einen hohen Anteil polyreaktiver Zellen besitzen. Im Gegensatz dazu ist die Frequenz von autoreaktiven Gedächtnis-B-Zellen und PC gegenüber naiven B-Zellen erhöht. Daneben konnten auch spezifische AK gegen bakterielle Antigene (insbesondere gegen Parodontitis-auslösende Bakterien) und gegen das Auto-Ag MCV identifiziert werden. Eine Affinitätsmessung des MCV-spezifischen Auto-AK zeigte, dass im SG sezernierte Auto-AK eine sehr hohe Affinität erreichen können. Die hier gewonnenen Daten verdeutlichen, dass B-Lymphozyten entscheidend an der Aufrechterhaltung oder gar Entstehung von RA beteiligt sind
Although B cells have an important impact on the pathogenesis of Rheumatoid arthritis (RA) still surprisingly little is known about their activation and differentiation within the inflamed synovial tissue (ST). A hallmark of RA is the presence of auto-antibodies (auto-Ab). However, still little is known about the frequency of self reactive synovial lymphocytes and it is unclear to which extent the inflamed ST contributes to auto-Ab production. These thesis deals with the characterization of the synovial B cell response and the specificity of synovial B lymphocytes. B and plasma cells (PC) from RA patients were isolated either by Laser Capture Microdissection or by FACS and their immunoglobulin(Ig)-repertoire was determined. The analysis of the VH-genes revealed that both naïve and memory B cells can immigrate the ST. A comparison of VDJ-rearrangements of B cells and PC showed that in ST without ectopic germinal centres mainly memory B cells become activated, expand clonally and differentiate into PC. During this process B cells can switch their Ig-class but do only hypermutate slightly. To determine the specificity of synovial B lymphocytes, recombinant Ab from synovial B cells and PC were generated. The polyreactivity assay showed that particularly naïve B cells were polyreactive. In contrast, the frequency of autoreactive memory B cells and PC was much higher than that of naïve B cells. In addition, Ab specific for bacterial antigens (especially for periodontal bacterias) and for the autoantigen MCV were identified. The affinity measurement of the MCV-specific autoantibody revealed that auto-Ab secreted in the ST can exhibit very high affinities. The data presented here show that B cells seem to play an important role in the maintenance and possibly the development of RA.

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Oliveira, Gabriela Pintar de. "Análise da participação das células neuronais e não-neuronais na Esclerose Lateral Amiotrófica em camundongos transgênicos para SOD1 humana utilizando técnicas de microdissecção a laser e PCR em tempo real." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5138/tde-06062014-101803/.

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A Esclerose Lateral Amiotrófica (ELA) é a doença neurodegenerativa do neurônio motor que acomete indivíduos adultos e promove a perda progressiva das funções motoras. A evolução é rápida (2 a 5 anos) e culmina na morte por complicações e falência dos músculos respiratórios. Descrições recentes sugerem a contribuição de tipos celulares não neuronais, particularmente o astrócito e a microglia, para a morte do neurônio motor. O camundongo transgênico SOD1G93A, que carrega a SOD1 humana mutada, foi utilizado neste trabalho. Estudos comportamentais apontaram alterações motoras importantes no animal transgênico a partir de 90 dias de vida e permitiram selecionar, então, as idades pré-sintomáticas de 40 dias e 80 dias para os estudos moleculares. A análise da expressão gênica nos animais transgênicos e selvagens destas duas idades foi realizada por microarray utilizando-se a plataforma que contém o genoma completo do camundongo e detectou 492 e 1105 transcritos diferencialmente expressos nos animais de 40 e 80 dias, respectivamente. Estes resultados foram validados por PCR quantitativa (qPCR). As análises bioinformáticas dos resultados identificaram 17 e 11 vias moleculares super-representadas nas idades de 40 dias e 80 dias, respectivamente. Destas, as vias endocitose, sinapse glutamatérgica, proteólise mediada por ubiquitina, via de sinalização de quimiocina, fosforilação oxidativa, processamento e apresentação de antígeno e junção oclusiva foram comuns a ambas as idades. Ainda, as vias sinapse glutamatérgica e fagossomo foram sugeridas como potencialmente mais importantes em animais transgênicos de 40 dias e 80 dias, respectivamente. Transcritos específicos foram analisados em amostras enriquecidas de células (astrócito, microglia e neurônio motor) microdissecadas a laser do corno anterior da medula espinal dos animais. Os transcritos Cxcr4, Slc1a2 e Ube2i foram avaliados por qPCR nas amostras enriquecidas de astrócitos dos animais de 40 dias, enquanto que Cxcr4 e Slc17a6 foram avaliados nas amostras de neurônios motores dos animais desta idade. Cxcr4 apresentou expressão diminuída nos astrócitos transgênicos e aumentada nos neurônios destes animais. Slc1a2, Ube2i e Slc17a6 estavam aumentados nos tipos celulares estudados nos animais transgênicos. Tap2 e Tuba1a foram avaliados nas amostras enriquecidas de microglias dos animais de 80 dias e mostraram-se aumentados nas amostras dos transgênicos. Finalmente, Akt1 apresentou expressão diminuída nos neurônios motores microdissecados dos animais transgênicos em comparação aos selvagens. Os resultados sugerem que alterações na sinalização glutamatérgica podem exercer papel essencial em fases pré-sintomáticas mais precoces da doença (40 dias), enquanto que em fases pré-clínicas mais próximas ao aparecimento dos sintomas (80 dias), as respostas mais importantes parecem estar relacionadas à neuroimmunomodulação. Dessa forma, este trabalho aponta para novas perspectivas para o estudo da ELA
Amyotrophic Lateral Sclerosis (ALS) is an adult onset motor neuron neurodegenerative disease that leads to the progressive loss of muscular functions. It is a fast progression disorder (2 to 5 years) culminating in death by respiratory failure. Recent findings suggest that non neuronal cell types, especially astrocytes and microglia, might contribute to the neuronal death. The transgenic mouse SOD1G93A, carring human mutant SOD1, was used in this study. Behavioral studies pointed to the onset of the clinical symptoms occurring at 90 days in the animal model, thus, allowing the selection of the pre-symptomatic ages of 40 and 80 days to the molecular studies. Gene expression analysis of transgenic mice and their non-transgenic littermates at those ages was performed by using a microarray platform containing the whole mouse genome and has detected 492 and 1105 differentially expressed genes at 40 days and 80 days old mice, respectively. These results were validated by quantitative PCR (qPCR). Bioinformatic analysis of the results identified 17 and 11 over-represented molecular pathways at 40 days and 80 days, respectively. Of these, endocytosis, glutamatergic synapse, ubiquitin-mediated proteolysis, chemokine signaling pathway, oxidative phosphorylation, antigen processing and presentation and also tight junction were common to both ages. Furthermore, glutamatergic synapse and fagosome were suggested as potentially more important at 40 and 80 days, respectively. Specific transcripts were analyzed on enriched samples of cells (astrocytes, microglia and motor neuron) obtained by laser microdissection from the ventral horn of mouse spinal cord. The transcripts Cxcr4, Slc1a2 and Ube2i were evaluated by qPCR in enriched samples of astrocytes of the 40 days old mice, and Cxcr4 and Slc17a6 were analyzed in motor neuron samples at this age. Cxcr4 has been found decreased in astrocytes from transgenic mice and increased in the motor neurons of these animals. Slc1a2, Ube2i and Slc17a6 have increased in the cell type in which they were evaluated in the transgenic mice. Tap2 and Tuba1a were evaluated at microglia enriched samples of 80 days old mice and were found to be increased. Finally, Akt1 has decreased in enriched samples of motor neurons from 80 days old mice. The results suggest that glutamatergic signaling might play essential role in early stages of the disease (40 days), while in phases closer to the appearance of the symptoms (80 days), the neuroimmunomodulation takes place. Thus, this study points to new perspectives for ALS study

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Rasiah, Krishan Kumar St Vincent's UNSW. "The identification of novel biomarkers in the development and progression of early prostate cancer." Awarded by:University of New South Wales. St Vincent's, 2006. http://handle.unsw.edu.au/1959.4/24187.

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ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

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Oeschger, Franziska M. "Subplate populations in normal and pathological cortical development." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:686d99bd-36e0-47f2-9680-9874f413d1bb.

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The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. Subplate plays a fundamental role in cortical development. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of corticofugal and thalamic axons. At later stages, these cells are involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. In my thesis, I aimed to further characterize the embryonic murine subplate by establishing a gene expression profile of this population at embryonic day 15.5 (E15.5) using laser capture microdissection combined with microarrays. I found over 250 transcripts with presumed higher expression in the subplate at E15.5. Using quantitative RT-PCR, in situ hybridization and immunohistochemistry, I have confirmed specific expression in the E15.5 subplate for 13 selected genes which have not been previously associated with this compartment. In the reeler mutant, the expression pattern of a majority of these genes was shifted in accordance with the altered position of subplate cells. These genes belong to several functional groups and likely contribute to the maturation and electrophysiological properties of subplate cells and to axonal growth and guidance. The roles of two selected genes - cadherin 10 (Cdh10) and Unc5 homologue c (Unc5c) - were explored in more detail. Preliminary results suggest an involvement of Cdh10 in subplate layer organization while Unc5c could mediate the waiting period of subplate corticothalamic axons in the internal capsule. Finally, I compared the expression of a selection of subplate-specific genes (subplate markers) between mouse and rat and found some surprising species differences. Confirmed subplate markers were used to monitor subplate injury in a rat model of preterm hypoxiaischemia and it appeared that deep cortical layers including subplate showed an increased vulnerability over upper layers. Further characterization of subplate-specific genes will allow us to broaden our understanding of molecular mechanisms underlying subplate properties and functions in normal and pathological development.

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Iyer, Chitra C. "The Role of Muscle and Nerve in Spinal Muscular Atrophy." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1451568269.

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Chen, Wen-Chi, and 陳文奇. "Bacterium Isolation Using Laser Capture Microdissection." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/yn2bvr.

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碩士
國立臺北科技大學
光電工程系研究所
96
Bacterial cultures are very important study issues in microbiology. Human can’t observe bacterial cultures by naked eyes, thus, the way to study bacterial cultures is totally unique. In traditional, to study unknown bacterial cultures must purify the target bacterium from complex sample conditions. In nature, bacterial cultures are mixed and coexisted. There are among of 99% of the sea bacterial cultures can’t be isolated by culture. In this study, we use laser capture microdissection to isolated a sea bacterial culture, named acinetobacter venetianus which already had been well purified by culture, and to ensure that LCM system can be used in bacterial cultures isolation. we set up LCM system on a headstand microscope, and isolate bacterial cultures under microscope view. Before isolation, we dye bacterial samples easier to be observed. We find out bacterium has been Gram’s dyed can’t be observed on translated film after isolaed in microscope view. Thus, we add DAPI on Gram’s dyed sample and redo experience again. In this study, we discover that bacterial cultures about 1μm in size can be isolated and observed under the condition that the distance between laser probe and film less than 1.0μm and laser light must irradiate translate film more than 400ms

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Tseng, Yu-Wei, and 曾昱瑋. "The Study of Multi-Layer Transfer Film for Laser Capture Microdissection." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/q6yw4a.

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碩士
國立臺北科技大學
光電工程系研究所
96
The purpose of the study is to provide the development of multi-layer transfer film for laser capture microdissection. Before developing single material of the transfer film thickness of 30 μm, it has been successfully applied to the LCM system. In the study, we hope could reduce the film’s thickness for LCM system, and uses support layer, multi-layer optical film and uses EVA solution of two melt index values to develop new hot-melt film for the usage of LCM system. By using EVA, polyester film, TiO2 and SiO2 as hot-melt film, the support layers, and major multi-layer optical materials. By way of mixing different MI value EVA solution, substrate preparation, and spin coating etc., we made various hot-melt film with different thickness. In this study, hot-melt film’s appraisal which used AFM, SEM, and LCM system, and found the best surface roughness, hot-melt depth, and different thickness and size of the melting point to provide LCM system in different sections of the target application.

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Lin, Chia-Ching, and 林佳慶. "Construction of a Electric Moving Stage for Laser Capture Microdissection System." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/gj984x.

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碩士
國立臺北科技大學
光電工程系研究所
96
We construct a new Laser Capture Microdissection (LCM) system by Using Electric Moving Stage. It spends a lot of time in positioning when the new LCM system with near-field fiber probe is integrated with microscopy. For overcoming the shortcoming, we designed a precision stage for moving the sample to adjust the target to be in the center of the scope. The fiber probe was set over the view center, and allowed to move only up and down. The designed stage includes a stepping motor and piezo actuators for moving fine in X-Y directions. We build a component of Human Machine Interface on electric moving stage control system and LCM system by Visual Basic. Then, we actually use electric moving stage on LCM experiments. By statistical analysis, we obtained the average displacement value of the stage is 3.265 μm and Relative standard deviation(RSD) is 0.00025. We successfully melted the EVA transfer film into figure by using the 70 nm fiber probe. The target can be moved to the center of scope in one minute by this new stage, then the process of laser capture be completed more quickly and higher precision control.

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Al-Sowaimel, Lujain Fawzi. "Regional podocyte isolation using laser capture microdissection for molecular profiling of glomerular disease." Thesis, 2014. https://hdl.handle.net/2144/15219.

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FSGS (focal segmental glomerulosclerosis) is a pathologic pattern that affects the glomerulus of the kidneys and is associated with progression toward end-stage kidney disease. It is characterized by destruction of the glomerular filtration system and replacement by scar tissue, which leads to chronic renal failure. An increase in glomerular capillary pressure may be an important factor in glomerulosclerosis. The podocyte is a major structural component of the filtration barrier. Since podocytes have an important role in maintaining the structural and functional integrity of glomerular filtration, a chronic increase in glomerular filtration pressure could ultimately damage and compromise the podocyte filtration apparatus. The segmental nature of FSGS suggests that podocyte damage may only occur at specific regions in the glomerulus. This poses the question as to whether differences in podocyte function, assessed by gene expression, may be dependent on their location in the glomerulus, and these regional differences might be responsible for FSGS development. Consequently, in order to evaluate this hypothesis there is a need to selectively isolate podocytes from various regions in a glomerulus. Therefore, the objective of this study was to determine the feasibility of selectively isolating podocyte cells from other renal cells using laser capture microdissection (LCM), and analyzing gene expression in these isolated cells using quantitative real-time polymerase chain reaction (qRT-PCR). If feasible, this method could be used to examine isolated cells for unique podocyte gene expression patterns that vary regionally throughout the glomerulus, and identify potential molecular and cellular mechanisms responsible for the initiation and progression of FSGS. Such information could then be used to identify new and pharmacologically accessible molecular targets. The specific goals of this project were to: 1) determine the feasibility of employment of the LCM system in the Boston University Department of Pathology for the capture of isolated mouse podocytes. 2) determine optimal tissue preservation and preparation methods for laser capture and mRNA analysis of isolated podocytes; and 3) determine the minimum number of isolated podocytes required to analyze gene expression using qRT-PCR. The results show that 1) liquid nitrogen was the preferred method of tissue freezing; 2) the use of Histogene stain improved cell identification during laser capture; 3) the LCM instrument parameters required for selective podocyte capture were identified; and 4) RNA quality obtained from the LCM samples was suboptimal. These results indicate that the possibility of using LCM for regional podocyte isolation and gene expression analysis is quite promising, and further optimization of the technique will likely yield an important new method for the study of kidney disease pathogenesis.

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Shen, Chuan-Yu, and 沈傳宇. "The Study of Transfer Film for Micron and Nano Laser Capture Microdissection System." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/5a47m5.

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碩士
國立臺北科技大學
光電工程系所
94
The purpose of the study is to provide the innovation of transfer film for Micron and Nano Laser Capture Microdissection (LCM) system. The principle of LCM comes from placing a transfer film on the top of dissected tissue. As laser beam pierce on the transfer film, the penetrated spot then converts and absorbs laser energy into heat to adhere the target cell after melting. Then the target cell will be identified after transfer film is removed from the dissected tissue. In order to make it flat and convenient to use, transfer film is coated on the bottom of a cap. But the cap may cause negative impacts such as power loss after the absorption and reflection of laser beam. Therefore, this study is aiming to find the new type EVA cap for the usage of traditional and probe type Laser Capture Microdissection. The melting point of the transfer film is 88℃, which may damage cells easily. We use local-made EVA, which melting point is 65℃, as major material and add near-infrared absorbing dyes to enhance the laser energy absorption. By combining different concentration of EVA solution, substrate preparation, and spin-coating etc., we made various transfer films with different concentration and thickness. In this study, we designed a hollow cap, which made the fiber probe exert close melting, and the optimal conditions of making the thermoplastic film adhere on the cap were also studied.

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Yen, Chi-Fu, and 顏畿府. "To Construct a Laser Capture Microdissection System in Nano-Scale by Using Near-Field Fiber Probe." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/3jeqp5.

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碩士
國立臺北科技大學
光電工程系所
93
Laser Capture Microdissection(LCM) is a technique that permits rapid and reliable procurement of pure population of cells from tissue sections. In LCM, the resolution of capture is directly related to the spot size on Ethyl Vinyl Acetate(EVA) transfer film. Microscope objective is used to focus laser beam and the resolution is 5μm in conventional system. In this paper, we try to update the resolution of capture by replacing objective with a near-field fiber probe. By using our new construct of LCM system, we successfully melted the EVA transfer film into spots, whose diameter ranged from 400nm to 1.4μm and the maximum droopy depth is 282.5nm. We also found the droopy depth will increase 9.53nm as laser pulsing time increases each 20ms. In the condition of laser pulsing time is 100ms and the distance between fiber probe and EVA transfer film is 1μm, we successfully captured monolayer of gold particles into spots and the minimal spot size is 400nm. These results prove that we have completed a new LCM system with higher resolution in this paper.

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Xu, Baogang Jonathan. "Combining laser capture microdissection and MALDI mass spectrometry for tissue protein profiling methodology development and clinical applications /." Diss., 2005. http://etd.library.vanderbilt.edu/ETD-db/available/etd-03092005-132210/.

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Chang, Chao-Yuah, and 張昭元. "Selective isolation of hepatocytes by laser capture microdissection for molecular analysis in a rat model of sepsis." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/69726388215659043218.

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碩士
高雄醫學大學
醫學研究所碩士班
92
It is known that proinflammatory cytokines: TNF-α, IL-1, IL-6 and adhesion molecules: P-selectin and E-selectin play roles in multiple organ dysfunction in sepsis. Carbon monoxide ( CO ) participates in regulation of cardiovascular physiology. CO is synthesized by heme-oxygenase ( HO ): inducible HO-1 or constitutive HO-2. To better understand gene expression correlated with patho-physiological functions of liver in sepsis, rats were subjected to cecal ligation and puncture ( CLP ) to induce sepsis. Rats were randomized into 4 groups of 6 animals each: CLP 9h ( early sepsis ), CLP 18h ( late sepsis ), sham 9h and sham 18h. Rat livers were excised and processed for frozen sections. To compare with conventional homogenization from liver slice, the techniques of laser capture microdissection ( LCM ) were used to isolate cells from liver sections. RNA was extracted from both liver slice and isolated specific cells by LCM. Gene expression of TNFα, IL-1β, IL-6, P-selectin, E-selectin, HO-1 and HO-2 was detected by using two-step techniques of reverse transcriptase-polymerase chain reaction ( RT-PCR ) . In situ hybridization ( ISH ) was also employed to examine gene expression in liver. In groups of LCM combined with RT-PCR, the results showed that HO-1 gene expression in early sepsis appeared significantly higher than that in both late sepsis and control ( sham 9h ). HO-1 gene expression in late sepsis appeared the same as control ( sham 18h ). IL-6 gene expression in early and late sepsis appeared significantly higher than that in sham control. In groups of tissue homogenization combined with RT-PCR, the results showed that IL-6 gene expression in early and late sepsis appeared significantly higher than that in sham control. No obvious change of HO-1, TNFα and HO-2 mRNA expression was detected among 4 groups of rats. Unlike the expression of the HO-1, IL-6, TNFα and HO-2, mRNA expression of P-selectin, E-selectin and IL-1was not detectable in any of the samples from either LCM or tissue homogenization. The results of ISH showed that gene expression of IL-6 appeared in both early and late sepsis but not in sham control. The results of ISH also showed that gene expression of HO-1 appeared in not only Kupper cells but hepatocytes in early sepsis. These studies indicate that detection sensitivity of LCM combined with RT-PCR is higher than tissue homogenization combined with RT-PCR. These studies also indicate that when rats were subjected to CLP to induce sepsis, IL-6 gene expression appeared significantly higher in early and late sepsis than sham control. HO-1 gene expression elevated in early sepsis and declined in late sepsis.

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Williams, Rachel, Irene Castellano-Pelicena, Aaiad H. A. Al-Rikabi, Stephen K. Sikkink, Richard Baker, Kirsten Riches-Suman, and M. Julie Thornton. "Laser capture microdissection on surgical tissues to identify aberrant gene expression in impaired wound healing in type 2 diabetes." 2020. http://hdl.handle.net/10454/18481.

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No
The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss.
European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no 607886. Aveda, Hair Innovation & Technology, USA

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McGrice, Hayley Ann. "Molecular characterisation of primary wool follicle initiation in Merino sheep." 2010. http://hdl.handle.net/2440/60986.

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Primary wool follicles are initiated in the skin of sheep foetuses at approximately day 50 of gestation as the result of complex reciprocal molecular interactions between the mesenchyme and overlying epithelium. The lifetime wool production potential and fibre diameter of the Merino sheep is dependent on the total number of follicles initiated in utero. Understanding the molecular events that surround primary wool follicle initiation may provide approaches to enhance or manipulate this process in order to maximise the profitability of wool production enterprises. In order to study the morphological and molecular changes occurring during early wool follicle development, a foetal skin series spanning primary follicle initiation was generated. Foetal skin was sampled from the shoulder, midside and rump of four foetuses at 8 time points between day 43 and day 68 of gestation. Histological characterisation of the shoulder skin samples revealed that primary epidermal placodes emerged at around day 53, dermal condensates were visible from day 57 and downgrowth of the follicle began at day 68. An equation relating age of the foetus (day of gestation post AI) and crown-rump length, specific to Merino foetuses, was developed for use in future studies of this nature. Molecular markers of fibroblast migration, epidermal and dermal stem cells and cell proliferation were selected to test the hypothesis that dermal condensates are initiated at discrete sites beneath the epidermis as a result of a combination of migration and arrangement of multipotent pre-papilla cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of RAC1 and RHOa (migration markers), β1-integrin and alkaline phosphatase (stem cell markers), proliferative nuclear cell antigen and cyclinB1 (proliferation markers), patched-1, selected tumor necrosis factor (TNF) signalling molecules and eleven reference genes was conducted using midside and rump skin samples from each of four foetuses from the 8 time points. geNorm analysis of the reference and target genes revealed that the migration markers RAC1 and RHOa along with GAPDH were the most stably expressed genes in this sample series. Significant changes in mRNA expression were detected for β1-integrin, alkaline phosphatase, patched-1 and the TNF members EDA, EDAR, TROY and TRAF6. Many of these significant differences in expression coincided with key morphological events. Significant differences in expression were also detected between the midside and rump samples for numerous transcripts. Laser capture microdissection (LCM) was implemented for analysis of the target transcripts within particular structures of foetal sheep skin. Frozen tissue sectioning, staining, LCM, RNA extraction and cDNA synthesis were optimised for qRT-PCR analysis of endogenous controls and selected TNF transcripts. Several RNA extraction methods and reverse transcription approaches were trialled to ensure optimum extraction and reverse transcription efficiency for this tissue type. Exogenous mRNA transcripts were also incorporated prior to RNA extraction and reverse transcription to track reaction efficiency between samples. A comparison of different slide types revealed that laser pressure catapulting from membrane slides was an absolute requirement for foetal skin tissue studies. Follicle regions (including the epidermal placode and dermal condensate) and the adjacent non-follicle regions were laser captured from foetal skin, and the mRNA expression levels of patched-1 and selected TNF members was compared. Preliminary qRT-PCR analysis using this technique revealed that EDAR, TROY and PTCH1 mRNA levels were higher in the follicle regions than the non-follicle regions. The TNF signalling pathway appears to play an important role in primary wool follicle initiation and patterning at different sites on the body. Spatial differences in expression of some of these regulators may be involved in initiating different types of follicles. The molecular events surrounding primary wool follicle initiation also show a high degree of conservation between sheep, humans, and mice. Considering the high degree of DNA sequence conservation as well as the histological, signalling and cycling similarities between sheep and humans, sheep may represent a better model for the study of human hair follicle initiation and disease than the currently used mice and rat models.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1523639
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010

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Braig, Ute Christine [Verfasser]. "Untersuchung genetischer Aberrationen am duktal invasiven Mammakarzinom mittels laser capture microdissection (LCM) und comparativer genomischer Hybridisierung : Etablierung einer neuen Methode / vorgelegt von Ute Christine Braig." 2003. http://d-nb.info/966347552/34.

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Coelho, Mafalda Maria Pereira. "Avaliação do conhecimento atual dos profissionais de saúde oral quanto ao diagnóstico do cancro oral." Master's thesis, 2016. http://hdl.handle.net/10284/5390.

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Introdução: Atualmente o cancro oral é o sexto cancro mais comum em todo o mundo. A sua elevada taxa de morbilidade e consequente mortalidade está relacionada com uma deteção da doença em estádios de malignidade avançados. Esta conjuntura acaba por levar ao aumento de complicações no tratamento, o que o torna mais agressivo consoante o grau de malignidade. Por isso o diagnóstico precoce combinado com o tratamento adequado é a chave fundamental para o controlo da doença. Face a esta realidade os profissionais de saúde têm um papel fulcral na deteção precoce de lesões potencialmente malignas ou malignas, sendo por isso um objetivo prioritário na saúde. Para avaliação deste tipo de lesões o diagnóstico definitivo, histopatologicamente fornecido pela biópsia convencional, continua a ser o método imprescindível e de eleição para os profissionais de saúde, no entanto novas técnicas não-invasivas têm surgido como auxiliares de diagnóstico. Objetivos: Esta monografia teve como objetivo obter um conhecimento detalhado acerca do cancro oral, nomeadamente as suas principais particularidades e especificidades de forma a obter uma deteção precoce e consequentemente um prognóstico favorável. Conjuntamente, foi realizado um estudo que recolheu informações sobre o conhecimento geral dos médicos dentistas e estomatologistas portugueses relativamente ao cancro oral, a utilização de modalidades auxiliares de diagnóstico hoje em dia disponíveis, assim como a colheita de dados face ao protocolo de atuação dos clínicos aquando suspeita de lesões potencialmente malignas ou malignas. Materiais e Métodos: Este trabalho teve como base as palavras-chave delineadas e critérios de inclusão e exclusão específicos, com o objetivo de angariar e debater o máximo de informação acerca desta grande associação entre oncologia e medicina dentária, o cancro oral. De forma a dar resposta aos objetivos enunciados associou-se a esta monografia um estudo quantitativo do tipo descritivo e transversal. A recolha de dados foi realizada através de um inquérito em que participaram 324 profissionais de saúde oral. O inquérito, com a totalidade de vinte e uma perguntas, teve como principal objetivo a recolha de informação sobre o conhecimento geral do médico dentista ou estomatologista relativamente ao cancro oral. Após colheita de dados, estes foram tratados e analisados recorrendo a programas informáticos como o Microsoft Office Excel (2010) e o programa Statistical Package for the Social Sciences (IBM© SPSS© Statistics) vs. 22.0 para Windows. Resultados: A amostra selecionada demonstrou ter os conhecimentos necessários e fundamentais quando abordados sobre este tema, estando conscientemente alerta caso suspeitem de uma lesão potencialmente maligna ou maligna. Cerca de 78% dos inquiridos preferem encaminhar o paciente quando perante uma lesão suspeita. Relativamente aos indivíduos que realizam biópsia, 66.7% prefere a técnica excisional. 96% discute com colegas quando suspeita de uma lesão maligna. A maioria dos profissionais (88.9%) refere que nunca utilizou outros métodos auxiliares para além da biópsia. Dos 11.1% que admitem ter utilizado outro método auxiliar de diagnóstico, 58.8% indica o azul de toluidina como método preferencial. 27.2% dos profissionais não considera a biópsia como elemento fundamental. Discussão e Conclusão: Através da pesquisa realizada verificou-se que é fundamental um conhecimento dos sinais e sintomas e a realização de rastreio em consultas de rotina para detetar precocemente lesões potencialmente malignas ou malignas. Por consenso geral dos inquiridos, constatou-se que a história clínica, o exame clínico e a biópsia são os métodos mais eficazes e comummente utilizados a fim de reduzir a morbilidade e mortalidade associada a esta doença. Ainda assim, 27.2% dos profissionais não considera a biópsia como elemento fundamental o que revela ser preocupante. Contudo corroborou-se que a maioria dos inquiridos, mesmo aptos teoricamente, prefere encaminhar ao invés de realizar biópsia.
Introduction: Currently oral cancer is the sixth most common cancer worldwide. Its high rate of morbidity and consequent mortality, is related with the detection of this disease already in advanced stages of malignancy. In this context, it ends up leading to increased complications in treatment that becomes more aggressive according to the degree of malignancy. Therefore early diagnosis along with suitable treatment is the fundamental key to control the disease. Due to this reality, health professionals have a key role in early detection of potential malignant or malignant lesions making it a priority goal in health. To evaluate this kind of lesions, the definitive histopathologic diagnosis provided by conventional biopsy, still remains the crucial method of choice for health professionals, although new non-invasive techniques have emerged as auxiliary diagnosis methods. Objective: This paper aims to obtain a detailed knowledge of oral cancer, including its main particularities and specificities in order to obtain an early detection and consequently a favorable prognosis. It has also have been conducted a study that collected information on the general knowledge of portuguese dentists and stomatologists regarding oral cancer, the use of auxiliary diagnostic tools nowadays available, as well as data collection concerning the clinical performance protocol when suspected potentially malignant or malignant lesions. Materials and Methods: This study was based on the keywords and outlined specific inclusion and exclusion criteria, in order to raise and discuss as much information about this great association between oncology and dentistry, which is oral cancer. In order to meet the stated goals, it was associated with this monography a quantitative study of a descriptive and cross-sectional type. Data collection was conducted through a survey involving 324 dental professionals. The survey, with a total of twenty-one questions, aimed to collect information on the general knowledge of dentists regarding oral cancer. After data collection, these were processed and analyzed using computer programs such as Microsoft Office Excel (2010) and the Statistical Package for Social Sciences (SPSS IBM © © Statistics) vs. 22.0 for Windows. Results: The selected sample has demonstrated the necessary and basic knowledge when approached about the subject, consciously being alert if suspected a potentially malignant or malignant lesion. About 78% of respondents prefer to refer the patient when faced with a suspected lesion. Regarding the individuals that perform biopsy, 66.7% prefer the excisional technique. 96% approaches other colleagues when suspects of a malignant lesion. Most professionals (88.9%) reported ever used other auxiliary methods in addition to biopsy. Of the 11.1% who admit to having used an auxiliary diagnostic tool, 58.8% indicates the toluidine blue as the preferred method. 27.2% of professionals do not consider the biopsy as a fundamental element. Discussion and Conclusion: Through the research made, it was found that it is essential to have knowledge of signs and symptoms and provide screening in routine consults aiming for an early detection of potentially malignant or malignant lesions. By general consensus of those surveyed, it was found that the clinical history, clinical examination and biopsy are the most effective methods and commonly used to reduce morbidity and mortality associated with this disease. Still, 27.2% of the respondents do not consider the biopsy as a fundamental element, which is concerning. However it was corroborated that the majority of respondents although theoretically able, prefer to refer instead of performing biopsy.

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Dissertations / Theses on the topic 'Laser capture microdissection'

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