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Highly Multiplexed Single Cell in situ Protein Analysis with Cleavable Fluorescent ProbesJanuary 2019 (has links)
abstract: Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019
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Detection of artefacts in FFPE-sample sequence dataSwenson, Hugo January 2019 (has links)
Next generation sequencing is increasingly used as a diagnostic tool in the clinical setting. This is driven by the vast increase in molecular targeted therapy, which requires detailed information on what genetic variants are present in patient samples. In the hospital setting, most cancer diagnostics are based on Formalin Fixed Paraffin Embedded (FFPE) samples. The FFPE routine is very beneficial for logistical purposes and for some histopathological analyses, but creates problems for molecular diagnostics based on DNA. These problems derive from sample immersion informalin, which results in DNA fragmentation, interstrand DNA crosslinking and sequence artefacts due to hydrolytic deamination. Distinguishing such artefacts from true somatic variants can be challenging, thus affecting both research and clinical analyses. In order to identify FFPE-artefacts from true variants in next generation sequencing data from FFPE samples, I developed the novelprogram FUSAC (FFPE tissue UMI based Sequence Artefact Classifier) for the facility Clinical Genomics in Uppsala. FUSAC utilizes UniqueMolecular Identifiers (UMI's) to identify and group sequencing reads based on their molecule of origin. By using UMI's to collapse duplicate paired reads into consensus reads, FFPE-artefacts are classified through comparative analysis of the positive and negative strand sequences. My findings indicate that FUSAC can succesfully classify UMI-tagged next generation sequencing reads with FFPE-artefacts, from sequencing reads with true variants. FUSAC thus presents a novel approach in bioinformatic pipelines for studying FFPE-artefacts.
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Essays on spatial point processes and bioinformaticsFahlén, Jessica January 2010 (has links)
This thesis consists of two separate parts. The first part consists of one paper and considers problems concerning spatial point processes and the second part includes three papers in the field of bioinformatics. The first part of the thesis is based on a forestry problem of estimating the number of trees in a region by using the information in an aerial photo, showing the area covered by the trees. The positions of the trees are assumed to follow either a binomial point process or a hard-core Strauss process. Furthermore, discs of equal size are used to represent the tree-crowns. We provide formulas for the expectation and the variance of the relative vacancy for both processes. The formulas are approximate for the hard-core Strauss process. Simulations indicate that the approximations are accurate. The second part of this thesis focuses on pre-processing of microarray data. The microarray technology can be used to measure the expression of thousands of genes simultaneously in a single experiment. The technique is used to identify genes that are differentially expressed between two populations, e.g. diseased versus healthy individuals. This information can be used in several different ways, for example as diagnostic tools and in drug discovery. The microarray technique involves a number of complex experimental steps, where each step introduces variability in the data. Pre-processing aims to reduce this variation and is a crucial part of the data analysis. Paper II gives a review over several pre-processing methods. Spike-in data are used to describe how the different methods affect the sensitivity and bias of the experiment. An important step in pre-processing is dye-normalization. This normalization aims to remove the systematic differences due to the use of different dyes for coloring the samples. In Paper III a novel dye-normalization, the MC-normalization, is proposed. The idea behind this normalization is to let the channels’ individual intensities determine the correction, rather than the average intensity which is the case for the commonly used MA-normalization. Spike-in data showed that the MC-normalization reduced the bias for the differentially expressed genes compared to the MA-normalization. The standard method for preserving patient samples for diagnostic purposes is fixation in formalin followed by embedding in paraffin (FFPE). In Paper IV we used tongue-cancer microRNA-microarray data to study the effect of FFPE-storage. We suggest that the microRNAs are not equally affected by the storage time and propose a novel procedure to remove this bias. The procedure improves the ability of the analysis to detect differentially expressed microRNAs.
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Expressão de receptores de EGF, inibidores e reguladores do ciclo celular em lesões proliferativas da próstata canina / Expression of EGF rececptors, cell cycle inhibitors and regulators in canine prostate with proliferative lesionsFaleiro, Mariana Batista Rodrigues 14 November 2014 (has links)
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Previous issue date: 2014-11-14 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Somedysplastic lesionsof prostate, such as proliferativeinflammatoryatrophy (PIA) and
prostatic intraepithelial neoplastic(PIN) are proliferative,consideredpremalignant andcan
progress toprostatic carcinoma (PC). Others, such asbenign prostatichyperplasia(BPH) are
proliferative, butare not related tomalignancy.The humanPC is the one ofcancers that more
kill,and dog`sthe onlyanimal species thatpresentsadiseasewith similarcharacteristics, thishas
been usedas a modelfor studyof prostatecancer. Thus, in order tobetter understandthe
molecular mechanisms involvedin repairand cell cycleevolution ofproliferative
lesionsincanine prostate, this study aimed to evaluatethe gene expression ofErbB1, ErbB2,
CDKN1A, CDKN1BandTP53andimmunostaining ofEGFR(Her1), Her2(c-erbB-2), p21,
p27and p53 inprostateswithPIAandPC. Also was evaluatedtheimmunostaining ofp21, p27,
p53, c-myc andcyclinD1inprostateswith BPH, PIA, PINandPC. For this purpose,wereobtained
from atissue microarray (TMA)block, 106samplesofcanineprostate tissue, which 15 normal,
16with BPH, 30 withPIA, 20 with PIN, and 25with CP. The RT-PCR andRT-qPCR
techniques wereused to evaluategene expression, as well as immunohistochemistrywas used
to determinetheimmunophenotypeofprostatic lesions. As forEGFRand Her2has beenobserved
that thesereceptorsactin canine lesions withPIA and with PCan importantaction ofHer2,
suggesting that theyare involvedin carcinogenesisandtumor development in canineprostate.
Regarding to cellcycleinhibitor concludethat whenoverexpressedin canine
prostateswithPIAandPC, p21andp27control cellproliferation, acting as a protective factorin
the evolution of PIA to PC, and in the PC development, even in the presence of altereted p53.
In assessing ofimmunostaining ofcell cycle regulators(p21, p27, p53, cyclinD1andc-myc) in
canineprostateswithproliferative lesionswas observedthatcarcinomas probably have
lowgrowth potentialwhenoverexpressed of p21andp27, reduced expression of cyclinD1and
unalteredexpression ofc-myc, even in the presence ofmutant p53type.Further,considering
thesimilarimmunophenotypein canine glands with PIA, PINandPC considered the
regulatorsofcell cycle progression, it is reinforce the pre-malignant potential of PIA andPINin
the canineprostate. / Algumas lesões displásicas da próstata, como a atrofia inflamatória proliferativa (PIA) e a
neoplasia intraepitelial prostática (PIN), são proliferativas, consideradas pré-malignas e
podem evoluir para carcinoma prostático (PC). Outras, como a hiperplasia prostática benigna
(HPB), apesar de proliferativas, não possuem relação com malignidade. Sendo o PC humano
uma das neoplasias que mais matam, e o cão aúnica espécie animal que apresenta a
enfermidade com características semelhantes, esta tem sido utilizada como modelo para o
estudo do câncer de próstata. Assim, com o intuito de melhor compreender os mecanismos
moleculares envolvidos na regulação do ciclo celular e evolução das lesões proliferativas na
próstata canina, este estudo teve por objetivo avaliar a expressão gênica de ErbB1, ErbB2,
CDKN1A, CDKN1B e TP53,e imunomarcação deEGFR (Her1), Her2 (c-erbB-2), p21, p27 e
p53 em próstatas com PIA e PC. Também foi avaliada a imunomarcação de p21, p27, p53, cmyc
e cyclin D1 em próstatas com HPB, PIA, PIN e PC. Para tal, foram obtidas de um bloco
de microarranjo tecidual (TMA) 106 amostras de tecido prostático canino, sendo 15 normais,
16com HBP, 30com PIA, 20com PIN, e25com PC. As técnicas de RT-PCR e RT-qPCR
foram empregadas na avaliação da expressão gênica, assim como a técnica de
imunoistoquímica foi utilizada para verificar o imunofenótipo das lesões prostáticas. Quanto
aos receptores EGFReHer2 foi observado que esses atuam naslesões de PIAePCcanino, com
ação importante do receptor Her2, sugerindoqueessesestão envolvidosna carcinogênese e no
desenvolvimentotumoral da prostata canina.Em relação aos inibidores do ciclo celular
councluiu-se que quando superexpressos em próstatas com PIA e PC, p21 e p27 controlam a
proliferação celular, atuando comofator protetivo na evolução da PIA para PC, e no
desenvolviemento do PC, mesmo quando da alteração de p53. Na avaliação da
imunoexpressão dos reguladores do ciclo celular (p21, p27, p53, ciclina D1 e c-myc) em
próstatas caninas com lesões proliferativas foi constatado que os carcinomas possivelmente
apresentam baixo potencial de crescimento quando da superexpressãop21 ep27, menor
subexpressão de ciclina D1e expressão inalterada de c-myc, mesmo na presença de p53 tipo
mutante. Ainda, considerando o imunofenótipo semelhante nas glândulas com PIA, PIN e PC
no que se refere aos reguladores daprogressão do ciclo celular, reforça o potencial prémaligno
da PIA e PIN na próstata canina.
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Optimering av DNA-extraktion inför Illumina metyleringsarray : Genomförd på formalinfixerad och paraffininbäddad lymfomvävnadPersson, Adam January 2023 (has links)
No description available.
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A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE SamplesYeo, Jiyoun 20 August 2014 (has links)
No description available.
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Framtagande av PCR-metod för diagnostik av Toxoplasma gondii- infektionWadenius, Tove January 2019 (has links)
Toxoplasma gondii är en encellig parasit som kan skapa svåra skador hos immunsupprimerade individer samt hos foster då mamman infekteras under graviditet. Genom framtagande av en realtids-PCR-metod för detektion av T. gondii ska denna diagnostik flyttas från Klinisk Patologi (KP) samt Karolinska Universitetslaboratoriet till Klinisk Mikrobiologi i Lund. Två regioner: REP-529 samt genen B1, som båda förekommer i ett flertal kopior i T. gondii-genomet, utgjorde mål för primerutprovning. Analyser utfördes både med realtids-PCR och konventionell PCR. De primerpar som gav lägst Ct-värde samt högst amplitud valdes ut för REP-529 respektive B1 för vidare analyser. Hybridiseringsprober användes för att öka specificiteten och metoden utvärderades på upprenat T. gondii DNA från olika stammar samt Formalin-Fixed Paraffin-Embedded (FFPE) -prover erhållna från KP. I alla provmaterial amplifierades REP-529 mer effektivt (lägre Ct-värde) än B1 vilket var förväntat då fler kopior finns av REP-529 än av B1. Nedre kvantifikationsgränsen för REP-529 och B1 visade sig dock vara samma på ca 1,8 genomkopior/µl. / Toxoplasma gondii is a unicellular organism that can cause severe damage in immunocompromised patients or in fetuses when the mother is infected during pregnancy. Through development of a real time-PCR based method for detection of T. gondii-DNA, the diagnostics is planned to be relocated from the department of Clinical Pathology in Lund and the Karolinska University Laboratory in Huddinge to the department of Clinical Microbiology in Lund. The method was built on amplification of REP-529 and the B1-gene of the T. gondii genome, both present in multiple copies. Primers were tested on purified T. gondii DNA with conventional PCR as well as real time PCR with EvaGreen dye. Primers with high amplitude and low Ct-value were selected for both REP-529 and B1. Hybridizing probes were applied to increase specificity and the method was evaluated on T. gondii-DNA extracted from Formalin-Fixed, Paraffine-Embedded (FFPE) tissue as well as purified DNA from ten different strains of T. gondii. The amplification of REP-529 yielded a higher amplitude and lower Ct-value compared to that of B1 but the lower limit of quantification seemed to correlate between the two, with 1,8 genome-copies/µl.
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APC, BRAF and KRAS mutations, and MLH1, MGMT and CDKN2A expression analysis in Nepalese colorectal cancer patients. : - / - : -Nourizadeh, Alireza January 2017 (has links)
Colorectal cancer (CRC) is a common malignancy which develops due to old age and lifestyle factors, low percent of patients afflicted by a genetic disorders. Half of all colorectal cancer patients are diagnosed after metastasis. The high rate of the late detection, emphasizes on the requirement of convenient and inexpensive diagnostic methods for comprehensive screening programs. The aim of this study was to discover proto-oncogenes mutation and assessment of tumor suppressor genes expression. Formalin fixed paraffin embedded (FFPE) histologically verified colorectal cancer samples were used. APC, KRAS and BRAF mutations were investigated using polymerase chain reaction (PCR) fragments and direct sequencing. Gene expression assessment of MLH1, MGMT and CDKN2A were achieved via quantitative polymerase chain reaction (qPCR). In the present study we could detect a novel transversion heterozygous mutation in APC gene codon 1365 in three patients. BRAF codon 600 mutation were detected in one patient. KRAS codon 12 mutation was discovered in one sample and also a novel transition mutation in codon 15 was detected in 6 patients. In 80% of cases, MLH1 and MGMT expression were undetectable, in remaining 20%, MLH1 expression were reduced, but MGMT showed both reduced and increased expression compared to control. In 100% of patients CDKN2A expression was undetectable. The rate of mutations in predetermined hotspot codons and amount of uncommon mutations into APC, BRAF and KRAS in Nepalese patients indicates the requirement of further investigation in CRC patients from that part of the world. Also, the expression rate of MLH1, MGMT, CDKN2A and deficiency of an information source emphasizes the necessity of whole genome CRC expression profiling data to comparison and conclusion. / <p>-</p> / -
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Prognostický význam PCA3, fúzního genu TMPRSS2:ERG a dalších markerů u karcinomu prostaty / The prognostic value of PCA3, the fusion gene TMPRSS2:ERG and other markers in prostate cancerHOLÁ, Hana January 2014 (has links)
The aim of this thesis was to assess the presence of fusion gene TMPRSS2:ERG and expressions of PCA3, miR23b, miR26 and miR221 in PCa. PSA was measured in peripheral blood and tumor tissue (FFPE samples). The presence of fusion gene TMPRSS2:ERG and expression of PCA3 gene and miRNA in FFPE tumor tissue was analysed by RT real-time PCR. This determination would help to identify patients with high-risk tumors.
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Genome-wide modeling of mutation spectra of human cancer-risk agents using experimental systems / Modélisation à l'échelle du génome des spectres de mutations des agents de risque de cancer humain en employant des systèmes expérimentauxZhivagui, Maria 30 November 2017 (has links)
Les génomes du cancer présentent une mosaïque de types de mutations. Trente signatures mutationnelles ont été identifiées à partir d'un grand nombre de tumeurs humaines primaires. Déchiffrer l'origine de ces signatures mutationnelles pourrait aider à identifier les causes du cancer humain. Environ 40% des signatures décrites sont d'origine inconnue, soulignant la nécessité de modèles expérimentaux contrôlés pour étudier l'origine de ces signatures. Au cours de mon travail de doctorat, j'ai caractérisé et utilisé des modèles in vitro et in vivo d'exposition aux cancérogènes, caractériser les signatures mutationnelles au niveau de génome entier de plusieurs composés cancérogènes pour lesquels le spectre de mutations n'était pas connu ou controversé. Tout d'abord, les conditions de cytotoxicités et genotoxicités pour chaque composé ont été établies et la formation d'adduits d'ADN a été évaluée. Suite au séquençage du gène TP53, on a effectué un séquençage au niveau du génome des clones MEF immortalisés dérivés de l'exposition à l'acrylamide, au glycidamide et à l'ochratoxine A. Le travail suggère une nouvelle signature mutationnelle unique pour l'acrylamide et médiée par son métabolite actif, le glycidamide. En fait, le motif des mutations de glycidamide, correspondant au profil de sa signature mutationnelle, a récapitulé les types de mutations attendus en fonction de l'analyse des adduits d'ADN. En outre, une analyse intégrée utilisant des modèles in vitro et in vivo suggère un manque de mutagénicité directe pour l'OTA avec une contribution potentielle d'un mode d'action lié à la production des radicaux libres à la signature mutationnelle OTA dans les MEF. Cette stratégie expérimentale simple et puissante peut faciliter l'interprétation des empreintes de mutations identifiées dans les tumeurs humaines, élucider l'étiologie du cancer et finalement soutenir la classification des cancers du CIRC en fournissant des preuves mécanistes / Cancer genomes harbour a mosaic of mutation patterns from which thirty mutational signatures have been identified, each attributable to a particular known or yet undetermined causal process. Deciphering the origins of these global mutational signatures in full could help identify the causes of human cancer, especially for about 40% of those signatures identified thus far that remain without a known etiological factor. Thus, well-controlled experimental exposure models can be used to assign particular mutational signatures to various mutagenic factors.During the time frame of my PhD work, I characterized and employed innovative in vitro and in vivo models of carcinogen exposure, namely, primary Hupki MEF cells, HepaRG and lymphoblastoid cell lines as well as rodent tumors. The cytotoxic and genotoxic conditions for each tested exposure compound were established and DNA adduct formation was assessed in select cases. Following a pre-screen by TP53 gene sequencing, genome-wide sequencing of immortalized Hupki MEF clones derived from exposure to acrylamide, glycidamide and ochratoxin A was performed, alongside whole genome sequencing of ochratoxin A induced rat renal tumors. The results reveal a novel mutational signature of acrylamide mediated by its active metabolite, glycidamide, a pattern that can be explained by the parallel analysis of individual glycidamide-DNA adducts. In addition, an integrative mutation analysis using in vitro and in vivo models suggests a lack of direct mutagenicity for OTA and possible indirect effects due to the ROS-mediated mode-of-action in MEF cells. The presented robust experimental strategy can facilitate the interpretation of mutation fingerprints identified in human tumors, thereby elucidating cancer etiology, elucidating the relationship between mutagenesis and carcinogenesis and ultimately providing mechanistic evidence for IARC’s carcinogen classification
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