Das hereditäre nicht-polypöse kolorektale Karzinom (HNPCC) bildet mit 2-3 % aller kolorektalen Karzinomen die häufigste Form der erblichen Darmkrebserkrankungen. Sie wird meist durch Mutationen in den Genen MLH1 und MSH2 verursacht. Beide Gene codieren zusammen mit anderen Genen (MSH6, MLH3, PMS1 und PMS) für Proteine des DNA-Mismatch-Repair-Systems, welches wichtig für die Stabilität des Genoms ist. Um Träger dieser Mutationen zu identifizieren und frühzeitig Vorsorgeprogrammen zuzuführen, ist ein zuverlässiges Auswahlverfahren notwendig. Aus diesem Grund wird in der vorliegenden Arbeit untersucht, ob die Methode der denaturierenden Hochdruckflüssigkeits-Chromatographie (DHPLC) geeignet ist, um nach Mutationen in den Genen MLH1 und MSH2 zu suchen. Bei 26 weiblichen Patienten und 2 männlichen Probanden im Alter von 23 bis 69 Jahren, die an einem Mammakarzinom (26), einer Dysplasie der Portio (1) sowie an einem Ovarialkarzinom (1) erkrankt waren, und bei deren Familienmitglieder gehäuft Tumoren auftraten, wurden die Gene MLH1 und MSH2 mittels DHPLC gescreent. Dabei wurden alle Exons von MLH1 außer 3, 5-7, 9-10, 14-15 und 18 sowie alle Exons von MSH2 außer Exon 1-2, 4, 9 und 14 einschließlich der flankierenden Exon-/Intron-Grenzen untersucht. Bei insgesamt 41 einzelnen Patientenkurven wich das Muster des DHPLC-Chromatogramms als Hinweis auf eine Genveränderung durch mehrfache oder verbreiterte Peaks vom normalen Kurvenverlauf ab. Die sich daran anschließende DNA-Sequenzanalyse ergab bei 35 der auffälligen Kurven polymorphe Sequenz-Varianten. Keine der gefundenen Sequenzveränderungen stellte eine krankheitsauslösende Mutation für HNPCC dar. Die in dieser Arbeit erreichte Sensitivität des DHPLC-Verfahrens liegt damit bei 85,4 %. Das DHPLC-Verfahren konnte als zuverlässige und vergleichsweise kostengünstige Voruntersuchung etabliert werden.

In humans, multi enzymatic processes are involved in maintaining DNA stability and cellular homeostasis. Cells undergo several episodes to survive and protect itself in daily basis. Accumulation of DNA errors and breaks are repaired by dynamic machinery, such as mismatch repair (MMR), replication-related process. In presented diploma thesis, we report the studied MMR pathway and its involvement in malignancy of epithelial ovarian cancer (EOC). Our working hypothesis postulated that core genes of MMR, such as MLH1 and MSH2 are down-regulated in malignant cells. Cells therefore become incapable to repair accumulating DNA damage, undergo apoptosis or most likely uncontrolled proliferation. Above mentioned genes may also be silenced in cancer patients at transcription, translation or epigenetic levels. Our aims were to clarify and to investigate the importance of MMR based on mRNA transcription, protein stability and promoter hypermethylation on a set of major MMR genes, particularly MLH1, MSH2, PMS1, MLH3, MSH6, MSH3, and PMS2. In our study, we analysed samples from 63 epithelial ovarian cancer patients and 12 non-malignant reference tissues using RT-qPCR, MS-HRM, and Western Blotting methods. Consequently, our results show down-regulation of all MMR genes except for MSH2 (up-regulated) in tumor...

Das hereditäre nicht-polypöse kolorektale Karzinom (HNPCC) bildet mit 2-3 % aller kolorektalen Karzinomen die häufigste Form der erblichen Darmkrebserkrankungen. Sie wird meist durch Mutationen in den Genen MLH1 und MSH2 verursacht. Beide Gene codieren zusammen mit anderen Genen (MSH6, MLH3, PMS1 und PMS) für Proteine des DNA-Mismatch-Repair-Systems, welches wichtig für die Stabilität des Genoms ist. Um Träger dieser Mutationen zu identifizieren und frühzeitig Vorsorgeprogrammen zuzuführen, ist ein zuverlässiges Auswahlverfahren notwendig. Aus diesem Grund wird in der vorliegenden Arbeit untersucht, ob die Methode der denaturierenden Hochdruckflüssigkeits-Chromatographie (DHPLC) geeignet ist, um nach Mutationen in den Genen MLH1 und MSH2 zu suchen. Bei 26 weiblichen Patienten und 2 männlichen Probanden im Alter von 23 bis 69 Jahren, die an einem Mammakarzinom (26), einer Dysplasie der Portio (1) sowie an einem Ovarialkarzinom (1) erkrankt waren, und bei deren Familienmitglieder gehäuft Tumoren auftraten, wurden die Gene MLH1 und MSH2 mittels DHPLC gescreent. Dabei wurden alle Exons von MLH1 außer 3, 5-7, 9-10, 14-15 und 18 sowie alle Exons von MSH2 außer Exon 1-2, 4, 9 und 14 einschließlich der flankierenden Exon-/Intron-Grenzen untersucht. Bei insgesamt 41 einzelnen Patientenkurven wich das Muster des DHPLC-Chromatogramms als Hinweis auf eine Genveränderung durch mehrfache oder verbreiterte Peaks vom normalen Kurvenverlauf ab. Die sich daran anschließende DNA-Sequenzanalyse ergab bei 35 der auffälligen Kurven polymorphe Sequenz-Varianten. Keine der gefundenen Sequenzveränderungen stellte eine krankheitsauslösende Mutation für HNPCC dar. Die in dieser Arbeit erreichte Sensitivität des DHPLC-Verfahrens liegt damit bei 85,4 %. Das DHPLC-Verfahren konnte als zuverlässige und vergleichsweise kostengünstige Voruntersuchung etabliert werden.:Inhaltsverzeichnis 1 Einleitung 8 1.1 Das hereditäre nicht-polypöse kolorektale Karzinom (hereditary non-polyposis colorectal Cancer; HNPCC) 8 1.1.1 Definition, Epidemiologie und Krankheitsbild 8 1.1.2 Klinische Diagnostik 9 1.1.3 Genetische Diagnostik 11 1.1.4 Bedeutung der Diagnosestellung bei HNPCC-Patienten für die Behandlung 11 1.1.5 Molekulargenetische Grundlagen des HNPCC 13 1.2 Die hMLH1- und hMSH2-Gene 15 1.2.1 Lokalisation, Struktur und Funktion der hMHL-Gene 16 1.3 Detektion von Struktur- und Sequenzveränderungen bei HNPCC 17 1.3.1 Die Einzelstrang-Konformationspolymorphismus-Analyse (SSCP) 18 1.3.2 Die denaturierende Gradientengelelektrophorese (DGGE) 18 1.3.3 Die denaturierende Hochdruckflüssigkeits-Chromatographie (DHPLC) 19 2 Ziele dieser Arbeit 23 3 Patienten, Material und Methoden 24 3.1 Patienten 24 3.2 Materialien und Geräte 27 3.2.1 Materialien 27 3.2.2 Technische Geräte 28 3.2.3 Reagenzien 29 3.2.4 Lösungen und Puffer 30 3.2.5 Standards und Farbstoffe 31 3.2.6 Molekulardiagnostische Kits 31 3.2.7 Enzyme 32 3.3 Primer 32 3.4 Softwareprogramme, Datenbanken und Sequenzquellen 34 3.4.1 Software zur Schmelzpunktbestimmung 34 3.4.2 ABI PRISMTM Software zur Sequenzanalyse 34 3.4.3 Mutationsdatenbanken (Online) 34 3.4.4 DNA-Sequenzen 34 3.5 Methoden 35 3.5.1 DNA-Isolierung aus Vollblut 35 3.5.2 DNA-Amplifikation mittels Polymerase-Kettenreaktion (PCR) 36 3.5.3 Denaturierende Hochdruckflüssigkeits-Chromatographie (DHPLC) 38 3.5.4 DNA-Sequenzierung 41 4 Ergebnisse 46 4.1 Quantität und Qualität der isolierten DNA 46 4.1.1 DNA-Schmelzpunkt- und Fließgradienten-Bestimmung mittels WAVE®Maker-Software 47 4.1.2 Fragmentanalyse mittels DHPLC 49 4.2 DNA-Sequenzierung 54 4.2.1 Polymorphismen 55 5 Diskussion 61 5.1 Genotyp- und Phänotyp-Beziehung 61 5.1.1 Erstmanifestationsalter und Tumorspektrum 61 5.1.2 Fehlender Mutationsnachweis 62 5.2 DHPLC-Methode als Screening-Verfahren 65 5.2.1 Zeitersparnis 65 5.2.2 Kostenersparnis 67 5.2.3 Nachweisgenauigkeit 67 5.2.4 Nachweis von Sequenzvarianten 68 5.2.5 Chromatogramm-Interpretation 70 5.2.6 Anfälligkeiten für Störfaktoren 71 6 Zusammenfassung der Arbeit 73 7 Literaturverzeichnis 75 7.1 Web-Adressen 86 8 Erklärung über die eigenständige Abfassung der Arbeit 89 9 Lebenslauf 90 10 Danksagung 92

The DNA Mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15-17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. MMR deficient tumours are often resistant to standard chemotherapies, therefore there is a critical need to identify new therapeutic strategies to treat MMR deficient disease. This study demonstrates that MLH1 deficient tumours are synthetically lethal with the mitochondrial-targeted agent Parthenolide which is known to induce reactive oxygen species (ROS) as one of its main mechanisms of action. Upon functional analysis, I show for the first time that loss of MLH1 is associated with deregulated mitochondrial function evidenced by a reduction in complex I expression and activity, reduced basal oxygen consumption rate and reduced spare respiratory capacity. This mitochondrial phenotype in the MLH1-deficient cell lines is accompanied by a reduction in mitochondrial biogenesis as evidenced by down regulation of pgc1β and decreased mitochondrial copy number. Furthermore, MLH1-deficient cancer cells have a decreased antioxidant defence capacity with reduced expression of the antioxidant genes NRF1, NRF2, Catalase, Glutathione peroxidase and SOD1 as well as increased ROS production when treated with Parthenolide. I further demonstrate that both MSH2- and MSH6-deficient cell lines also display deficiencies in complex I compared to their MMR-proficient counterparts. Taken together, the results of this study show a novel role for MLH1 in mitochondrial function and biogenesis. The MMR proteins MSH2 and MSH6 are also likely to have a role in the mitochondria. My results suggest that targeting the mitochondria may be a potential therapeutic strategy for the treatment of MMR and specifically MLH1 deficient disease.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

While the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility, concerns have been raised regarding the risk of chromosomal abnormalities in pregnancies derived from ICSI. Studies on sperm from infertile men have suggested that this population may produce higher rates of aneuploid sperm. Thus, we hypothesized that defects in early meiotic events may contribute to both male infertility and the production of aneuploid sperm. We used immunofluorescent techniques to observe the synapsis and recombination of chromosomes during meiosis, and fluorescent in-situ hybridization (FISH) to assess sperm aneuploidy. We analyzed testicular tissue from thirty-one men (10 fertile and 21 infertile men). We observed that ~36% (5/14) of men with impaired spermatogenesis displayed reduced genome-wide recombination. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. We combined immunofluorescent and FISH techniques to study recombination patterns on chromosomes 13, 18 and 21 in fifteen men (5 fertile and 10 infertile men). Four of the infertile men displayed altered recombination distributions on at least one of the chromosome arms studied. Finally, we examined early meiotic events in two biopsies from an azoospermic t(8;13) carrier. While global recombination rates were not altered, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and γH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes, suggesting that regions of autosomal chromosomes that fail to synapse undergo transcriptional silencing in humans. In summary, we observed that a subset of infertile men display alterations in the number and position of meiotic crossovers, which may contribute to both infertility and an increased risk of sperm aneuploidy. The fidelity of synapsis is also a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

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Orientador: Carmen Sílvia Bertuzzo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T00:44:00Z (GMT). No. of bitstreams: 1 Rueda_LidianeCamila_D.pdf: 2777204 bytes, checksum: dc2eb94ba9de0c91e1c7a3fed2fbd3e4 (MD5) Previous issue date: 2013 / Resumo: O câncer colorretal tem importância elevada frente a sua incidência e morbidade. Dentre os casos hereditários, o câncer colorretal hereditário sem polipose (HNPCC), ou Síndrome de Lynch, é responsável por cerca de 5% do total de casos. No HNPCC, a alteração genética herdada é a inativação de um dos alelos dos genes envolvidos em reparo do DNA, sendo os principais os genes hMLH1 e hMSH2. O objetivo deste trabalho foi investigar, em indivíduos com diagnóstico clínico de HNPCC, a presença de mutações nos genes MLH1 e MSH2, associar as variáveis clínicas com o gene mutado e investigar os familiares de portadores de HNPCC aos quais tivemos acesso, com relação a mutações germinativas. A investigação das mutações foi realizada por meio de sequenciamento direto dos éxons, região promotora e regiões de junção. Foram analisados 65 indivíduos divididos em três grupos, sendo (I) 46 pacientes portadores de câncer colorretal inclusos nos Critérios de Amsterdã, (II) dois familiares portadores de câncer colorretal e (III) 17 familiares sem câncer, todos da região metropolitana de Campinas, atendidos no Hospital de Clínicas da UNICAMP. Em 21 (45,65%) dos pacientes foram encontradas mutações deletérias. As mutações deletérias nos genes MLH1 e MSH2 estavam na proporção de 34,78% (16 pacientes) e 10,86% (5 pacientes), respectivamente. As mutações não deletérias nos genes MLH1 e MSH2 estavam na proporção de 65,22% dos pacientes (30 alterações) e 50% dos pacientes (23 alterações), respectivamente. Foi possível identificar 23 mutações potencialmente deletérias entre os pacientes com HNPCC por meio de sequenciamento dos genes MLH1 e MSH2, com uma porcentagem de detecção de 50%. Parece não haver variações nas características clínicas do tumor quando a mutação germinativa ocorre no gene MLH1 ou MSH2, com exceção da relação entre presença de mutação no gene MLH1 e idade de manifestação da doença. Como ocorre no resto do mundo a doença mostrou-se extremamente heterogênea em termos moleculares, pois apenas duas mutações se repetiram em dois pacientes. A partir da análise das duas famílias foi possível mostrar a dificuldade para estabelecer a presença da mutação germinativa deletéria que poderia levar à predisposição ao HNPCC, bem como a importância da analise familial no diagnóstico molecular dessa alteração / Abstract: Colorectal cancer has high importance because of its incidence and morbidity. Among the hereditary cases, the hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, accounts for about 5% of cases. In HNPCC, the genetic alteration inherited is the inactivation of one of the alleles of genes involved in the DNA repair, being hMSH2 and hMLH1 the main genes. The objective of this study is to investigate the presence of mutations in MLH1 and MSH2 in patients with clinical diagnosis of HNPCC, correlate clinical variables with the mutated gene, and investigate the relatives of patients with HNPCC who we had access to, in relation to germline mutations. Investigation of the mutations was performed by éxons direct sequencing, the promoter and junction regions. Sixty-five individuals, divided into three groups, were studied: (I) 46 patients with colorectal cancer included in the Amsterdam Criteria, (II) two family members of colorectal cancer patients and (III) 17 relatives without cancer, all of them treated at Hospital das Clínicas at UNICAMP and living in the Campinas metropolitan area. Deleterious mutations were found in 21 patients (45.65%). The ratio of deleterious mutations in MLH1 and MSH2 was 34.78% (16 patients) and 10.86% (5 patients) respectively. The ratio of non deleterious mutations in genes MLH1 and MSH2 was 65.22% of patients (30 alterations) and 50% of patients (23 alterations) respectively. Among patients with HNPCC, 23 potentially deleterious mutations were identified, via sequences of MLH1 and MSH2 with a 50% detection rate. It doesn't seem to appear variations in the clinical characteristics of the tumor when a germline mutation occurs in MLH1 or MSH2, with the exception of the relationship between the presence of mutation in the MLH1 gene and age of disease onset. As it occurs throughout the world, the disease present a his molecular extremely heterogeneoty, where only two mutations were repeated in two patients. The analysis of the two families demonstrated not only the difficulty to establish the presence of deleterious germline mutation that could lead to a predisposition to of HNPCC, but also the importance of familial analysis in molecular diagnostics of this alteration / Doutorado / Clinica Medica / Doutora em Ciências

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-24T17:10:12Z No. of bitstreams: 1 Arquivototal.pdf: 1560244 bytes, checksum: 121c5ccc4c7de435dbb28f4f19393b40 (MD5) / Made available in DSpace on 2018-04-24T17:10:13Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 1560244 bytes, checksum: 121c5ccc4c7de435dbb28f4f19393b40 (MD5) Previous issue date: 2017-05-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Basal cell carcinoma (BCC) is considered a tumor involving genetic, epigenetic and environmental factors. UVB radiation is considered to be one of the main physical agents involved in the carcinogenesis of the epidermis promoting DNA damage. In response to DNA damage the DNA repair mechanisms are activated, among them, the mismatch repair mechanism (MMR). The MMR system is an extremely important to maintain the fidelity of replication, however single nucleotide polymorphisms (SNPs) in genes involved in MMR should be considered an important factor for CBC carcinogenesis. The present study carried out the genotyping of SNPs rs560246973 (T> C), rs2303425 (-118 T> C) in the MSH2 gene and rs565410865 (G> T) in the MLH1 gene, in 100 samples of paraffin-embedded tissue from patients diagnosed with basal cell carcinoma. The results were obtained by means of the Dideoxy Single Genotype Allele Specific PCR-DSASP genotyping method. SNPs rs565410865 MLH1 and rs560246973 (C> T) MSH2 showed a statistically significant association with the susceptibility and risk of developing BCC. The results suggest that SNPs rs565410865 MLH1 and rs560246973 (C> T) MSH2 are potential molecular markers associated with susceptibility to the development of BCC in the analyzed samples. . / O carcinoma basocelular (CBC) é considerado um tumor que envolve fatores genéticos, epigenéticos e ambientais. Sendo a radiação UVB considerada um dos principais agentes físicos envolvido na carcinogênese da epiderme promovendo danos ao DNA. Em resposta aos danos no DNA os mecanismos de reparo do DNA são ativados, entre eles, o Mecanismo de Reparo de Mal Pareamento (MMR). O sistema MMR é uma via extremamente importante para manter a fidelidade da replicação, no entanto polimorfismos de nucleotídeo único (SNPs) em genes envolvidos no MMR deve ser considerado fator importante para carcinogênese do CBC. O presente estudo realizou a genotipagem dos SNPs rs560246973 (T>C), rs2303425 (-118 T>C) no gene MSH2 e rs565410865 (G>T) no gene MLH1, em 100 amostras de tecido parafinado de pacientes diagnosticados com carcinoma basocelular. Os resultados foram obtidos por meio do método de genotipagem Didesoxi Único Alelo Específico PCR- DSASP. Os SNPs rs565410865 MLH1 e rs560246973 (C>T) MSH2 apresentaram associação estatisticamente significativa à susceptibilidade e o risco de desenvolver CBC. Os resultados sugerem que SNPs rs565410865 MLH1 e rs560246973 (C>T) MSH2 são potenciais marcadores moleculares associados à susceptibilidade ao desenvolvimento do CBC nas amostras analisadas.