| 1. |
- Ahlgren, Kerstin M.
(författare)
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Immunological Studies using Human and Canine Model Disorders
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The studies presented in this thesis focus on human and canine models for autoimmune disease, with the main aim to gain new knowledge about disease mechanisms and to further evaluate the dog as a model for autoimmune disease. Autoimmune Polyendocrine Syndrome type 1 (APS-1) is a hereditary human multiorgan disease caused by mutations in the autoimmune regulator (AIRE) gene. Hallmarks of APS-1 are chronic mucocutaneous candidiasis caused by Candida albicans, together with the autoimmune endocrine disorders hypoparathyroidism and adrenocortical failure. Many human diseases have an equivalent disease in dogs. Because humans share environment, and in part life style with the dogs they provide an interesting model for further genetic studies. Immune responses to Candida albicans in APS-1 patients displayed an increased secretion of the proinflammatory cytokine IL-17A and similar results were also found in AIRE deficient mice. Anticytokine autoantibodies to IL-17A, IL-17F and IL-22 were detected in APS-1 patients, and a radioligand binding assay for measuring these autoantibodies was developed and evaluated. In the canine studies we investigated whether canine diabetes mellitus could serve as a model for human autoimmune diabetes mellitus. Furthermore, we investigated type I IFN responses in Nova Scotia duck tolling retriever dogs with a systemic autoimmune disease resembling human SLE. Four assays were used in search for signs of humoral autoimmunity in diabetic dogs. However, no evidence for a type 1 diabetes-like phenotype in dogs was found. Sera from Nova Scotia duck tolling retrievers suffering from steroid-responsive meningitis arteritis elicited an increased expression of IFN-inducible genes in the canine MDCK cell line. This suggests that these dogs have an IFN signature, as seen in human SLE.
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| 2. |
- Berglund, Jonas, 1983-
(författare)
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Meiotic Recombination in Human and Dog : Targets, Consequences and Implications for Genome Evolution
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Understanding the mechanism of recombination has important implications for genome evolution and genomic variability. The work presented in this thesis studies the properties of recombination by investigating the effects it has on genome evolution in humans and dogs.Using alignments of human genes with chimpanzee and macaque orthologues we studied substitution patterns along the human lineage and scanned for evidence of positive selection. The properties mirror the situation in human non-coding sequences with the fixation bias ‘GC-biased gene conversion’ (gBGC) as a driving force in the most rapidly evolving regions. By assigning candidate genes to distinct classes of evolutionary forces we quantified the extent of those genes affected by gBGC to 20%. This suggests that human-specific characters can be prompted by the fixation bias of gBGC, which can be mistaken for selection.The gene PRDM9 controls recombination in most mammals, but is lacking in dogs. Using whole-genome alignments of dog with related species we examined the effects of PRDM9 inactivation. Additionally, we analyzed genomic variation in the genomes of several dog breeds. We identified that non-allelic homologous recombination (NAHR) via sequence identity, often GC-rich, creates structural variants of genomic regions. We show that these regions, which are also found in dog recombination hotspots, are a subset of unmethylated CpG-islands (CGIs). We inferred that CGIs have experienced a drastic increase in biased substitution rates, concurrent with a shift of recombination to target these regions. This enables recurrent episodes of gBGC to shape their distribution.The work presented in this thesis demonstrates the importance of meiotic recombination on patterns of molecular evolution and genomic variability in humans and dogs. Bioinformatic analyses identified mechanisms that regulate genome composition. gBGC is presented as an alternative to positive selection and is revealed as a major factor affecting allele configuration and the emergence of accelerated evolution on the human lineage. Characterization of recombination-induced sequence patterns highlights the potential of non-methylation and establishes unmethylated CGIs as targets of meiotic recombination in dogs. These observations describe recombination as an interesting process in genome evolution and provide further insights into the mechanisms of genomic variability.
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| 3. |
- Imsland, Freyja
(författare)
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Monogenic Traits Associated with Structural Variants in Chicken and Horse : Allelic and Phenotypic Diversity of Visually Appealing Traits
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Domestic animals have rich phenotypic diversity that can be explored to advance our understanding of the relationship between molecular genetics and phenotypic variation. Since the advent of second generation sequencing, it has become easier to identify structural variants and associate them with phenotypic outcomes. This thesis details studies on three such variants associated with monogenic traits.The first studies on Rose-comb in the chicken were published over a century ago, seminally describing Mendelian inheritance and epistatic interaction in animals. Homozygosity for the otherwise dominant Rose-comb allele was later associated with reduced rooster fertility. We show that a 7.38 Mb inversion is causal for Rose-comb, and that two alleles exist for Rose-comb, R1 and R2. A novel genomic context for the gene MNR2 is causative for the comb phenotype, and the bisection of the gene CCDC108 is associated with fertility issues. The recombined R2 allele has intact CCDC108, and normal fertility.The dominant phenotype Greying with Age in horses was previously associated with an intronic duplication in STX17. By utilising second generation sequencing we have examined the genomic region surrounding the duplication in detail, and excluded all other discovered variants as causative for Grey.Dun is the ancestral coat colour of equids, where the individual is mostly pale in colour, but carries intensely pigmented primitive markings, most notably a dorsal stripe. Dun is a dominant trait, and yet most domestic horses are non-dun in colour and intensely pigmented. We show that Dun colour is established by radially asymmetric expression of the transcription factor TBX3 in hair follicles. This results in a microscopic spotting phenotype on the level of the individual hair, giving the impression of pigment dilution. Non-dun colour is caused by two different alleles, non-dun1 and non-dun2, both of which disrupt the TBX3-mediated regulation of pigmentation. Non-dun1 is associated with a SNP variant 5 kb downstream of TBX3, and non-dun2 with a 1.6 kb deletion that overlaps the non-dun1 SNP. Homozygotes for non-dun2 show a more intensely pigmented appearance than horses with one or two non-dun1 alleles. We have also shown by genotyping of ancient DNA that non-dun1 predates domestication.
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| 4. |
- Jiang, Lin
(författare)
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Functional Studies of Genes Associated with Muscle Growth in Pigs and Hair Greying in Horses
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Domestic animals have become very different from their wild ancestors during domestication and animal breeding. This provides a good model to unravel the molecular mechanisms underlying phenotypic variation. In my thesis I have studied genes affecting two important traits, leanness in pigs and hair greying-associated melanoma in horses.In the first part of the thesis, I focused on an intronic mutation leading to more muscle growth and less fat deposition in domestic pigs to identify a transcription factor (TF) that binds to the regulatory element overlapping with the mutation. The aim has been to further study the function of the previously unknown TF in mouse myoblast cells and in insulin-producing cells (Paper I-III). We discovered a new TF ZBED6 binding to intron 3 of the IGF2 gene, in which a single nucleotide substitution in pigs abrogates the binding and causes increased leanness in domestic pigs. Silencing of ZBED6 expression in mouse myoblasts increased Igf2 expression, cell proliferation and migration, and myotube formation. This result is in line with the increased leanness phenotype in mutant pigs. Chromatin Immunoprecipitation-sequencing (ChIP-seq) using an anti-ZBED6 antibody identified 1200 ZBED6 target genes besides IGF2 and many are TFs controlling fundamental biological processes. In the first follow-up study we found ZBED6 mainly affected the expression of muscle protein genes by directly regulating Igf2 and Twist2 expression, in agreement with our previous observation of faster myotube formation in ZBED6-silenced cells. ChIP-seq with antibodies against six different histone modifications revealed that ZBED6 preferentially binds to active promoters and modulates transcriptional activity by a novel mechanism rather than by recruiting repressive histone modifications. The second follow-up study revealed that ZBED6 affects the morphology and insulin content and release in pancreatic ß cells.In the second part (Paper IV), we investigate the functional significance of an intronic duplication in the Syntaxin 17 (STX17) gene causing hair greying and melanoma in horses. We found two Microphtalmia-associated transcription factor (MITF) binding sites within the duplication and showed that the duplicated sequence up-regulates reporter gene expression in a melanocyte-specific manner both by reporter assays in mouse melanocytes and in transgenic zebrafish. These results established that the intronic duplication acts as a melanocyte-specific enhancer that becomes much stronger when it is duplicated.
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| 5. |
- Mathioudaki, Argyri, Ph.D student, 1986-
(författare)
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Complex disease genetics : Utilising targeted sequencing and homogeneous ancestry
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The complex disease investigations presented in this thesis aimed to provide new information regarding underlying genetics by using targeted sequencing and ethnically homogeneous cohorts. This work moved past current methodologies and addressed data stratification issues, that might have been hindering new findings. The results contribute to a more comprehensive view of the genetics of ankylosing spondylitis (AS) and breast cancer (BC), in Sweden.Paper-I presents a sex-stratified analysis of a Swedish AS cohort that incorporated both common and rare variants. Single variant and aggregate tests both showed different signals in AS male and female patients, previously masked. Specifically, the RUNX3 locus in males (univariate test: rs7414934, OR=2.58, p=1.7x10-5) and MICB in females (SKAT: 27 variants, p=1.2x10-6; rs3828903, OR=4.62, p=6.2x10-13) exceeded discovery thresholds. In the functional follow up of these loci, risk alleles appear to regulate the expression of genes in multiple tissues. Also, the results highlight the importance of disease regulation from different haplotypes and loci breakdown proved that Sweden’s genetic architecture might be critical for AS studies.Paper-II is a replication study, in our modest-sized Swedish cohort, of AS associations, previously discovered in populations of British origin, Initially, power calculations assessed that the Swedish cohort had the power to replicate only published associated markers with high effect (OR > 7), e.g., HLA-B but the replication analysis revealed three associated loci (ORrange:1.9-2.7). Notably, the multiplicated HLA-B marker (rs4349859) was not in HWE equilibrium. Population structure differences could not explain this replication pattern. However, sequencing resolution revealed fine-scale differences with repositioned association signals in the known loci. Specifically, the identification of two CCHCR1 protective haplotypes (OR: 0.14/0.3) that affect other MHC gene expression through eQTLs, provided the first suggestion of the differential function of known associated loci with cis gene regulation.Paper-III provides the first fingerprint of the somatic mutation profile of Swedish BC. The significantly mutated genes were PIK3CA (28%), TP53 (21%) and CDH1 (16%) while histone-modifying genes (e.g., KMT2C and ARID1A: together 28%) exhibited an increased somatic mutation prevalence, not observed previously. Additionally, within the patients that did not receive neoadjuvant treatment, there were distinct age groups with different mutational profiles and differential APOBEC signature driving genes.Taken together, these studies emphasize the contribution to the underlying genetics deriving from smaller ethnic populations, when assessed with a shift in methodology to account for biological bias, like sex and age. The results will hopefully assist and guide other genetic studies of human complex disease.
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| 6. |
- Megquier, Kate
(författare)
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Analysis of inherited and somatic variants to decipher canine complex traits
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis presents several investigations of the dog as a model for complex diseases, focusing on cancers and the effect of genetic risk factors on clinical presentation.In Papers I and II, we performed genome-wide association studies (GWAS) to identify germline risk factors predisposing US golden retrievers to hemangiosarcoma (HSA) and B-cell lymphoma (BLSA). Paper I identified two loci predisposing to both HSA and BLSA, approximately 4 megabases (Mb) apart on chromosome 5. Carrying the risk haplotype at these loci was associated with separate changes in gene expression, both relating to T-cell activation and proliferation.Paper II followed up on the HSA GWAS by performing a meta-analysis with additional cases and controls. This confirmed three previously reported GWAS loci for HSA and revealed three new loci, the most significant on chromosome 18. This locus contains several candidate genes with a clear role in carcinogenesis, including KMT5B and LRP5. Overall, carriers of the risk alleles at the top six loci are diagnosed with HSA earlier in life.In Paper III we investigated the somatic mutations which occur in HSA tumor tissue by performing tumor-normal exome sequencing of 47 golden retrievers. We identified 7 recurrently mutated genes, including the tumor suppressor TP53 (mutated in 59.6% of tumors) and oncogene PIK3CA (mutated in 29.8% of tumors). Additional somatically mutated genes overlap those found in human angiosarcomas, suggesting that angiosarcomas in dogs and humans are genetically very similar.In Paper IV, we investigated the variable penetrance of a SOD1 mutation in Pembroke Welsh corgis causing degenerative myelopathy (DM), a model of the human motor neuron disease amyotrophic lateral sclerosis (ALS). We discovered that regulatory variants near the SP110 gene were associated with an increased risk of DM and an earlier age at diagnosis, suggesting a role for immune response in the pathogenesis of the disease.Taken together, these findings provide new insight into the pathophysiology of both hemangiosarcoma and degenerative myelopathy, which could guide future diagnostics and therapeutic strategies both in humans and veterinary patients. In addition, they demonstrate the power of the dog as a biomedical model for human complex diseases.
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| 7. |
- Nordin, Jessika
(författare)
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Human leukocyte antigen in sickness and in health : Ankylosing spondylitis and HLA in Sweden
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human leukocyte antigen (HLA) plays a major role in keeping us healthy, but some of the HLA alleles can contribute to disease susceptibility. One example is HLA-B*27, which confers increased susceptibility of ankylosing spondylitis and represents one of the strongest genetic associations found in any common human disease. Ankylosing spondylitis shows a strong sex ratio skew (2-3:1 male to female) and studies confirm the existence of sexual-dimorphism in the presentation of this disease. The genetic predisposition for this, however, has not previously been studied. A Swedish ankylosing spondylitis population was sequenced with a targeted array to investigate the existence of sex-specific associations. RUNX3 was revealed to be associated in males by a univariate test, while aggregate tests revealed the HLA gene MICB to be associated in females. Functional validation demonstrated that the risk variants in RUNX3 increase expression, and MICB changed the transcription factor binding sites. Interestingly, since the disease involves bone changes, both RUNX3 and one of the MICB variants had effect in the bone cell line, SaOS-2.In order to help researchers obtain more controls for HLA analysis, an HLA allele bioresource (SweHLA) was generated from 1,000 Swedish genomes. The alleles were typed with three to four HLA typing software programs and results were combined by an n-1methodology. This produced high quality alleles where the bias from each software program was diminished.The methodology from SweHLA was utilised to study HLA in ankylosing spondylitis. To investigate both sex-specific predisposition and HLA-B*27 independence, samples were subdivided into two populations (one population with mixed HLA-B*27 positive and negative samples and one with only HLA-B*27 positive samples) that in turn were grouped by sex. In the mixed population, several alleles were replicated from previous studies. This study also revealed three female-specific alleles, two of which were new and one that had previously been associated to the severity of radiological changes. The HLA-B*27 population revealed a previously unknown protective allele, HLA-A*24:02. Through deeper examination of the HLA-B*27 population, two amino acids in HLA-A, position 119 in the whole set and position 180 in the male set, were revealed to be protective.This thesis brings new insight into the genetic predisposition for a sex-skewed disease, demonstrating how sexual-dimorphism can be reflected in the genetic predisposition, hopefully leading to more similar studies. It also highlights the importance of methodology and demonstrate the drastic biases that can be imparted by software programs.
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| 8. |
- Olsson, Mia, 1978-
(författare)
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Uncovering a Novel Pathway for Autoinflammation : With a Little Help from a Wrinkled Friend
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A major challenge in medical genetics is to identify the mutations underlying heritable diseases. Dogs are excellent genetic models in the search for causative mutations, as they constitute a large library of naturally occurring heritable diseases many of which are analogous to those suffered by man. In addition, these animals have a genome structure well suited to gene mapping. The Shar-Pei dog has two breed-specific features; a strongly selected for wrinkled skin and a high predisposition to an autoinflammatory disease (AID). Abnormalities in the innate immune system cause this type of disease, presenting as spontaneous attacks of inflammation. Persistent inflammation puts an affected Shar-Pei at risk of amyloidosis, organ failure and premature death. In humans, similar AIDs occur and for a majority of cases, no underlying genetic cause has yet been identified. The aim of this thesis was to use the Shar-Pei as a genetic model for autoinflammation in order to find new genes and signalling pathways involved in disease. In paper I, a pleiotropic mutation was identified that could explain both the wrinkled skin and autoinflammation in Shar-Pei. The mutation is associated with an up-regulation of Hyaluronic Acid Synthase 2 (HAS2). Increased expression of HAS2 leads to abnormal depositions of hyaluronic acid (HA) in the skin, resulting in the wrinkled appearance. When fragmented, HA also function as a damage signal sensed by the innate immune system which then responds with inflammation. By selecting for the wrinkled skin, the autoinflammatory disease has inadvertently been enriched in the breed. In paper II, five different inflammatory signs could be associated with the same genetic risk factor, allowing the introduction of a new terminology: Shar-Pei autoinflammatory disease (SPAID) to describe the whole disease complex. In addition, a modifying locus containing several biologically attractive genes was suggested to contribute to varying incidence of amyloidosis in Shar-Pei. In paper III, signs of pathological changes in HA metabolism were investigated in human AID. HA concentration was found to be both higher in subjects with no molecular diagnosis and also associated to disease activity and severity. Taken together, this suggests HA is also involved in human AID.
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| 9. |
- Ratnakumar, Abhirami
(författare)
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Detecting Signatures of Selection within the Dog Genome
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Deciphering the genetic basis of phenotypic diversity is one of the central aims of biological research. Domestic animals provide a unique opportunity for making substantial progress towards this goal. Intense positive selection has lead to a rich reservoir of phenotypes and underlying genotypes that can be interrogated using genetic tools to gain insight into the genetic basis of phenotypic diversity.The dog is the most phenotypically diverse mammal. It was domesticated from the grey wolf 11-30,000 years ago. After domestication, a period of intense breeding has lead to the massive phenotypic diversity seen amongst dog breeds today. These two phases of strong positive selection at domestication and at breed creation are likely to have left their signature on the genome. In this thesis, we have analysed genome-wide patterns to detect genomic regions involved in selection in both of these phases. We used whole genome sequences from 60 dogs and 12 wolves, to detect dog domestication selective sweeps. We find evidence for genes involved in memory formation, neurotransmission and starch digestion.To decipher the genetic signals underlying breed diversity, we used genome-wide genotype data from >170,000 SNPs in 509 dogs from 46 different breeds. We find evidence for genes under selection in many breeds, and only a few breeds. In addition, we identify novel sweeps underlying morphology and behavior.Recombination can influence the configuration of alleles present on a haplotype, and can thus increase or decrease the efficiency of selection. The PRDM9 protein has been shown to be important for determining recombination hotspot locations in humans and other mammals, but of all the mammals studied so far the dog is the only one to have a non-functional PRDM9.We used the genome-wide genotype data described above to characterise the fine scale recombination map in dogs. We find that recombination hotspots exist in dogs despite the absence of PRDM9. Moreover, we show that these hotspots are enriched for GC rich peaks and that these peaks are getting stronger over time. Our results show that the absence of PRDM9 has lead to the stabilisation of the recombination landscape in dogs.
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| 10. |
- Sakthikumar, Sharadha
(författare)
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Characterizing the spectrum of somatic alterations in canine and human cancers
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancers arise as a result of deleterious somatic alterations accumulating in the genome during the process of cell division. These alterations arise either via exposure to mutagens or due to errors occurring during DNA replication. In this thesis, a systematic exploration, from discovery to analyses of somatic alterations in three diverse cancers that affect dogs and humans, was undertaken.In Studies I and II, whole-exome sequencing of dogs affected by the cancers of osteosarcoma and hemangiosarcoma were done to delineate coding mutations that can contribute to their carcinogenesis. Besides, as these cancers mirror the corresponding human disease in clinical manifestation and histological features, a secondary objective was to confirm the molecular drivers found in the canines were also influencing factors in the human cancer(s).In the osteosarcoma investigations with three breeds, we found that tumors show a high frequency of somatic copy-number alterations, affecting key cancer genes. TP53 was the most frequently altered gene, akin to human osteosarcoma. The second most mutated gene, histone methyltransferase SETD2, has known epigenetic roles in multiple cancers but not in osteosarcoma. Our study highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine disease may serve as an excellent model for developing treatment strategies in both species.In the hemangiosarcoma study in golden retrievers, putative driver alterations were identified in the tumor suppressor TP53 and in genes involved in the cell cycle regulating PI3K pathway, including PIK3CA and PIK3R1. Furthermore, we find several somatic alterations between the dog hemangiosarcoma and human angiosarcoma overlap, indicating we can use the canine model to apprise the infrequently occurring human disease.In Study III, we implemented whole-genome sequencing methodologies to define both coding and non-coding alterations in the glioblastoma cancer genome. We find the coding somatic alterations recapitulate what has been previously seen for the cancer, including driver alterations in the genes of EGFR, PTEN, and TP53. Significantly though, using the concept of evolutionary constraint, we find an enrichment of non-coding mutations in regulatory regions, around GBM-implicated genes. The mutated regions include splice sites, promoters, and transcription factor binding sites, suggesting the importance of regulatory mutations for the pathogenesis of glioblastoma.Overall, the insights garnered from the above exome- and genome-wide surveys provide novel insights into unraveling some of the complexities associated with somatic genomic alterations in cancer genomes. It also convincingly underscores the benefits of using sequencing technologies to comprehend complex biological diseases.
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