Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method

BACKGROUND: Neural stem cells (NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. RESULTS: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. CONCLUSIONS: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis

Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)

Background: Tumor heterogeneity in breast cancer tumors is today widely recognized. Most of the available knowledge in genetic variation however, relates to the primary tumor while metastatic lesions are much less studied. Many studies have revealed marked alterations of standard prognostic and predictive factors during tumor progression. Characterization of paired primary- and metastatic tissues should therefore be fundamental in order to understand mechanisms of tumor progression, clonal relationship to tumor evolution as well as the therapeutic aspects of systemic disease. Methods: We performed full exome sequencing of primary breast cancers and their metastases in a cohort of ten patients and further confirmed our findings in an additional cohort of 20 patients with paired primary and metastatic tumors. Furthermore, we used gene expression from the metastatic lesions and a primary breast cancer data set to study the gene expression of the AKAP gene family. Results: We report that somatic mutations in A-kinase anchoring proteins are enriched in metastatic lesions. The frequency of mutation in the AKAP gene family was 10% in the primary tumors and 40% in metastatic lesions. Several copy number variations, including deletions in regions containing AKAP genes were detected and showed consistent patterns in both investigated cohorts. In a second cohort containing 20 patients with paired primary and metastatic lesions, AKAP mutations showed an increasing variant allele frequency after multiple relapses. Furthermore, gene expression profiles from the metastatic lesions (n = 120) revealed differential expression patterns of AKAPs relative to the tumor PAM50 intrinsic subtype, which were most apparent in the basal-like subtype. This pattern was confirmed in primary tumors from TCGA (n = 522) and in a third independent cohort (n = 182). Conclusion: Several studies from primary cancers have reported individual AKAP genes to be associated with cancer risk and metastatic relapses as well as direct involvement in cellular invasion and migration processes. Our findings reveal an enrichment of mutations in AKAP genes in metastatic breast cancers and suggest the involvement of AKAPs in the metastatic process. In addition, we report an AKAP gene expression pattern that consistently follows the tumor intrinsic subtype, further suggesting AKAP family members as relevant players in breast cancer biology.Peer reviewe

Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)

Abstract Background Tumor heterogeneity in breast cancer tumors is today widely recognized. Most of the available knowledge in genetic variation however, relates to the primary tumor while metastatic lesions are much less studied. Many studies have revealed marked alterations of standard prognostic and predictive factors during tumor progression. Characterization of paired primary- and metastatic tissues should therefore be fundamental in order to understand mechanisms of tumor progression, clonal relationship to tumor evolution as well as the therapeutic aspects of systemic disease. Methods We performed full exome sequencing of primary breast cancers and their metastases in a cohort of ten patients and further confirmed our findings in an additional cohort of 20 patients with paired primary and metastatic tumors. Furthermore, we used gene expression from the metastatic lesions and a primary breast cancer data set to study the gene expression of the AKAP gene family. Results We report that somatic mutations in A-kinase anchoring proteins are enriched in metastatic lesions. The frequency of mutation in the AKAP gene family was 10% in the primary tumors and 40% in metastatic lesions. Several copy number variations, including deletions in regions containing AKAP genes were detected and showed consistent patterns in both investigated cohorts. In a second cohort containing 20 patients with paired primary and metastatic lesions, AKAP mutations showed an increasing variant allele frequency after multiple relapses. Furthermore, gene expression profiles from the metastatic lesions (n = 120) revealed differential expression patterns of AKAPs relative to the tumor PAM50 intrinsic subtype, which were most apparent in the basal-like subtype. This pattern was confirmed in primary tumors from TCGA (n = 522) and in a third independent cohort (n = 182). Conclusion Several studies from primary cancers have reported individual AKAP genes to be associated with cancer risk and metastatic relapses as well as direct involvement in cellular invasion and migration processes. Our findings reveal an enrichment of mutations in AKAP genes in metastatic breast cancers and suggest the involvement of AKAPs in the metastatic process. In addition, we report an AKAP gene expression pattern that consistently follows the tumor intrinsic subtype, further suggesting AKAP family members as relevant players in breast cancer biology

Role of endogenous and exogenous female sex hormones in arthritis and osteoporosis development in B10.Q-ncf1*/* mice with collagen-induced chronic arthritis

<p>Abstract</p> <p>Background</p> <p>Collagen-induced arthritis (CIA) is an often-used murine model for human rheumatoid arthritis (RA). Earlier studies have shown potent anti-arthritic effects with the female sex hormone estradiol and the selective estrogen receptor modulator (SERM) raloxifene in CIA in DBA/1-mice. B10.Q-ncf1^*/*mice are B10.Q mice with a mutated Ncf1 gene. In B10.Q-ncf1^*/*mice, CIA develops as a chronic relapsing disease, which more accurately mimics human RA. We investigated the role of endogenous and exogenous sex steroids and raloxifene in the course of this model of chronic arthritis. We also examined whether treatment would prevent the development of inflammation-triggered generalized osteoporosis.</p> <p>Methods</p> <p>Female B10.Q-ncf1^*/*mice were sham-operated or ovariectomized, and CIA was induced. 22 days later, when 30% of the mice had developed arthritis, treatment with raloxifene, estradiol or vehicle was started, and the clinical disease was evaluated continuously. Treatment was continued until day 56 after immunization. At termination of the experiment (day 73), bone mineral density (BMD) was analyzed, paws were collected for histological examination, and sera were analyzed for markers of cartilage turnover and pro-inflammatory cytokines.</p> <p>Results</p> <p>Raloxifene and estradiol treatment, as well as endogenous estrogen, decreased the frequency of arthritis, prevented joint destruction and countered generalized osteoporosis. These effects were associated with lower serum levels of the pro-inflammatory cytokine IL-6.</p> <p>Conclusions</p> <p>This is the first study to show that raloxifene and estradiol can ameliorate established erosive arthritis and inflammation-triggered osteoporosis in this chronic arthritis model. We propose that treatment with raloxifene could be a beneficial addition to the treatment of postmenopausal RA.</p

”Genusteorier och radikal ideologi maskerad som vetenskap” : En diskursteoretisk analys av förståelser av genusvetenskap i Genusdoktrinen och Om könets existens.

Under de senaste decennierna har en antigenusrörelse etablerats på allvar i Europa. En rörelse som motarbetar genusvetenskap och jämställdhetsarbete. I Sverige har vi hittills sett relativt lite av denna rörelse. Nu formulerar sig kritiken mot genusvetenskapen även i Sverige. Ordet genusdoktrin fogas till det engelska genderism och tyska Genderismus som beteckning på en konspiration mot samhället och dess institutioner. I denna uppsats har jag genomfört en diskursanalys av Ivar Arpi och Anna-Karin Wyndhamns bok Genusdoktrinen (2020) och Kajsa Ekis Ekmans bok Om könets existens (2021). Med hjälp av diskursteori enligt Laclau och Mouffe analyserar jag vilken syn på genusvetenskap, kön och jämställdhet som artikuleras i de två böckerna. En grundläggande fråga är om det handlar om en gemensam diskurs eller om artikulationerna skiljer sig åt på väsentliga punkter. De tecken som artikuleras i texterna analyserades med avseende på den vikt de har för diskursen och relationerna till varandra. I min analys framträder en gemensam grund i de två artikulationerna. Båda artikulerar en positivistisk vetenskapssyn. De menar att genusvetenskapen ifrågasätter den biologiska grunden för kön med hjälp av social-konstruktivism. De menar vidare att det är politiska beslut som lett till genusvetenskapens etablering och att ett binärt hierarkiskt kön är en förutsättning för jämställdhetsarbetet. De två artikulationerna skiljer sig åt på ett par punkter. Ekman menar i motsats till Arpi och Wyndhamn att jämställdhetsarbetet måste fortsätta. Där Arpi och Wyndhamn nöjer sig med att hävda ett binärt biologiskt kön som vetenskaplig slutpunkt menar Ekman att diskussionen fortgår och att alla feminister inte står bakom genus-vetenskapen.There is a growing anti gender movement in Europe that fights gender science and gender equality. It has not been that prominent in Sweden. Recently a new word genusdoktrin has been added to the English genderism and German genderismus as a label for a conspiracy against society and its institutions. In this thesis I have carried out a discourse analysis of the two books Genusdoktrinen (2020) by Ivar Arpi and Anna-Karin Wyndhamns and Om könets existens (2021) by Kajsa Ekis Ekman. Using theory of discourse by Laclau and Mouffe I analyze what view of gender studies that is articulated in the two books. An important question is whether they differ or not. The articulated signs were analyzed with regard to the weight carried they in the discourse. The two books articulate a positivist science view and claim that gender science is unscientific. They argue that gender science question the biology of sex from a social constructivist point of view. Moreover, they claim that political decisions made the establishment of gender science possible in Sweden and that a binary hierarchic sex is a prerequisite for a continued gender equality work. The articulations differ in a couple points. Ekman argues contrary to Arpi and Wyndhamn that we have not yet reached gender equality and that the work must continue. Arpi and Wyndhamn says that a binary biological sex is an unquestionable scientific fact. For Ekman, the political discussion continues, and she argues that not all feminists support gender science.2022-06-02</p

Molecular genetics of kidney cancer : Chromosomal regions of importance in the development of renal cell carcinoma

Molecular genetics of kidney cancer: Chromosomal regions of importance in tbe developmentof renal cell carcinoma by Rikard Erlandsson. Microbiology and Tumor Biology Center (MTC),Karolinska Institute, Box 280, S-171 77 STOCKHOLM, SwedenKeywords: Carcinoma, renal cell; Chromosomes, human, pair 3; Chromosome deletion; Genes, suppressor,tumor; Sequence analysis, DNA. Renal cell carcinoma (RCC) is a malignancy of the human kidney, occurring most commonlyduring the seventh and eighth decades of life. The incidence is high in the Nordic countries and thefive year survival is 40-50%. Smoking is a risk factor inferring a minor increase in risk. The bestestablished risk factor is obesity. It is usually sporadic and unilateral but can also occur in hereditaryforms characterized by an earlier age of onset, multiple primary, bilateral tumors. The bestestablished prognostic factor for RCC is stage. Other factors like cell type, age at diagnosis, andtumor size, have been implicated to affect survival. We wanted to establish, what chromosomal abnormalities are associated with the development ofRCC. Tumor and normal kidney tissues were examined from sporadic, non-hereditary RCC cases.86% of the examined patients had a detectable anomaly of chromosome 3p, manifested as a deletion,distal to band 3pl 1.2-pl3 combined with the non reciprocal translocation of a segment from anotherchromosome or monosomy 3. Restriction fragment length polymorphism (RFLP) analysis showedloss of D3F15S2 heterozygosity in 76%. D3S2 hetaozygosity was lost in 18%. The variability ofthebreakpoint between 3pl 1.2 and 3pl3 and the absence of a consistently translocated segment fromanother chromosome suggested a genetic loss mechanism. With the previously demonstratedinvolvement of the 3pl4.2 region in a familial case, these findings suggested that RCCs arise by thedeletion of a tumor suppressor gene located at the short arm of chromosome 3. Physical maps were constructed around loci D3F15S2, D3S2 and c-rafl on the short arm ofhuman chromosome 3 using pulsed field gradient gel electrophoresis. The normal restriction patternwas not altered by the t(3;8)(pl4.2;q24.1) characteristic for a hereditary form of renal cellcarcinoma, indicating that the breakpoint itself is not included in any of the mapped areas. We founda CpG island within the D3F 15S2 locus, suggesting,the presence of a functional gene in the region. The screening of a human placenta cDNA library with DNA probes derived from D3F15S2 led tothe isolation of several cDNA clones. They identified a 2.9 Kb long message in human placenta andkidney. A DNA homology search in GENBANK revealed that the gene encoded human acylpeptidehydrolase (APEH). The nucleotide identity between the rat and the human genes was 88%. Wefound an APEH expression below 20%, compared to normal kidney, in 73% of the studied primaryRCCs. No tumor had an expression above half of that in normal kidney. It was expressed in all celllines and normal tissues examined, except in one Burkitt's Iymphoma cell line (DG75). RCC appearsto be associated with a decrease of this enzyme activity. This reduction may reflect the existence of asmall, acetylated growth factor of importance in renal tumors. Since tumors in animal models are easier to manipulate we wanted to determine the chromosomallocation of APEH in rat and mouse. It is localized at mouse chromosome 9 and rat chromosome 8.This increases the number of conserved genes in this region. Other regions, beside chromosome 3, have also been found to harbour non-random losses inRCC. They might be associated with the progression of the tumor. Tumor and constitutional DNApairs were analysed with RFLP markers from all chromosomal arms and compared with clinicalclassifications and survival. Allelic imbalance (Al) for loci on 5q was found to be a negativeprognostic factor for RCC. Grade correlated with fractional allelic loss, loss of 14q and 17p Al butnot with specific aberrations on other chromosomes.ISBN 91-628-1863-5 Stockholm 199

”Genusteorier och radikal ideologi maskerad som vetenskap” : En diskursteoretisk analys av förståelser av genusvetenskap i Genusdoktrinen och Om könets existens.

Under de senaste decennierna har en antigenusrörelse etablerats på allvar i Europa. En rörelse som motarbetar genusvetenskap och jämställdhetsarbete. I Sverige har vi hittills sett relativt lite av denna rörelse. Nu formulerar sig kritiken mot genusvetenskapen även i Sverige. Ordet genusdoktrin fogas till det engelska genderism och tyska Genderismus som beteckning på en konspiration mot samhället och dess institutioner. I denna uppsats har jag genomfört en diskursanalys av Ivar Arpi och Anna-Karin Wyndhamns bok Genusdoktrinen (2020) och Kajsa Ekis Ekmans bok Om könets existens (2021). Med hjälp av diskursteori enligt Laclau och Mouffe analyserar jag vilken syn på genusvetenskap, kön och jämställdhet som artikuleras i de två böckerna. En grundläggande fråga är om det handlar om en gemensam diskurs eller om artikulationerna skiljer sig åt på väsentliga punkter. De tecken som artikuleras i texterna analyserades med avseende på den vikt de har för diskursen och relationerna till varandra. I min analys framträder en gemensam grund i de två artikulationerna. Båda artikulerar en positivistisk vetenskapssyn. De menar att genusvetenskapen ifrågasätter den biologiska grunden för kön med hjälp av social-konstruktivism. De menar vidare att det är politiska beslut som lett till genusvetenskapens etablering och att ett binärt hierarkiskt kön är en förutsättning för jämställdhetsarbetet. De två artikulationerna skiljer sig åt på ett par punkter. Ekman menar i motsats till Arpi och Wyndhamn att jämställdhetsarbetet måste fortsätta. Där Arpi och Wyndhamn nöjer sig med att hävda ett binärt biologiskt kön som vetenskaplig slutpunkt menar Ekman att diskussionen fortgår och att alla feminister inte står bakom genus-vetenskapen.There is a growing anti gender movement in Europe that fights gender science and gender equality. It has not been that prominent in Sweden. Recently a new word genusdoktrin has been added to the English genderism and German genderismus as a label for a conspiracy against society and its institutions. In this thesis I have carried out a discourse analysis of the two books Genusdoktrinen (2020) by Ivar Arpi and Anna-Karin Wyndhamns and Om könets existens (2021) by Kajsa Ekis Ekman. Using theory of discourse by Laclau and Mouffe I analyze what view of gender studies that is articulated in the two books. An important question is whether they differ or not. The articulated signs were analyzed with regard to the weight carried they in the discourse. The two books articulate a positivist science view and claim that gender science is unscientific. They argue that gender science question the biology of sex from a social constructivist point of view. Moreover, they claim that political decisions made the establishment of gender science possible in Sweden and that a binary hierarchic sex is a prerequisite for a continued gender equality work. The articulations differ in a couple points. Ekman argues contrary to Arpi and Wyndhamn that we have not yet reached gender equality and that the work must continue. Arpi and Wyndhamn says that a binary biological sex is an unquestionable scientific fact. For Ekman, the political discussion continues, and she argues that not all feminists support gender science.2022-06-02</p