No description available.

The major cell types in the canine colon musculature are interstitial cells of Cajal (ICC), circular muscle (CM) cells and longitudinal muscle (LM) cells. In isolated muscle strip studies, spontaneous membrane potential oscillations (slow waves) are generated in the submucosal border of the circular muscle where a gap junctionally well-coupled network of ICC and CM is found. CM devoid of LM and submucosal pacemaker region (CM preparations) are spontaneously quiescent. The research undertaken was to understand the mechanism of slow wave propagation into the circular muscle and to investigate the consequences to the electrical activity in CM after coupling with different electrical activities from different cells types. Our results show that CM cells, although spontaneously quiescent because of high K+ conductance, are excitable and can actively participate in slow wave generation. The electrical oscillations induced in the CM preparations could easily be potentiated by an L-type Ca2+ channel activator, Bay K 8644, and abolished by a L-type Ca2+ antagonist, D600, suggesting involvement of the conductance in the induced activity. The induced oscillations are similar to the SLAPs in the longitudinal muscle which shows that it is not necessary to have a specialized pacemaker cells for generating SLAPs. Using a cross sectioned preparation with all intact muscle layers, we also showed that the heterogeneity in the electrical activity of CM, such as: the resting membrane potential gradient, depolarization of plateau potential in the myenteric border and "apparent" decay in slow wave amplitude, is due to electrical interactions between different intrinsic activities from different cell types. Morphological evidence was obtained for the possible communication pathways in the submucosal and the myenteric borders of the circular muscle. Different coupling mechanisms in different areas were hypothesized. In addition, the 3-dimensional aspects of the submucosal ICC network in the ca.nine colon were clarified. / Thesis / Master of Engineering (ME)

No description available.

Somatic cell nuclear reprogramming (SCNR) by eggs is a way to forcibly transform the nuclei of terminally differentiated somatic cells to an embryonic state and gain totipotency (Gurdon et al., 1958). Additionally, induced pluripotency is applied to transform identities of somatic cells to induced pluripotent stem cells by overexpression of combinatorial Yamanaka factors (iPS, Takahashi et al., 2006). Although both approaches aim to derive cells with highest plasticity, the mechanisms and differences between these procedures are not yet clear. In my thesis, I used quantitative polymerase chain reaction (QPCR) and RNAseq plus 5-bromouridine 5'-triphosphate (BrUTP) pulldown to evaluate the transcriptional reprogramming by maternal factors and overexpressed transcription factors during SCNR by Xenopus oocytes, which are inactive in DNA replication and cell division. QPCR measures changes in the steady-state levels of transcripts within 2 days of nuclear transfer to Xenopus oocytes (Oocyte-NT). Three pairs of Yamanaka factor homologs were tested by QPCR and Yamanaka factor homologues regulated similar sets of pluripotency genes in mouse embryonic fibroblasts (MEFs). Pioneer factor mFoxA1 could not up-regulate most pluripotency genes and their binding targets of neurogenic genes in MEFs while pioneer factors are proposed to bind to their targets even if they may reside in inaccessible chromatin. This shows that the existence of other factors is needed at specified developmental stages. Hence, gene activation by transcription factors in the Oocyte-NT system requires not only corresponding binding on regulatory elements of linked genes but transcription cooperators to exert effective gene activation. Additionally, RNA-seq plus BrUTP pulldown measures the extent to which oocytes change the transcriptional activity of nuclei transplanted to oocytes. Through RNA-seq plus BrUTP pulldown, I compared the reprogrammed transcriptomes of embryonic and somatic cells, including mouse embryonic stem cells, mouse embryonic fibroblasts and mouse myoblasts, to demonstrate the effects of maternal factors and overexpression of transcription factors on gene activities during SCNR by oocytes. Importantly, I find that maternal factors of oocytes and the overexpression of transcription factors exert different strategies to reprogram somatic cells. Oocyte factors reprogram the donor cell nuclei to an oocyte-steady state except for the SCNR resistance genes and xklf2-HA overexpression enhances expression of reprogrammable genes and activates SCNR resistance genes.

In a case-control study comprising 589 cases of male lung cancer in northern Sweden longitudinal data concerning occupations, employments and smoking habits were collected by questionnaires. Pipe smoking was as common as cigarette smoking and gave very similar relative risk. The pipe smoking cases, however, had significantly higher mean age and mean smoking years at the time of diagnosis than the cigarette smoking cases. In ex- smokers, the relative risk gradually decreased from 5 years after smoking cessation but this decrease was much less pronounced in ex-pipe smokers than in ex-cigarette smokers. High relative risks were obtained for small cell and squamous cell carcinomas. For adenocarcinoma the relative risk was considerably lower but still significantly increased. The population etiologic fraction attributable to smoking was about 80% in this series. Some occupational groups (underground miners, copper smelter workers, electricians and plumbers) exposed to previously known lung carcinogenic agents had considerably increased odds ratios, which persisted after adjustment for smoking. A slightly elevated odds ratio was observed in a group of blue collar workers potentially exposed to lung carcinogenic agents but this elevation generally disappeared after adjustment for smoking. For two specific subgroups, asphalt and concrete workers and pulp workers, the overrisk persisted after adjustment for smoking. Farmers and foresters had strikingly low odds ratios, which could only partly be explained by their more moderate smoking habits. The population etiologic fraction attributable to occupation was in the reported material assessed to 9 per cent. Professional drivers had higher average tobacco consumption than non-drivers, which explained the slightly increased crude odds ratio found for the occupational group as a whole. Smoking drivers in an upper age group (70 and over), however, had a high relative risk of lung cancer, while in a lower age group (under 70) no significant increase was found. The results in the older age group suggested a multiplicative effect between smoking and the occupational exposure. The study clearly verified the increased lung cancer risk in underground miners. An obvious dose-response relation was found with high risk after long time exposure. All analyses concerning underground miners suggested an interaction of a multiplicative type between underground mining and smoking in the causation of lung cancer. The cases of small cell carcinoma among the underground miners had shorter average latency time and in contrast to the other part of the material, shorter average age than the cases with epidermoid cancer. / <p>S. 1-40: sammanfattning, s. 41-136: 6 uppsatser</p> / digitalisering@umu

No description available.

No description available.

Single-cell RNA-sequencing makes possible to study the gene expression at the level of individual cells. However, one of the main challenges of the single-cell RNA-sequencing analysis today, is the identification and annotation of cell types. The current method consists in manually checking the expression of genes using top differentially expressed genes and comparing them with related cell-type markers available in scientific publications. It is therefore time-consuming and labour intensive. Nevertheless, in the last two years,numerous automatic cell-type identification and annotation tools which use different strategies have been created. But, the lack of specific comparisons of those tools in the literature and especially for immuno-oncologic and oncologic purposes makes difficult for laboratories and companies to know objectively what are the best tools for annotating cell types. In this project, a review of the current tools and an evaluation of R tools were carried out.The annotation performance, the computation time and the ease of use were assessed. After this preliminary results, the best selected R tools seem to be ClustifyR (fast and rather precise) and SingleR (precise) for the correlation-based tools, and SingleCellNet (precise and rather fast) and scPred (precise but a lot of cell types remains unassigned) for the supervised classificationtools. Finally, for the marker-based tools, MAESTRO and SCINA are rather robust if they are provided with high quality markers.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 &mug/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 &mug/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-&kappaB were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-&kappaB was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-&kappaB are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.