| 1. |
- Behboudi, Afrouz, 1967, et al.
(author)
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Molecular classification of mucoepidermoid carcinomas-prognostic significance of the MECT1-MAML2 fusion oncogene.
- 2006
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In: Genes, chromosomes & cancer. - : Wiley. - 1045-2257 .- 1098-2264. ; 45:5, s. 470-81
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Journal article (peer-reviewed)abstract
- Mucoepidermoid carcinomas (MECs) of the salivary and bronchial glands are characterized by a recurrent t(11;19)(q21;p13) translocation resulting in a MECT1-MAML2 fusion in which the CREB-binding domain of the CREB coactivator MECT1 (also known as CRTC1, TORC1 or WAMTP1) is fused to the transactivation domain of the Notch coactivator MAML2. To gain further insights into the molecular pathogenesis of MECs, we cytogenetically and molecularly characterized a series of 29 MECs. A t(11;19) and/or an MECT1-MAML2 fusion was detected in more than 55% of the tumors. Several cases with cryptic rearrangements that resulted in gene fusions were detected. In fusion-negative MECs, the most common aberration was a single or multiple trisomies. Western blot and immunohistochemical studies demonstrated that the MECT1-MAML2 fusion protein was expressed in all MEC-specific cell types. In addition, cotransfection experiments showed that the fusion protein colocalized with CREB in homogeneously distributed nuclear granules. Analyses of potential downstream targets of the fusion revealed differential expression of the cAMP/CREB (FLT1 and NR4A2) and Notch (HES1 and HES5) target genes in fusion-positive and fusion-negative MECs. Moreover, clinical follow-up studies revealed that fusion-positive patients had a significantly lower risk of local recurrence, metastases, or tumor-related death compared to fusion-negative patients (P = 0.0012). When considering tumor-related deaths only, the estimated median survival for fusion-positive patients was greater than 10 years compared to 1.6 years for fusion-negative patients. These findings suggest that molecularly classifying MECs on the basis of an MECT1-MAML2 fusion is histopathologically and clinically relevant and that the fusion is a useful marker in predicting the biological behavior of MECs.
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| 2. |
- Flaberg, Emilie
(author)
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Development of automated fluorescence microscopy methods to assist the diagnosis and treatment of human malignancies
- 2011
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Doctoral thesis (other academic/artistic)abstract
- Fluorescence microscopy is a powerful technique used in many biological laboratories. It is often used as an illustrative tool but in combination with manual efforts, valuable quantitative results can also be obtained. The potential of automated fluorescence microscopy has been clearly shown by the high-content and high throughput studies performed in the pharmaceutical industry and more recently in specialized high throughput imaging facilities. Computer controlled image capture and analysis not only scales up the actual image capture process and data production it also brings an important level of objectiveness into microscopy, a field otherwise suffering from an inherent human bias in the selection of which objects to study. There is a growing need for the technique to be available for “everyone”, as a regular microscopy tool in biological laboratories. This thesis illustrates that the combination of available and affordable techniques allow us to develop automated microscopy methods, including automated capture and image analysis routines that can be applied to answer different biological questions. We show that a visual programming language enable us to make flexible applications for automated microscopy, without detailed knowledge of complex computer languages (Paper I, Paper III - Paper VI). Using our automated fluorescence imaging and analysis method, in combination with a fluid dispenser robot for drug printing, we have developed an assay for high throughput drug sensitivity testing. In Paper III, we studied the effect of 29 different cytostatic drugs on 17 different lymphoblastoid cell lines. These cell lines are good in vitro models for the post-transplant lymphoproliferative disease (PTLD), and the aim of this study was to characterize which drugs would be optimal in the treatment of these patients. Summarizing the effect of each drug on all 17 LCLs, we identified epirubicin and paclitaxel as drugs that were highly effective (even at low concentrations). The same microscopy method was applied in combination with an analysis program that could identify and count differentially labelled cells in co-cultures. Using this approach, we could characterize which cytostatic drugs affect NK cell cytotoxicity negatively (Paper IV). Our data suggested that chemotherapy protocols including proteosome inhibitors (such as bortezomib) or anti-microtubule drugs (paclitaxel, docetaxel and vinblastine) may interfere with NK cell-based immunotherapy, if applied simultaneously. Taken together, these two studies suggested that our drug sensitivity test might prove useful in assisting the design of optimal and individualized treatment protocols for cancer patients. In Paper V and VI, we have applied the technique in an approach to study neighbour suppression by fibroblasts on tumor cell proliferation, in an attempt to mimic in vitro a possible microenvironmental control function in vivo. These two studies demonstrated that the culture of tumor cells on monolayer of primary fibroblasts, might lead to either growth stimulation or growth inhibition of tumor cells. Fibroblasts derived from the prostate of patients diagnosed with prostate carcinoma (potential CAFs) were the least inhibiting, and occasionally even promoting tumor cell proliferation. However, fibroblasts derived from the skin of pediatric patients were highly represented in the group of the most inhibitory fibroblasts. Our high-throughput study, with over 500 heterotypic cell combinations, with four independent measurements for each sample and individual counting of each tumor cell, indicated that the effect of fibroblasts on tumor cell proliferation was predominantly inhibitory. The technique allowed us to identify fibroblasts with consistently high and with consistently low inhibitory capacity. These are valuable fibroblasts to use in further studies to understand the mechanism behind inhibition and its possible clinical relevance. In Paper VI we specifically investigated the role of the structure of the fibroblast monolayer. We found that it was clearly not sufficient to have the inhibitory fibroblasts present in the mixed cell culture but that they also had to form a confluent and sufficiently matured, intact monolayer to exert the inhibitory effect. Our data suggested that structured accumulation and deposition of extracellular matrix molecules might provide orientation dependent behavioral cues to the tumor cell in an un-manipulated, inhibitory monolayer. Preliminary gene profiling suggested multiple differences in the signature of inhibitory and non-inhibitory fibroblasts.
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| 3. |
- Ribacke, Ulf, et al.
(author)
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Improved in vitro culture of plasmodium falciparum permits establishment of clinical isolates with preserved multiplication, invasion and rosetting phenotypes
- 2013
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In: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 8:7
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Journal article (peer-reviewed)abstract
- To be able to robustly propagate P. falciparum at optimal conditions in vitro is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved P. falciparum isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different in vitro growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of multiple infections were observed during static growth conditions and outgrowth and multiplication rates were inferior. Serum, as compared to Albumax, was found necessary for optimal presentation of PfEMP1 at the pRBC surface and/or for binding of serum proteins (immunoglobulins). Optimal in vitro growth conditions of P. falciparum therefore include orbital shaking (50 rev/min), human serum (10%) and a fixed gas composition (5% O2, 5% CO2, 90% N2). We subsequently established 100% of 76 frozen patient isolates and found rosetting with schizont pRBCs in every isolate (>26% schizont rosetting rate). Rosetting during schizogony was often followed by invasion of the bound RBC as seen by regular and time-lapse microscopy as well as transmission electron microscopy. The peripheral parasitemia, the level of rosetting and the rate of multiplication correlated positively to one another for individual isolates. Rosetting was also more frequent with trophozoite and schizont pRBCs of children with severe versus uncomplicated malaria (p<0.002; p<0.004). The associations suggest that rosetting enhances the ability of the parasite to multiply within the human host.
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| 4. |
- Vasquez, Alejandra, et al.
(author)
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Symbionts as major modulators of insect health: lactic Acid bacteria and honeybees.
- 2012
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:3
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Journal article (peer-reviewed)abstract
- Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.
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