| 1. |
- Aarnio, Riina
(författare)
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Self-sampling for HPV testing in primary cervical screening : Including clinical and health economic aspects
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Persistent infection with high-risk human papillomavirus (HPV) is a prerequisite for the development of cervical cancer. HPV testing has higher sensitivity for high-grade cervical intraepithelial neoplasia (CIN2+) than cytology, resulting in more effective screening. As HPV testing also offers an opportunity for self-sampling, it could serve as an even more effective and cost-effective method of cervical screening.First, we compared repeated self-sampling for HPV testing with Pap smear cytology in detection of CIN2+ in primary cervical screening for women aged 30–49 years (n=36 390). We found a more than twofold higher detection rate of CIN2+ and a fourfold higher detection rate of CIN2 with self-sampling compared with cytology. However, no difference was seen between the arms in the detection rate of CIN3+. It thus seems that CIN is detected at an earlier stage with self-sampling than with cytology, but the impact of this needs to be further explored.Second, as management of HPV-positive women with normal cytology results is a challenge, we wanted to evaluate the proportion of cases of histological CIN2+ in these women. In this prospective study we performed LEEP and found that 15% (6/40) of the women had undetected CIN2+. These findings can be used in counseling women about the risk of cervical cancer and helping clinicians in decisions on management.Third, we performed a cost-effectiveness analysis on the same study population as in Study I. Self-sampling for HPV testing resulted in a higher participation rate and more detected cases of CIN2+ at a lower cost and was regarded as more cost-effective than Pap smear cytology in cervical screening. These results can guide policy-makers when planning future screening programs.Fourth, we compared self-sampling with sampling by medical professionals for HPV testing in detection of CIN2+, using a combination of an FTA card as storage medium and a PCR-based HPV test (hpVIR) in women aged 30–60 years (n=11 951). No difference in the detection rates of histological CIN2+ was found between the arms.Taken together, self-sampling resulted in a higher participation rate than sampling by medical professionals in cervical screening and that triage with repeated self-sampling resulted in high compliance and detection rate of CIN2+. As repeated self-sampling for HPV testing was also cost-effective, it could serve as an attractive alternative in the development of future cervical screening programs. More research is needed on how to refine the management of HPV-positive women by self-sampling only.
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| 2. |
- Abdalaal, Hind
(författare)
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Deciphering molecular mechanisms in the evolution of new functions
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The evolution of new genes and functions is considered to be a major contributor to biological diversity in organisms. Through de novo origination, “duplication and divergence”, and horizontal gene transfer, organisms can acquire new genetic material that can evolve to perform novel functions. In this thesis, we investigate how functional trade-offs, “gene duplication and amplification”, and neutral divergence contribute to the emergence of a new function from a preexisting gene. In Paper i, we investigated the ability of Salmonella enterica to compensate for the loss of peptide release factor 1 (RFI) and the potential of peptide release factor 2 (RF2) to gain a new function to replace RFI. The amplification of RF2 and accumulated mutations within RF2 were the main evolutionary routes by which the fitness cost was restored. However, further characterization of the evolved RF2 showed a toxic effect to the cell due to the termination on tryptophan codon (UGG). This evolutionary trade-off - which we named “collateral toxicity” - might present a serious barrier for evolving an efficient RF2 to replace RF1.In Paper ii, we determined whether we could evolve a generalist enzyme with two functions (HisA + TrpF) from the specialist enzyme HisA, which can only synthesize histidine. In a previous study, we showed that HisA evolved a TrpF activity through strong trade-off trajectories. Here, we developed a selection scheme in which we constantly selected for keeping the original function (HisA), while intermittently selecting for the new function (TrpF). Our results showed that all evolved lineages shared the same “stepping stone” mutations in the hisA gene, which enabled them to grow well in the absence of both histidine and tryptophan. Additional accumulated mutations in the hisA gene gave the strains an increased ability to grow without both amino acids, indicating that the HisA enzyme evolved to be an efficient generalist. In Paper iii, we explored how differences between diverged orthologs influence evolvability. We generated artificial orthologs using a random mutagenesis approach. First, we screened for orthologs with a lower HisA activity and then selected for orthologs with a higher HisA activity; these steps were repeated in alternating rounds. We then tested the ability of each ortholog to evolve TrpF activity. As expected, the orthologs showed varying abilities to evolve the new function. In particular, orthologs with higher HisA activity levels showed both a higher potential to evolve the new function and a higher TrpF activity when they acquired the new function.
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| 3. |
- Abdulla, Maysaa
(författare)
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Prognostic signficance of tumor cell markers in diffuse large B-cell lymphoma with special emphasis on lymphoma localization
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diffuse large B-cell lymphoma (DLBCL) is the most common type of high-grade B-cell lymphoma with different clinical, morphological, immunophenotypical, and molecular features. DLBCL is curable in 60-70% of patients when treated with standard immunochemotherapy R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone).The main aim of this thesis is to identify prognostic factors in DLBCL by studying tumor markers (paper I and II), site of disease (paper III) and tumor microenvironment markers in primary DLBCL of the CNS (PCNSL) (paper IV) in order to better identify different risk groups of DLBCL patients.In papers I-III, we studied DLBCL patients treated homogeneously with R-CHOP. The negative prognostic impact of double protein expression of MYC and BCL2 so called “double-expressor lymphoma” (DEL) was a common finding in the three papers. In paper I, we detected DEL in 27% of patients, distributed with no significant difference between the germinal center derived B-cell subgroup (GCB) in 52% of cases and the non-GCB subgroup in 37% of cases. There was no significant difference in survival between GCB and non-GCB patients. The diagnosis in most of the patients with DEL was made on core needle biopsy in this paper. This finding was more thoroughly investigated in paper III with attention paid to the site of biopsy. In paper II, we evaluated the concordance of cell of origin (COO) assignment between gene expression profile (GEP) and immunohistochemistry (IHC) to identify the best predictor of survival in a DLBCL cohort including patients from Sweden and Denmark. The overall concordance between the two methods was 83%. We found that ABC/non-GCB subtype identified by both GEP and IHC is associated with the worst outcome. This finding indicates the importance of precise risk stratification in the era of precision medicine. DEL was more common in ABC patients categorized by GEP. In paper III, we identified abdominal lymph node involvement by radiological examination in 63% of DLBCL patients with an inferior survival, adverse clinical characteristics and significantly more frequent DEL. These findings may indicate a distinct biological behavior in patients with abdominal nodal disease. In paper IV, we demonstrated a significant association between IDO1 and PD-L1 in PCNSL patients. This finding indicates the crucial immunosuppressive role of these two molecules. In addition, in PCNSL low frequencies of MYC and BCL2 translocations and high frequency of BCL6 translocation was observed and DEL was detected in 49% of cases. Contrary to our results in systemic DLBCL in papers I-III, there was no significant prognostic impact of DEL in PCNSL.
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| 4. |
- Almstedt, Elin, 1988-
(författare)
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New targeted therapies for malignant neural tumors : From systematic discovery to zebrafish models
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancers in the neural system presents a major health challenge. The most aggressive brain tumor in adults, glioblastoma, has a median survival of 15 months and few therapeutic options. High-risk neuroblastoma, a childhood tumor originating in the sympathetic nervous system, has a 5-year survival under 50%, despite extensive therapy. Molecular characterization of these tumors has had some, but so far limited, clinical impact. In neuroblastoma, patients with ALK mutated tumors can benefit from treatment with ALK inhibitors. In glioblastoma, molecular subgroups have not yet revealed any subgroup-specific gene dependencies due to tumor heterogeneity and plasticity. In this thesis, we identify novel treatment candidates for neuroblastoma and glioblastoma. In paper I, we discover novel drug targets for high-risk neuroblastoma by integrating patient data, large-scale pharmacogenomic profiles, and drug-protein interaction maps. Using a novel algorithm, TargetTranslator, we identify more than 80 targets for this patient group. Activation of cannabinoid receptor 2 (CNR2) or inhibition of mitogen-activated protein kinase 8 (MAPK8) reduces tumor growth in zebrafish and mice models of neuroblastoma, establishing TargetTranslator as a useful tool for target discovery in cancer. In paper II, we screen approximately 1500 compounds across 100 molecularly characterized cell lines from patients to uncover heterogeneous responses to drugs in glioblastoma. We identify several connections between pathway activities and drug response. Sensitivity to proteasome inhibition is linked to oxidative stress response and p53 activity in cells, and can be predicted using a gene signature. We also discover sigma receptors as novel drug targets for glioblastoma and find a synergistic vulnerability in targeting cholesterol homeostasis.In paper III, we systematically explore novel targets for glioblastoma using an siRNA screen. Downregulation of ZBTB16 decreases cell cycle-related proteins and transcripts in patient-derived glioblastoma cells. Using a zebrafish assay, we find that ZBTB16 promotes glioblastoma invasion in vivo. In paper IV, we characterized the growth of seven patient-derived glioblastoma cell lines in orthotopic zebrafish xenografts. Using automated longitudinal imaging, we find that tumor engraftment strongly correlates with tumor initiation capacity in mice xenografts and that the heterogeneous response to proteasome inhibitors is maintained in vivo. In summary, this thesis identifies novel targets for glioblastoma and neuroblastoma using systematic approaches. Treatment candidates are evaluated in novel zebrafish xenograft models that are developed for high-throughput glioblastoma and neuroblastoma drug evaluation. Together, this thesis provides promising evidence of new therapeutic options for malignant neural tumors.
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| 5. |
- Álvarez-Aznar, Alberto
(författare)
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Cdc42, orchestrator of vascular morphogenesis in the retina
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cdc42 is a small GTPase that controls many cellular functions related to cytoskeletal dynamics, such as migration, polarity, and proliferation. Despite what we know of Cdc42 in other cell types, not much research has been done on the vasculature. This thesis describes the consequences of Cdc42 deletion in two vascular cell types—endothelial and mural cells—during developmental angiogenesis.In paper I, we demonstrate through a combination of in vitro, in silico, and in vivo assays, that Cdc42-deficient endothelial cells migrate less and fail to distribute normally in areas of naturally occurring high proliferation during angiogenesis, causing vascular malformations with enlarged lumens. In addition, these cells present impaired filopodia formation, a disadvantage for the tip cell position, disturbed axial polarity and altered junctions.With an in vivo approach, in paper III we demonstrate that the deletion of Cdc42 in mural cells has consequences on the morphogenesis of the retinal vasculature. Cdc42-deficient mural cells proliferate less and cannot keep up with the nascent angiogenic vasculature, which results in a complete pericyte loss at the sprouting front. Furthermore, we describe that mural cells contribute to the remodeling of the vasculature, also after the initial phases of angiogenesis.The CreERT2 system is frequently used for conditional gene deletion and lineage tracing. Tamoxifen administration allows spatiotemporally controlled recombination of fluorescent reporters, and tracing of the labeled cells. However, in the course of our studies, we observed tamoxifen-independent recombination. In paper II, we describe this phenomenon in detail, using different combinations of CreERT2 and fluorescent reporter lines. We conclude that tamoxifen-independent recombination is a widespread occurrence, and that fluorescent reporter lines present varying levels of susceptibility to it.In summary, the work presented here sheds new light on the role of Cdc42 in the vasculature. Additionally, this thesis describes in detail an important feature of CreERT2 and reporter lines that should be taken into account when performing lineage-tracing experiments.
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| 6. |
- Attwood, Misty M.
(författare)
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Membrane-bound proteins : Characterization, evolution, and functional analysis
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alpha-helical transmembrane proteins are important components of many essential cell processes including signal transduction, transport of molecules across membranes, protein and membrane trafficking, and structural and adhesion activities, amongst others. Their involvement in critical networks makes them the focus of interest in investigating disease pathways, as candidate drug targets, and in evolutionary analyses to identify homologous protein families and possible functional activities. Transmembrane (TM) proteins can be categorized into major groups based the same gross structure, i.e., the number of transmembrane helices, which are often correlated with specific functional activities, for example as receptors or transporters. The focus of this thesis was to analyze the evolution of the membrane proteome from the last holozoan common ancestor (LHCA) through metazoans to garner insight into the fundamental functional clusters that underlie metazoan diversity and innovation. Twenty-four eukaryotic proteomes were analyzed, with results showing more than 70% of metazoan transmembrane protein families have a pre-metazoan origin. In concert with that, we characterized the previously unstudied groups of human proteins with three, four, and five membrane-spanning regions (3TM, 4TM, and 5TM) and analyzed their functional activities, involvement in disease pathways, and unique characteristics. Combined, we manually curated and classified nearly 11% of the human transmembrane proteome with these three studies. The 3TM data set included 152 proteins, with nearly 45% that localize specifically to the endoplasmic reticulum (ER), and are involved in membrane biosynthesis and lipid biogenesis, proteins trafficking, catabolic processes, and signal transduction due to the large ionotropic glutamate receptor family. The 373 proteins identified in the 4TM data set are predominantly involved in transport activities, as well as cell communication and adhesion, and function as structural elements. The compact 5TM data set includes 58 proteins that engage in localization and transport activities, such as protein targeting, membrane trafficking, and vesicle transport. Notably, ~60% are identified as cancer prognostic markers that are associated with clinical outcomes of different tumour types. This thesis investigates the evolutionary origins of the human transmembrane proteome, characterizes formerly dark areas of the membrane proteome, and extends the fundamental knowledge of transmembrane proteins.
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| 7. |
- Backman, Samuel, 1994-
(författare)
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Molecular studies of endocrine tumors : Insights from genetics and epigenetics
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Endocrine tumors may be benign or malignant and may occur in any of the hormone producing tissues. They share several biological characteristics, including a low mutation-burden, and may co-occur in several hereditary tumor syndromes. The aim of this thesis was to identify genetic and epigenetic aberrations in endocrine tumors.In paper I we performed a comprehensive DNA methylation analysis of 39 pheochromocytomas/paragangliomas as well as 4 normal adrenal medullae on the HumanMethylation27 BeadChip array. We validated two previously described clusters based on DNA methylation with distinct genetic associations.In Paper II we performed a transcriptomic analysis of 15 aldosterone producing adenomas. CTNNB1-mutated tumors were found to form a distinct subgroup based on gene expression and to share gene expression similarities with non-aldosterone producing adrenocortical tumors with CTNNB1 mutations, including overexpression of AFF3 and ISM1.In paper III we used whole genome sequencing to identify germline genetic variants in 14 patients with Multiple Endocrine Neoplasia type 1 previously found to be wildtype for the MEN1 gene on routine clinical testing. Three patients were found to carry previously undetected MEN1 mutations. Two patients were confirmed to have phenocopies caused by variants affecting CASR or CDC73. In total 9/14 patients were not found to have a disease-causing germline variant, suggesting that the syndrome may in some cases be due to chance co-occurrence of several sporadic tumors.In paper IV RNA-Seq and whole genome sequencing of a cohort of SI-NETs selected on the basis of unusually short or long survival was performed in order to identify disease causing genes and potential prognostic factors. We confirmed known genetic aberrations and found rare variants in known cancer driver genes. Based on gene expression two clusters that differ in prognosis were detected. Moreover, through integration of copy number variation data and gene expression, we identied novel potential disease causing genes.
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| 8. |
- Bergfelt Lennmyr, Emma, 1984-
(författare)
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Registry-Based Studies in Adult Acute Lymphoblastic Leukemia in Sweden : Survival and Quality of Life
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Acute lymphoblastic leukemia (ALL), a common child malignancy, also constitutes a minor fraction of adult cancer with approximately 50 new cases per year in Sweden. While the five-year overall survival (OS) in pediatric ALL is more than 90%, the prognosis in adults is dismal. Using the Swedish ALL quality registry, this thesis investigates treatment and outcome of adult ALL according to national guidelines. In addition, the introduction of patient-reported outcome in the ALL and Acute Myeloid Leukemia registries is evaluated. In Paper I, measurement of minimal residual disease by flow cytometry was found to be feasible but not consistently applied in the 35 patients with Philadelphia (Ph)-negative B-ALL investigated. In Paper II, treatment, toxicity and outcome of 155 patients, 55-85 years (y) with ALL diagnosis between 2005 and 2012 were studied in detail by patient charts review. An age-adopted protocol recommended from 2009 did not result in better outcome. In Paper III, disease recurrence in the same cohort as Paper II was studied. The median overall survival (OS) after ALL relapse was 3.6 months. In Paper IV, the whole ALL registry was studied and OS was estimated in 930 adult patients diagnosed in the periods 1997-2006 and 2007-2015. Five year OS improved in patients 18-45y from 50% to 65%, in patients 46-65y from 25% to 46%, and in patients >65y from 7% to 11%. This demonstrates that young patients have the best prognosis, in part due to the introduction of a dose-intense “pediatric-like” chemotherapy protocol. Compared to women, middle-aged men were found to have a worse outcome.Historically, Philadelphia-positive (Ph-pos) ALL has a poor prognosis compared to Ph-negative ALL. In this material, the frequency of Ph-pos ALL was 34% of examined B-ALL. Analysis of the whole registry revealed that in 2007-2015, i.e. after the introduction of the tyrosine kinase inhibitor imatinib, Ph-pos ALL was no longer associated with inferior OS. In Paper V, ALL and Acute Myeloid Leukemia patients, six months after diagnosis, completed a web or paper questionnaire regarding quality of life, symptoms and experience with care. The response rate was 64%. Depression symptoms were frequent (18%), especially in young women who reported worrying about fertility.In summary, although OS in adult ALL has improved, more effective and less toxic therapies in upfront treatment are highly warranted. Collection of patient-reported outcome in a national quality registry is feasible and can add important aspects of cancer care that are not usually addressed.
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| 9. |
- Berggrund, Malin, 1989-
(författare)
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Identification and clinical implementation of biomarkers for cervical cancer
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Introduction of organised screening programs and prophylactic vaccination against human papilloma virus (HPV) have successfully reduced the incidence of cervical cancer globally. In Sweden, the incidence has been reduced by about 50 % since the introduction of the national screening programme in the late 1960’s. Despite these efforts, cervical cancer is still a major cause of cancer deaths globally.In order to reduce cervical cancer, the screening program should have a high participation rate and be based on a sensitive and specific screening test. About 20 % of women in Sweden do not participate in the organised screening program, and during the last years we have also seen a rise in cervical cancer cases in Sweden among women who participate in the screening program. Thus, there is a need to develop improved screening strategies that result in a higher participation rate, and are based on tests that more precisely identify women with high risk of developing cervical cancer. This includes searching for novel biological markers (biomarkers) that can be used to more accurately identify women with a high risk of developing cervical cancer.By offering women self-sampling for HPV analysis through direct mailing of sample kits with a chemically treated paper card, the FTA card, we were able to increase the participation rate in the screening program. We also found that the use of repeated self-sampling for women that were HPV positive in the primary screening sample increased the number of women detected with higher risk of cervical cancer (Paper II). Self-sampling was shown to be non-inferior to assisted sampling by midwife (Paper III). Using this sample collection device, we further investigated the association between increased risk of cervical cancer and HPV viral load (Paper V) as well as the vaginal microbiota (Paper VI). We also showed that proteins in the vaginal fluid can be studied using self-sampling and the FTA card (Paper I). Lastly, we identified plasma proteins that are associated with cervical cancer and could represent future biomarkers (Paper IV).This thesis has provided novel aspects on the present screening strategy, explored opportunities to increase the participation rate as well as examined possible future biomarkers for screening of cervical cancer.
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| 10. |
- Bergmann, Astrid, 1972-
(författare)
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Remote Ischemic Preconditioning and its Effects on the Respiratory System
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mechanical ventilation in itself can lead to pulmonary damage, and one-lung ventilation (OLV), necessary for thoracic surgery, accentuates this injury. Remote ischemic preconditioning (RIP) is a potential tool to reduce lung injury after mechanical ventilation, including OLV. However, current data on pulmonary RIP-effects are contradictory. Therefore, the overall purpose of this Ph.D. project was to assess the effects of RIP on the respiratory system. In Study I, in healthy spontaneously breathing volunteers, oxygenation was impaired early after RIP, which was possibly induced by transient ventilation-perfusion inequality. Studies II, III, and IV were performed in a porcine OLV model. In Study II, we found that RIP possibly enhances alveolar injury, but attenuates the immune response. In Study III, we confirmed that an immune response to RIP takes place, which shows a different time pattern in each cytokine, depending on the site of measurement as well. In Study IV, we studied the porcine model for eight hours and found that RIP improved oxygenation after two hours of OLV and impeded the decline of exhaled nitric oxide (NO) during and after OLV. These findings indicate that RIP mitigates hypoxic pulmonary vasoconstriction (HPV).In summary, RIP has a complex effect on the respiratory system, which partly explains the previous contradictory findings.
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