| 1. |
- Gohel, Priya, 1988-
(författare)
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The Drosophila POU/Oct factors: multifaceted proteins
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Dysregulation of physiological and cellular processes underlies various pathological conditions, including cancer and inflammatory disorders. Unraveling the molecular mechanisms driving these processes is crucial. The aim of this thesis was to investigate the roles of evolutionarily conserved POU/Oct transcription factors using Drosophila melanogaster as a model organism. The thesis highlights the functions of Nubbin (Nub) protein isoforms (Nub-PB and Nub-PD) in the regulation of cellular proliferation and mitosis, epithelial regeneration, and innate immune responses.In paper I, we demonstrate that Nub-PB acts as a potent transcriptional activator of immune and stress response genes, while Nub-PD represses their expression, indicating transcriptional antagonism by these Nub isoforms. Overexpression of Nub-PB in midgut cells effectively cleared local infections. However, prolonged Nub-PB overexpression caused a hyperactive immune response, leading to pro-inflammatory reactions, apoptosis, and reduced adult lifespan. These findings emphasize the importance of Nub protein isoforms in fine-tuning immune responses. In Paper II, we generated and phenotypically characterized a Nub-PB-specific mutant revealing impaired gut morphology, disorganized visceral muscles, and aberrant lineage specification in the midgut. In addition, it displays impaired immune gene activation, shortened lifespan, and enhanced reactive oxygen species (ROS) expression, which correlates with increased numbers of gut microbiota, featuring an important role of Nub-PB in intestinal epithelium homeostasis. In Paper III, we show that Nub-PD is necessary for proper nuclear divisions in transcriptionally silent pre-blastoderm embryos. The Nub-PD protein is enriched around the mitotic spindles in metaphase, requiring intact spindle microtubules. Live imaging of mitotic divisions revealed that Nub-PD is involved in the maintenance of spindle organization and its dynamics. We also infer similar mitotic roles for Nub-PD in S2 cells and for Oct1/POU2F1 in human cell culture. Our findings unveil a direct role of POU/Oct factors in proper mitotic progression, which may be evolutionarily preserved among insects and mammals.In Paper IV, we study how the loss of Nub and Pdm2 proteins affects wing growth and development. We found that Nub-PD is specifically required for cell proliferation, while balanced Nub-PB and Nub-PD expression levels at the dorso-ventral boundary are essential for correct wing margin formation. Overall, this thesis elucidates crucial roles of Drosophila POU proteins in maintaining immune and tissue homeostasis and aditionally uncovers mitotic roles of POU/Oct factors, suggesting new functions in regulation of cell proliferation and development.
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| 2. |
- Seyedoleslami Esfahani, Shiva, 1973-
(författare)
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Signaling pathways in Drosophila immunity
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Drosophila relies on innate immunity to protect itself from its hostile environment throughout its life cycle. Despite the remarkable progress in understanding many aspects of Drosophila immunity, there are still big gaps in our knowledge. The general aim of this thesis was to gain a better understanding about the regulatory mechanisms controlling gene expression in Drosophila, with a focus on immunity. To enable isolation of Drosophila genes involved in immunity, we developed a method that allows visualization of immune gene expression in large number of embryos. Reporter gene expression in wild type and mutant embryos was used to validate this approach, which should be a valuable complement to existing genetic and RNAi screens. Cactus, the Drosophila IκB protein, is known as a cytoplasmic inhibitor of Dif and Dorsal. We discovered that Cactus is also present in the cell nucleus. In response to Toll pathways signaling, cytoplasmic Cactus degrades rapidly in a proteasome-dependent manner, while a nuclear form of Cactus is stable and persists throughout signaling. This suggests that Cactus also has a function in the nucleus.A genome-wide RNAi-based screen was performed in cultured S2 cells. Several novel components of NF-κB pathways were isolated as putative regulators of Drosophila immunity. One of them, the G protein-coupled receptor kinase-2 (Gprk2), was shown to be required for Drosomycin expression and for resistance to infection. Gprk2 interacts with Cactus, but is not required for Cactus degradation upon Toll pathway activation. The dpld/wech gene was previously found to affect periferal nervous system development. Here, we show that wech belongs to the LIN-41 subclade of the TRIM protein superfamily, and contains target sites for microRNAs. Genetic and cell transfection assays confirmed that wech expression is regulated by the microRNA let-7. This seems to be a conserved regulatory mechanism throughout the LIN-41 subclade.
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| 3. |
- Sundström, Johan, Professor, 1971-, et al.
(författare)
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Risk factors for subarachnoid haemorrhage : a nationwide cohort of 950 000 adults
- 2019
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Ingår i: International Journal of Epidemiology. - : Oxford University Press. - 0300-5771 .- 1464-3685. ; 48:6, s. 2018-2025
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Subarachnoid haemorrhage (SAH) is a devastating disease, with high mortality rate and substantial disability among survivors. Its causes are poorly understood. We aimed to investigate risk factors for SAH using a novel nationwide cohort consortium.METHODS: We obtained individual participant data of 949 683 persons (330 334 women) between 25 and 90 years old, with no history of SAH at baseline, from 21 population-based cohorts. Outcomes were obtained from the Swedish Patient and Causes of Death Registries.RESULTS: During 13 704 959 person-years of follow-up, 2659 cases of first-ever fatal or non-fatal SAH occurred, with an age-standardized incidence rate of 9.0 [95% confidence interval (CI) (7.4-10.6)/100 000 person-years] in men and 13.8 [(11.4-16.2)/100 000 person-years] in women. The incidence rate increased exponentially with higher age. In multivariable-adjusted Poisson models, marked sex interactions for current smoking and body mass index (BMI) were observed. Current smoking conferred a rate ratio (RR) of 2.24 (95% CI 1.95-2.57) in women and 1.62 (1.47-1.79) in men. One standard deviation higher BMI was associated with an RR of 0.86 (0.81-0.92) in women and 1.02 (0.96-1.08) in men. Higher blood pressure and lower education level were also associated with higher risk of SAH.CONCLUSIONS: The risk of SAH is 45% higher in women than in men, with substantial sex differences in risk factor strengths. In particular, a markedly stronger adverse effect of smoking in women may motivate targeted public health initiatives.
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| 4. |
- Tang, Xiongzhuo, 1985-
(författare)
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Isoform-specific regulation of Drosophila gut immunity and regeneration by the POU/Oct transcription factor Nub/Pdm1
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Innate immune reactions protect organisms against a variety of infections. In metazoans, these reactions involve both cellular and humoral responses. The immune responses have to be well-tuned, as excessive immune activation is associated with tissue-specific pathologies. However, the transcriptional regulatory mechanisms underlying how immune responses are balanced are still not well understood. The aim of this study was to investigate the role of the Drosophila POU/Oct transcription factor Nubbin (Nub) in regulating Drosophila innate immunity, with a special focus on intestinal immune and epithelium homeostasis.In Paper I, we show that the nub gene encodes two independent transcription factor isoforms, Nub-PB and Nub-PD. The short isoform, Nub-PD, acts as a repressor of NF-κB/Relish-dependent antimicrobial peptide (AMP) gene expression in healthy flies. Furthermore, we demonstrate that Nub-PD directly binds to Oct sequence motifs located in the distal promoter region of several AMP genes, thereby inhibiting gene transcription. In addition, loss of Nub-PD diminishes the number of cultivatable gut bacteria, possibly due to high expression levels of AMP genes. In Paper II, we show that the large isoform, Nub-PB, in a sharp contrast to Nub-PD, activates AMP gene expression, both independently of and together with Relish. Importantly, Nub-PB and Nub-PD regulated the same target AMP gene expression antagonistically. In addition, Nub-PB expression in gut enterocytes (ECs) negatively correlated with gut microbial loads and host lifespan. Finally, we found that enforced Nub-PB expression in ECs promotes a pro-inflammatory signature and stimulated epithelium renewal. In Paper III, we show that Nub-PB and Nub-PD are not only expressed in differentiated gut ECs, but also present in midgut progenitor cells. Depletion of Nub-PB in gut progenitor cells results in hyperproliferation of intestinal stem cells (ISCs), via direct or indirect de-repression of Escargot expression. Furthermore, enforced Nub-PB expression in ISCs and enteroblasts (EBs) inhibited Notch RNAi-induced tumor formation. In addition, Nub-PD was necessary for both basal and infection-induced ISC proliferation. Strikingly, Nub-PB and Nub-PD regulated ISC proliferation in antagonistic manners. In Paper IV, we created a Nub-PB-specific mutant and found that this mutant impairs normal gut development, giving rise to short and wide anterior midguts. Furthermore, loss of Nub-PB promoted rapid ISC proliferation, increased EC delamination, and increased numbers of enteroendocrine cells in the anterior midgut.Taken together, we have characterized a novel isoform-specific regulatory mechanism, involved in maintaining Drosophila intestinal immune homeostasis and epithelial regeneration.
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| 5. |
- Antonsson, Åsa, 1972-
(författare)
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Regulation of NF-κB by Calmodulin
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cells experience numerous external signals which they must respond to. Such signals arriving at the cell surface are transduced via various signal transduction pathways and often ultimately result in regulation of transcription. NF-κB is a family of transcription factors involved in the regulation of genes important for processes such as immune and inflammatory responses, cell growth, development and cell survival. NF-κB proteins are normally kept inactive in the cytoplasm due to masking of their nuclear localisation signal (NLS) by inhibitory IκB proteins. A large number of stimuli lead to the activation of IκB-kinase (IKK). Active IKK phosphorylates IκB and thereby labels it for ubiquitination and, subsequently, degradation by the proteasome. Liberated NF-κB enters the nucleus, where it takes part in the regulation of its target genes.Calmodulin (CaM) is a ubiquitous Ca2+-binding protein which is considered to be the predominant intracellular Ca2+ sensor. CaM plays a major role in the Ca2+-dependent regulation of a wide variety of cellular processes, including transcription. CaM regulates transcription both indirectly through CaM-dependent kinases and phosphatases and directly through interaction with transcription factors.CaM was found to bind directly and in a Ca2+-dependent fashion to the two NF-κB family members c-Rel and RelA. The CaM-NF-κB interactions were strongly enhanced by NF-κB activating stimuli and this enhancement was blocked by the addition of IκB, suggesting that c-Rel and RelA can bind CaM after their signal-induced release from IκB. Compared to wild-type c-Rel, CaM binding-deficient mutants were shown to exhibit an increased nuclear accumulation and transcriptional activity on Ca2+-regulated cytokine promoters. The results suggest that CaM can inhibit transport of c-Rel, but not of RelA, to the nucleus and thereby differentially regulate the activation of NF-κB proteins following cell stimulation. CaM was also found to affect NF-κB activity indirectly through the action of a CaM-dependent kinase (CaMK). Studies of the events leading to IκBα phosphorylation revealed that CaM and CaMKII inhibitors blocked phorbol ester induced activation of IKK. Furthermore, CaM and CaMKII inhibitors also blocked T cell receptor/CD3 induced IκBα degradation, and expression of an inhibitor-resistant derivative of the γ isoform of CaMKII caused the inhibitors lose their effect on phorbol ester induced IκBα degradation. Finally, expression of a constitutively active CaMKII resulted in the activation of NF-κB. These results identify CaMKII as a mediator of IKK activation, specifically in response to T cell receptor/CD3 and phorbol ester stimulation.In conclusion, this thesis describes the identification of CaM as a dual regulator of NF-κB proteins, acting both directly and indirectly to affect the activity of this family of transcription factors.
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| 6. |
- Beskow, Anne, 1983-
(författare)
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The Path to Destruction : Understanding the mechanism and regulation of proteasomal degradation
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A majority of intracellular proteins are degraded by the ubiquitin proteasome system (UPS). In this thesis, both the mechanism of the degradation and the regulation of UPS have been investigated. The importance of the p97 ATPase for proteasomal degradation of cytosolic substrates was examined. It was shown that tightly folded model substrates were dependent on p97 for their degradation. In addition to this, it was shown that an extended flexible peptide sequence on the substrate allowed degradation to occur directly by the proteasome. We propose that p97 works as an unfoldase on substrates that lack initiation regions. These results were originally achieved with experiments using Drosophila melanogaster S2 cell culture. Corresponding experiments were carried out in human cell lines. We observed that the human proteasome also needed assistance from the human p97 protein complex when model substrates lacked unfolded tagged regions. To identify the transcription factor(s) that regulate the expression of proteasomal genes, a large scale RNAi screen was performed. A library consisting of dsRNA to all known and predicted transcription factors in Drosophila was used. Drosophila S2 cells expressing the cytosolic UbG76V-GFP substrate were used in the screen. Since thisfusion protein isdependent on the UPS for its degradation,failure in UPS can easily be detected viafluorescent stabilization.When dsRNA targeted the bZIP transcription factor Cnc-C,it lead to a reduction of the proteasome subunit protein levels as well as decreased mRNA levels. Phylogenetic analysis together with sequence alignments were used to learn how Cnc-C is related to the bZIP CNC genes in other metazoans and in particular mammalian cells. In mammalian cells, NF-E2, Nrf1, Nrf2 and Nrf3 are present and we propose that Cnc-C is related to a common ancestor transcription factor for all these four genes. This contradicts earlier studies proposing that Cnc-C is a homolog of the mammalian Nrf2 protein.In the last study, theproteasome recovery pathway was examined tounderstand which bZIPtranscription factor in human cells is responsible for the expression of proteasome genes after proteasome inhibition.Different cancer cell lines were used to examine theexpression level of proteasome genes after treating the cells with proteasome inhibitors when either the bZIP protein Nrf1 or Nrf2 wereknocked down. It was shown that Nrf1-/- cellslacked the ability toupregulate proteasome genes after proteasomeinhibition. In contrast, Nrf2-/- cells still had the capacity to restore proteasome levels. This lead to the conclusion thatNrf1 is responsible for the proteasome recovery pathway in mammalian cells.
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| 7. |
- Byhamre, Marja Lisa, et al.
(författare)
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Swedish snus use is associated with mortality : a pooled analysis of eight prospective studies
- 2020
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Ingår i: International Journal of Epidemiology. - : Oxford University Press. - 0300-5771 .- 1464-3685. ; 49:6, s. 2041-2050
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The health consequences of the use of Swedish snus, including its relationship with mortality, have not been fully established. We investigated the relationship between snus use and all-cause and cause-specific mortality (death due to cardiovascular diseases, cancer diseases and all other reasons, respectively) in a nationwide collaborative pooling project.METHODS: We followed 169 103 never-smoking men from eight Swedish cohort studies, recruited in 1978-2010. Shared frailty models with random effects at the study level were used in order to estimate adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs) of mortality associated with snus use.RESULTS: Exclusive current snus users had an increased risk of all-cause mortality (aHR 1.28, 95% CI 1.20-1.35), cardiovascular mortality (aHR 1.27, 95% CI 1.15-1.41) and other cause mortality (aHR 1.37, 95% CI 1.24-1.52) compared with never-users of tobacco. The risk of cancer mortality was also increased (aHR 1.12, 95% CI 1.00-1.26). These mortality risks increased with duration of snus use, but not with weekly amount.CONCLUSIONS: Snus use among men is associated with increased all-cause mortality, cardiovascular mortality, with death from other causes and possibly with increased cancer mortality.
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| 8. |
- Dantoft, Widad, 1982-
(författare)
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Role of POU/Oct transcription factors in Drosophila immunity
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In response to infection, Drosophila melanogaster relies on the ability to mount a robust and potent innate immune response, characterized by induction of cellular and humoral processes. While rapid immune induction is of utmost importance, it is equally important to restrict and repress immune gene expression, where and when it is not needed. The aim of my studies was to elucidate the in vivo role of the Oct1/POU2F1 homolog Nubbin (Nub), an evolutionarily conserved transcription factor, in innate immunity.The transcription factor Nub-PD, a product of the nub gene, was studied in wild type and mutant (nub1) flies, as well as in transgenic flies that either overexpress or down-regulate Nub-PD. Infection-studies were conducted after both oral and septic infection. Expression analysis of target genes using quantitative mRNA measurements and microarray analysis of wild type and nub1, reveal that Nub-PD acts as a bona fide repressor of NF-κB/Relish-dependent expression of immune defense genes, e.g. antimicrobial peptides. I show that Nub-PD represses transcription in uninfected flies by binding to oct DNA sequence motifs located upstream of a number of immune genes. In the event of infection, repression by Nub-PD is alleviated through rapid signal-dependent degradation, in a proteasome- and Imd-dependent manner, allowing initiation of antimicrobial peptide gene expression in both fat body and intestine. Lastly, we show that nub1 mutants also exhibit a shortened lifespan as well as elevated loads of gut bacteria with altered composition. We suggest that the aberrant immune gene activation, elevated bacterial loads, and altered bacterial composition in nub1 can collectively explain the shortened lifespan.In conclusion, this work reveals a novel function of Nub-PD as a negative regulator of immune and stress response genes in vivo. Furthermore, my work sheds light on the importance of Nub-PD in host survival and in supporting a balanced composition of the gut microbiota.
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| 9. |
- Junell, Anna, 1972-
(författare)
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Regulation of antimicrobial peptide gene expression in Drosophila melanogaster : Involvement of POU and NF-kB/Rel factors in innate immunity
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The fruit fly, Drosophila melanogaster, has a well-developed immune response, and microbial assault induces a rapid production of potent antimicrobial peptides (AMPs). The aim of this thesis work was to gain deeper knowledge of the regulation of AMPs in Drosophila, by the isolation and characterization of transcription factors involved in AMP gene expression. A yeast screen was designed and used to isolate Drosophila cDNAs coding for novel regulators of the CecropinA1 (CecA1) gene. Three transcription factors belonging to the POU domain (Pdm) family were isolated, Pdm1, Pdm2, and Drifter (Dfr), and subsequently verified as regulators of CecA1 in Drosophila cells. POU proteins are known to regulate a range of developmental processes, but this is the first finding of POU factors controlling AMP gene expression. Dfr and Pdm1 were further analyzed with respect to their in vivo function as AMP gene regulators. Over-expression of Dfr activated several AMP genes in non-infected flies, suggesting that Dfr is involved in constitutive expression of AMP genes. Dfr was shown to bind to a CecA1 upstream enhancer, to which the homeodomain protein Caudal (Cad) previously had been shown to bind. Co-expression of Dfr and Cad promoted very high CecA1 expression, indicating that these two transcription factors act synergistically on CecA1 in tissues where both are expressed. In Pdm1 mutant flies, several AMP genes were highly expressed prior to infection, indicating that Pdm1 functions as a repressor of those genes. However, at least one gene, AttacinA, required Pdm1 for its expression suggesting that Pdm1 has dual functions, acting both as a repressor and activator. Finally, the post-translational activation of the NF-κB/Rel protein Relish in response to infection was investigated in detail. Deletion mapping revealed different functional domains of Relish, and site-directed mutagenesis was used to exactly determine the residues required for endoproteolytic cleavage by a caspase.
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| 10. |
- Pålsson, Ylva, 1971-
(författare)
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A pathway into the profession : The use, feasibility and outcomes of a peer learning intervention for nursing students and newly graduated nurses
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overall aim of present thesis was to study the use, feasibility and outcomes of a peer learning intervention for nursing students and new graduates, including studies using a quasi-experimental (Study I and III), descriptive (Study II) and mixed-methods (Study IV) design. Data were collected using questionnaires, observations, checklists for intervention fidelity, individual interviews and group interviews. When studying peer learning outcomes among nursing students, peer learning seems to have a significant interaction effect on self-efficacy, based on a comparison of changes over time between the intervention (n=42) and comparison (n=28) groups. Studying each group separately over time, significant improvements were found in the intervention group on thirteen of the twenty variables, whereas the comparison group improved on four (Study I). Observations of how nursing students (n=16) used peer learning revealed that the student pairs collaborated to different extents and in different ways. All students were observed practicing several competencies together (Study II). Testing the peer learning model in new graduates’ workplace introduction (n=10) revealed that new graduates’ descriptions of peer learning were consistent with the theoretical description (Study III). Feasibility was tested in relation to compliance and acceptability, and lessons were learned. In Study IV, fidelity to the intervention was generally good. When first-line managers (n=8) described their perception of using the peer learning intervention with new graduates, predominantly positive outcomes were expressed. When examining the effect of peer learning in workplace introduction for newly graduated nurses (n=35), it was difficult to draw any conclusions due to recruitment problems (Study IV). The conclusions is that peer learning is a useful model for nursing students’ that seems to improve self-efficacy more than traditional supervision does. The model gives nursing students opportunities to practice several competencies on each other, and these competencies, e.g., leadership and organizational skills are useful in their future profession. The students practice teaching and supervision skills on each other, which seems to be a natural part of the peer relationship. Peer learning in the context of new graduates’ workplace introduction describes in a way consistent with the theoretical description of peer learning outcomes thus, also here it seems as a useful model. When developing and testing new interventions such as peer learning, it is important to do so systematically to minimize problems when conducting an evaluation, where the MRC framework can be useful. First-line managers generally expressed a positive attitude toward the peer learning model.
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