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  • Public defence: 2018-12-15 11:00 Ihresalen, Uppsala
    Odyniec, Pawel
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Languages, Department of Linguistics and Philology.
    Engaging Advaita: Conceptualising liberating knowledge in the face of Western modernity2018Doctoral thesis, monograph (Other academic)
    Abstract [en]

    This dissertation is a study of modern Indian philosophy. It examines three engaging articulations of the Advaitic notion of liberating knowledge or brahmajñāna provided by three prominent Indian philosophers of the twentieth century, namely, Badrīnāth Śukla (1898-1988), Krishnachandra Bhattacharyya (1875-1949), and Sarvepalli Radhakrishnan (1888-1975). Particular attention is paid to the existing relation between their distinctive conceptualisations of liberating knowledge and the doxastic attitudes that these authors professed towards the Sanskrit intellectual past of South Asia and the presence of the Western Other.

    In the main, it argues that the profound differences to be found, on the one hand, in Śukla’s elucidation of this key Advaitic notion and, on the other, in Bhattacharyya’s and Radhakrishnan’s take on the same, betray their commitment to two radically different doxastic attitudes. Classifying these into (a) non-dialogical and (b) dialogical in relation to the Western Other as well as into (c) exegetic and (d) hermeneutic in relation to the Sanskrit intellectual past of South Asia, it contends that, in comparison to the conceptual scheme and the parameters of intelligibility that shaped and underpinned the precolonial Advaitic discourse on brahmajñāna in Sanskrit, there is a certain kind of epistemic discontinuity in the dialogical cum hermeneutic stance taken by Bhattacharyya and Radhakrishnan that is not to be found in the non-dialogical cum exegetic engagement enacted by Śukla. It suggests that this particular sort of discontinuity, absent as it is from Śukla’s elucidation of the process of knowing Brahman, reflects the far-reaching commitment of Krishnachandra Bhattacharyya and Sarvepalli Radhakrishnan to appropriate the precolonial Advaitic notion of liberating knowledge hermeneutically and to conceptualise it in the face of Western modernity, that is, rendering it meaningful in terms and within the parameters of intelligibility of the Western Other in order to contest what they took to be a troublesome predicament of Western modernity.

    By examining their ways of engaging with the Advaitic notion of liberating knowledge, this dissertation contributes to the on-going debate about the nature and the driving forces of modern Indian philosophy.

  • Public defence: 2018-12-15 13:00 Auditorium minus, Uppsala
    Grip, Olivia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Vascular Surgery. Välj ....
    Acute limb ischaemia: Treatment, outcome and time trends2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Acute limb ischaemia (ALI) is a frequent emergency associated with high rates of amputation and death. Traditionally, patients with ALI were treated with open surgical removal of the occlusion or bypass surgery. During the past few decades, new endovascular techniques developed.  

    No larger studies have investigated the optimal contemporary treatment for patients with ALI. Today, there are no international consensus for recommendations for the treatment of ALI, leaving it open to every surgeon or department to decide the best treatment option. 

    This thesis aimed to study patients with ALI as a means to extend the understanding of this group of patients, as well as to investigate treatment options. Data sources included hospital charts or information was gathered from the Swedish nationwide Vascular Registry (Swedvasc), the Swedish Population Registry for deaths and the Swedish Patient Registry for amputations.

    Paper I compared the results from thrombolysis with and without continuous heparin infusion in 749 thrombolytic procedures, concluding that both treatment strategies were equally successful in achieving revascularisation, with acceptable complication rates for both strategies. Continuous heparin infusion during intra-arterial thrombolysis offered no advantage. Although the regime with continuous heparin infusion was associated with a higher frequency of bleeding complications (p<0.001), this difference disappeared after adjustment for confounders.

    Paper II studied long-term outcome after thrombolysis and showed that thrombolytic therapy achieves good medium- and long-term clinical outcome, which reduces the need for open surgical treatment in most patients. More than half of the patients in paper II did not require any surgical reintervention or amputation in their remaining lifetime or during a mean of 6.2 years of follow-up. Long-term outcome differed between the aetiological groups. This information is valuable when deciding on the optimal treatment strategy for patients with ALI.

    Paper III compared outcomes after open and endovascular revascularisation for the treatment of ALI in 16,229 patients treated in 1994-2014. The large propensity score-matched nationwide cohort study revealed that endovascular treatment of ALI was associated with significantly better short-term survival and amputation-free survival compared with open revascularisation.

    Paper IV investigated acute aortic occlusion (AAO) and subsequent ALI. This study showed that mortality after AAO is high but has improved in the past 20 years. The proportion of AAO secondary to occluded graft/stent/stentgrafts increases over time as a result of the endovascular shift in treating aortic diseases and the proportion of AAO secondary to native artery thrombosis decreases.

    Taken together, the main findings of this thesis demonstrate a gradual improvement in survival and that endovascular techniques are becoming more frequently used as a first- line treatment of patients with ALI.

    List of papers
    1. Outcome and complications after intra-arterial thrombolysis for lower limb ischaemia with or without continuous heparin infusion
    Open this publication in new window or tab >>Outcome and complications after intra-arterial thrombolysis for lower limb ischaemia with or without continuous heparin infusion
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    2014 (English)In: British Journal of Surgery, ISSN 0007-1323, E-ISSN 1365-2168, Vol. 101, no 9Article in journal (Refereed) Published
    Abstract [en]

    Background

    Thrombolysis is a common treatment for acute leg ischaemia. The purpose of this study was to evaluate different thrombolytic treatment strategies, and risk factors for complications

    This was a retrospective analysis of prospective databases from two vascular centres. One centre used a higher dose of heparin and recombinant tissue plasminogen activator (rtPA).

    Results

    Some 749 procedures in 644 patients of median age 73 years were studied; 353 (47·1 per cent) of the procedures were done in women. The aetiology of ischaemia was graft occlusion in 38·8 per cent, acute arterial thrombosis in 32·2 per cent, embolus in 22·3 per cent and popliteal aneurysm in 6·7 per cent. Concomitant heparin infusion was used in 63·2 per cent. The mean dose of rtPA administered was 21·0 mg, with a mean duration of 25·2 h. Technical success was achieved in 80·2 per cent. Major amputation and death within 30 days occurred in 13·1 and 4·4 per cent respectively. Bleeding complications occurred in 227 treatments (30·3 per cent). Blood transfusion was needed in 104 (13·9 per cent). Three patients (0·4 per cent of procedures) had intracranial bleeding; all were fatal. Amputation‐free survival was 83·6 per cent at 30 days at both centres. In multivariable analysis, preoperative severe ischaemia with motor deficit was the only independent risk factor for major bleeding (odds ratio (OR) 2·98; P <0·001). Independent risk factors for fasciotomy were severe ischaemia (OR 2·94) and centre (OR 6·50). Embolic occlusion was protective for major amputation at less than 30 days (OR 0·30; P = 0·003). Independent risk factors for death within 30 days were cerebrovascular disease (OR 3·82) and renal insufficiency (OR 3·86).

    Conclusion

    Both treatment strategies were successful in achieving revascularization with acceptable complication rates. Continuous heparin infusion during intra‐arterial thrombolysis appeared to offer no advantage.

    Place, publisher, year, edition, pages
    Uppsala: , 2014
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-360872 (URN)10.1002/bjs.9579 (DOI)
    Available from: 2018-09-19 Created: 2018-09-19 Last updated: 2018-12-12Bibliographically approved
    2. Long-term Outcome after Thrombolysis for Acute Lower Limb Ischaemia
    Open this publication in new window or tab >>Long-term Outcome after Thrombolysis for Acute Lower Limb Ischaemia
    2017 (English)In: European Journal of Vascular and Endovascular Surgery, ISSN 1078-5884, E-ISSN 1532-2165, Vol. 53, no 6, p. 853-861Article in journal (Refereed) Published
    Abstract [en]

    Objectives: The purpose was to study long-term outcome after thrombolysis for acute arterial lower limb ischaemia, and to evaluate the results depending on the underlying aetiology of arterial occlusion.

    Methods: This was a retrospective study of patients entered into a prospective database. Patients were identified in prospective databases from two vascular centres, including a large number of variables. Case records were analysed retrospectively. Through cross linkage with the Population Registry 100% accurate survival data were obtained. Between January 2001 and December 2013, 689 procedures were included. The aetiology of ischaemia was graft/stent/stent graft occlusion in 39.8%, arterial thrombosis in 27.7%, embolus in 25.1% and popliteal aneurysm in 7.4%.

    Results: The mean follow-up was 59.4 months (95% CI, 56.1-62.7), during which 32.9% needed further re interventions, 16.4% underwent amputation without re-intervention, and 50.7% had no re-intervention. The need for re-intervention during follow-up was 48.0% in the graft/stent occlusions group, 34.0% of the popliteal aneurysm group, 25.4% in the thrombosis group, and 16.3% in the embolus group (p < .001). The overall primary patency rates were 69.1% and 55.9% at 1 and 5 years, respectively. Primary patency at 5 years was higher for the embolus group (83.3%, p = .002) and lower for the occluded graft/stent group (43.3%, p < .001). Secondary patency rates were 80.1% and 75.2% at 1 and 5 years, respectively, without difference between the subgroups. The amputation rate was lower in the embolic group at 1 and 5 years (8.1% and 11.1%, respectively, p = .001). Survival was higher in the group with occluded.popliteal aneurysms at 5 years (83.3%, p = 0.004). Amputation free survival was 72.1% and 45.2% at 1 and 5 years; lower in the occluded graft/stent group at five years (37.9%, p = .007).

    Conclusion: Intra-arterial thrombolytic therapy achieves good medium and long-term clinical outcome, reducing the need of open surgical treatment in most patients.

    Keywords
    Thrombolysis, Acute limb ischaemia, Long-term outcome, Long-term follow-up, Amputation free survival, Aetiological subgroups
    National Category
    Cardiac and Cardiovascular Systems Surgery
    Identifiers
    urn:nbn:se:uu:diva-329713 (URN)10.1016/j.ejvs.2017.02.003 (DOI)000403518800018 ()28291676 (PubMedID)
    Available from: 2017-10-03 Created: 2017-10-03 Last updated: 2018-10-24Bibliographically approved
    3. Open versus endovascular revascularization in the treatment of acute lower limb ischaemia
    Open this publication in new window or tab >>Open versus endovascular revascularization in the treatment of acute lower limb ischaemia
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    2018 (English)In: British Journal of Surgery, ISSN 0007-1323, E-ISSN 1365-2168, Vol. 105, no 12, p. 1598-1606Article in journal (Refereed) Published
    Abstract [en]

    Background: Consensus is lacking regarding intervention for patients with acute lower limb ischaemia (ALI). The aim was to study amputation-free survival in patients treated for ALI by either primary open or endovascular revascularization.

    Methods: The Swedish Vascular Registry (Swedvasc) was combined with the Population Registry and National Patient Registry to determine follow-up on mortality and amputation rates. Revascularization techniques were compared by propensity score matching 1:1.

    Results: Of 9736 patients who underwent open surgery and 6493 who had endovascular treatment between 1994 and 2014, 3365 remained in each group after propensity score matching. Results are from the matched cohort only. Mean age of the patients was 74⋅7 years; 47⋅5 per cent were women and mean follow-up was 4⋅3 years. At 30-day follow-up, the endovascular group had better patency (83⋅0 versus 78⋅6 per cent; P < 0⋅001). Amputation rates were similar at 30 days (7⋅0 per cent in the endovascular group versus 8⋅2 per cent in the open group; P = 0⋅113) and at 1 year (13⋅8 versus 14⋅8 per cent; P = 0⋅320). The mortality rate was lower after endovascular treatment, at 30 days (6⋅7 versus 11⋅1 per cent; P < 0⋅001) and after 1 year (20⋅2 versus 28⋅6 per cent; P < 0⋅001). Accordingly, endovascular treatment had better amputation-free survival at 30 days (87⋅5 versus 82⋅1 per cent; P < 0⋅001) and 1 year (69⋅9 versus 61⋅1 per cent; P < 0⋅001). The number needed to treat to prevent one death within the rst year was 12 with an endovascular compared with an open approach. Five years after surgery, endovascular treatment still had improved survival (HR 0⋅78, 99 per cent c.i. 0⋅70 to 0⋅86) but the difference between the treatment groups occurred mainly in the rst year.

    Conclusion: Primary endovascular treatment for ALI appeared to reduce mortality compared with open surgery, without any difference in the risk of amputation.

    National Category
    Medical and Health Sciences Surgery
    Research subject
    Medical Science; Surgery
    Identifiers
    urn:nbn:se:uu:diva-363353 (URN)10.1002/bjs.10954 (DOI)000447124200007 ()
    Available from: 2018-10-17 Created: 2018-10-17 Last updated: 2018-12-12Bibliographically approved
    4. Time-trends and management of acute aortic occlusion: a 21-years´ experience
    Open this publication in new window or tab >>Time-trends and management of acute aortic occlusion: a 21-years´ experience
    (English)In: Article in journal (Other academic) Submitted
    Abstract [en]

    Background: Acute aortic occlusion (AAO) is a rare and potentially catastrophic event. The aim was to study epidemiology and outcome of surgical treatment of AAO in a population-based cohort.

    Method: The Swedish nationwide vascular database (Swedvasc) was used to identify cases, and the Population Registry to study long-term survival.

    Results: During the 21-year study-period (1994-2014), 715 cases of AAO were included with a yearly incidence of 3.8 per million inhabitants. Mean age was 69.7 years, 50.5% were women and mean follow-up was 5.2 years. Most patients presented with bilateral acute limb ischemia. The aetiology for AAO was in-situ thrombosis in 64.1%, saddle embolus in 21.3% and occluded graft/stent/stentgrafts in 14.7%. The proportion of occluded grafts/stent/stentgrafts increased during the study period with a simultaneous reduction in the proportion of in-situ thrombosis.

    The most commonly used methods for revascularization were thromboembolectomy (32.0%), thrombolysis (22.4%), axillary-bifemoral bypass (18.9%) and aorto-biiliacal/bifemoral bypass (18.2%). The choice of revascularization technique depended on the aetiology of the occlusion.

    Amputation was preformed in 8.6%, and 19.9% of the patients died within 30-days after surgery. The 30-days mortality rate was lower after occluded grafts/stents/stentgrafts (9.5%) and higher after saddle embolus (30.9%, p<0.001). There was a reduction in overall 30-days mortality over time (25.0% 1994-2000 versus 15.3% 2008-2014, p=0.008). Long-term survival revealed significant differences between the subgroups, although the difference occurred early after the event (log-rank, p<0.001).

    Conclusion: Mortality after AAO is improving over time, but remains high. The proportion of AAO secondary to occluded grafts/stents/stentgrafts increased over time.

    National Category
    Medical and Health Sciences
    Research subject
    Surgery
    Identifiers
    urn:nbn:se:uu:diva-363356 (URN)
    Available from: 2018-10-17 Created: 2018-10-17 Last updated: 2018-10-24Bibliographically approved
  • Public defence: 2018-12-17 13:00 Häggsalen, Uppsala
    Edin, Elle
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Composite Regenerative Scaffolds2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Regenerative medicine and tissue engineering solutions of heavily innervated tissues are at this point lacklustre. This thesis expands our knowledge of appropriate acellular scaffolds for tissue repair in general and nerve regeneration in particular. The optimal surgical procedure for the implantation of artificial extracellular matrix (ECM) was evaluated for recombinant human collagen (RHCIII) implants. Suturing techniques, as well as the usage of human amniotic membrane “bandages” were evaluated. While complete regeneration of corneal tissues occurred, only slight differences in effects of surgical technique could be found.

    The safety and efficacy of clinical trials using mesenchymal stromal cells (MSCs) was evaluated by conducting a systematic review and meta-analysis. MSC therapy was shown to be safe, with no increases mortality, rehospitalization or adverse events. There was also an indication of efficacy, as the overall mortality in the studies included was significantly smaller in the MSC treated group.

    Multicomponent hydrogel capsules encapsulating single cells were developed. Capsules manufactured from gelatin, agarose and fibrinogen were compared to pure gelatin capsules. The composite capsules successfully delayed cell release and prolonged cell survival.

    Surface patterning of collagen based biomimetic corneas was performed by microcontact printing. The ability of different sizes of fibronectin stripes to stimulate cell adhesion and proliferation was compared. The patterned surfaces improved cell adhesion, as well as proliferation markers.

    Conductive polymer composites were manufactured for use as nerve guides. The guides were created from electrospun polycaprolactone fibers coated with a series of different poly(3,4-ethylenedioxythiophene) films. A comparison of nerve progenitor growth and differentiation on the composite fibers was performed. Both the effects of fiber composition and MSC co-culture was investigated, with or without electrostimulation. MSC treatments and polymer coating was both important for nerve cell differentiation and growth.

    List of papers
    1. Effect of Surgical Technique on Corneal Implant Performance.
    Open this publication in new window or tab >>Effect of Surgical Technique on Corneal Implant Performance.
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    2014 (English)In: Translational vision science & technology, ISSN 2164-2591, Vol. 3, no 2, article id 6Article in journal (Refereed) Published
    Abstract [en]

    PURPOSE: Our aim was to determine the effect of a surgical technique on biomaterial implant performance, specifically graft retention.

    METHODS: Twelve mini pigs were implanted with cell-free, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross-linked recombinant human collagen type III (RHCIII) hydrogels as substitutes for donor corneal allografts using overlying sutures with or without human amniotic membrane (HAM) versus interrupted sutures with HAM. The effects of the retention method were compared as well as the effects of collagen concentration (13.7% to 15% RHCIII).

    RESULTS: All implanted corneas showed initial haze that cleared with time, resulting in corneas with optical clarity matching those of untreated controls. Biochemical analysis showed that by 12 months post operation, the initial RHCIII implants had been completely remodeled, as type I collagen, was the major collagenous protein detected, whereas no RHCIII could be detected. Histological analysis showed all implanted corneas exhibited regeneration of epithelial and stromal layers as well as nerves, along with touch sensitivity and tear production. Most neovascularization was seen in corneas stabilized by interrupted sutures.

    CONCLUSIONS: This showed that the surgical technique used does have a significant effect on the overall performance of corneal implants, overlying sutures caused less vascularization than interrupted sutures.

    TRANSLATIONAL RELEVANCE: Understanding the significance of the suturing technique can aid the selection of the most appropriate procedure when implanting artificial corneal substitutes. The same degree of regeneration, despite a higher collagen content indicates that future material development can progress toward stronger, more resistant implants.

    Keywords
    biomaterials, biosynthetic cornea, corneal regeneration, corneal transplantation, recombinant human collagen
    National Category
    Surgery
    Identifiers
    urn:nbn:se:uu:diva-364450 (URN)10.1167/tvst.3.2.6 (DOI)24749003 (PubMedID)
    Available from: 2018-10-29 Created: 2018-10-29 Last updated: 2018-10-29
    2. Conductive PEDOT based coatings on microfibrous scaffolds: a nerve guide component
    Open this publication in new window or tab >>Conductive PEDOT based coatings on microfibrous scaffolds: a nerve guide component
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    2018 (English)Manuscript (preprint) (Other (popular science, discussion, etc.))
    National Category
    Biomaterials Science
    Identifiers
    urn:nbn:se:uu:diva-364452 (URN)
    Available from: 2018-10-29 Created: 2018-10-29 Last updated: 2018-10-29
    3. Mesenchymal Stromal Cells for the Treatment of Ischemic Injury and Vascular Trauma: A Systematic Review and Meta-Analysis
    Open this publication in new window or tab >>Mesenchymal Stromal Cells for the Treatment of Ischemic Injury and Vascular Trauma: A Systematic Review and Meta-Analysis
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    2018 (English)Manuscript (preprint) (Other (popular science, discussion, etc.))
    National Category
    Other Clinical Medicine
    Identifiers
    urn:nbn:se:uu:diva-364451 (URN)
    Available from: 2018-10-29 Created: 2018-10-29 Last updated: 2018-10-29
    4. Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications.
    Open this publication in new window or tab >>Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications.
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    2015 (English)In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 12, p. 70-80, article id S1742-7061(14)00486-3Article in journal (Refereed) Published
    Abstract [en]

    The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes for the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500μm thick RHCIII-MPC constructs comprising over 85% water are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, unlike the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30μm wide fibronectin stripes enhanced cell attachment and showed the highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas.

    Keywords
    Collagen, Cornea, Hydrogel, Laser profiling, Surface modification
    National Category
    Biomaterials Science
    Identifiers
    urn:nbn:se:uu:diva-364449 (URN)10.1016/j.actbio.2014.10.035 (DOI)25448347 (PubMedID)
    Available from: 2018-10-29 Created: 2018-10-29 Last updated: 2018-10-29
    5. Controlled Delivery of Human Cells by Temperature Responsive Microcapsules.
    Open this publication in new window or tab >>Controlled Delivery of Human Cells by Temperature Responsive Microcapsules.
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    2015 (English)In: Journal of Functional Biomaterials, ISSN 2079-4983, E-ISSN 2079-4983, Vol. 6, no 2, p. 439-53Article in journal (Refereed) Published
    Abstract [en]

    Cell therapy is one of the most promising areas within regenerative medicine. However, its full potential is limited by the rapid loss of introduced therapeutic cells before their full effects can be exploited, due in part to anoikis, and in part to the adverse environments often found within the pathologic tissues that the cells have been grafted into. Encapsulation of individual cells has been proposed as a means of increasing cell viability. In this study, we developed a facile, high throughput method for creating temperature responsive microcapsules comprising agarose, gelatin and fibrinogen for delivery and subsequent controlled release of cells. We verified the hypothesis that composite capsules combining agarose and gelatin, which possess different phase transition temperatures from solid to liquid, facilitated the destabilization of the capsules for cell release. Cell encapsulation and controlled release was demonstrated using human fibroblasts as model cells, as well as a therapeutically relevant cell line-human umbilical vein endothelial cells (HUVECs). While such temperature responsive cell microcapsules promise effective, controlled release of potential therapeutic cells at physiological temperatures, further work will be needed to augment the composition of the microcapsules and optimize the numbers of cells per capsule prior to clinical evaluation.

    Keywords
    cell delivery, cell encapsulation, human fibroblast, human umbilical vein endothelial cells, hydrogel, microcapsules, temperature responsive
    National Category
    Biomaterials Science
    Identifiers
    urn:nbn:se:uu:diva-364448 (URN)10.3390/jfb6020439 (DOI)26096147 (PubMedID)
    Available from: 2018-10-29 Created: 2018-10-29 Last updated: 2018-10-29
  • Public defence: 2018-12-17 13:00 B42, Uppsala
    Papoutsoglou, Panagiotis
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Regulation of TGFβ signaling by long non-coding RNAs and ADP-ribosylation2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transforming growth factor β (TGFβ) signaling pathways participate in embryonic development and tissue homeostasis and have a dual role in cancer. TGFβ acts as a tumor suppressor that promotes cell cycle arrest and apoptosis at initial stages of tumorigenesis. In contrast, TGFβ, induces epithelial to mesenchymal transition (EMT), a normal embryonic process which is employed by advanced cancers, in order to acquire mesenchymal traits and metastasize.

    Bone morphogenetic protein (BMP) family members belong to the TGFβ superfamily and are involved in cell differentiation, development and bone formation.

    Non-coding RNAs (ncRNAs) are not translated into proteins, are important regulators of gene expression and physiological processes and are often de-regulated in cancer. They control gene expression through physical association with chromatin, DNA, RNA molecules or proteins.

    Poly(ADP-ribose) polymerases (PARPs) catalyze the poly (ADP)-ribosylation of proteins, whereas the enzyme poly(ADP-ribose) glycohydrolase (PARG) removes ADP-ribose units. Members of the PARP family function in the DNA damage response, regulation of transcription and cell death.

    In this thesis, we investigated the importance of the TGFβ signaling pathway in regulating the expression of long non-coding RNAs (lncRNAs). We identified TGFβ-regulated lncRNAs and observed that a substantial number of them act in a feedback loop to modulate the magnitude of TGFβ signaling. Interestingly, the nuclear lncRNA TGFB2-antisense RNA 1 (TGFB2-AS1) is induced by TGFβ and negatively regulates expression of members of the TGFβ and BMP pathways, through interaction with EED, a protein of the polycomb repressor complex 2 (PRC2). Also, TGFβ signaling promoted the expression of mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG), which enhanced TGFβ signaling and affected TGFβ-mediated cell cycle arrest. The MIR100HG-derived miRNAs let-7a-2-3p, miR125b-5p and miR-125b-1-3p, were also induced by TGFβ. In contrast, the long intergenic non-protein coding RNA 707 (LINC00707), was reduced in response to TGFβ and affected the expression of a group of genes related to inflammatory responses and interferon-γ (IFN-γ) signaling.

    We also report that TGFβ and BMP pathways are regulated by ADP-ribosylation of Smad proteins, the signaling mediators of these pathways. We observed that PARP1 and PARP2 attenuated, while PARG favored TGFβ signaling. Furthermore, PARP1 negatively regulated BMP signaling, by ADP-ribosylating Smad1 and Smad5, whereas PARG enhanced BMP signaling by de-ADP-ribosylating Smads.

    Collectively, we provide evidence that lncRNAs and ADP-ribosylating enzymes modulate TGFβ and BMP signaling pathways and propose models for their molecular mechanisms and functional roles.

    List of papers
    1. The TGFB2-AS1 lncRNA regulates TGFβ signaling by modulating corepressor activity
    Open this publication in new window or tab >>The TGFB2-AS1 lncRNA regulates TGFβ signaling by modulating corepressor activity
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    2018 (English)Article in journal (Refereed) Submitted
    Abstract [en]

    LncRNAs regulate cell function through many physiological processes. We have identified lncRNAs whose expression is regulated by transforming growth factor β (TGFβ), by a transcriptomic screen. We focused on TGFB2-antisense RNA1 (TGFB2-AS1), which was induced by TGFβ through Smad and protein kinase pathways, and exhibited predominantly nuclear localization. Depleting TGFB2-AS1 enhanced TGFβ/Smad-mediated transcription and expression of the TGFβ-target genes FN1 and SERPINE1. Overexpression of TGFB2-AS1 reduced expression of these genes, attenuated TGFβ-induced cell growth arrest, and altered BMP and Wnt pathway gene profiles. Mechanistically, TGFB2-AS1 mainly via its 3’ terminal region, bound to EED, an adaptor of the Polycomb repressor complex 2 (PRC2), promoting repressive histone H3K27me3 modifications at TGFβ-target gene promoters. Silencing EED or inhibiting PRC2 methylation activity, partially rescued TGFB2-AS1 mediated gene repression. Our observations support the notion that TGFB2-AS1 is a TGFβ-induced lncRNA with inhibitory functions on TGFβ and BMP pathways output, constituting an auto-regulatory negative feedback mechanism that balances TGFβ- and BMP-mediated responses.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-363700 (URN)
    Available from: 2018-10-20 Created: 2018-10-20 Last updated: 2018-12-03
    2. TGFβ signaling down-regulates LINC00707 to inhibit inflammatory responses
    Open this publication in new window or tab >>TGFβ signaling down-regulates LINC00707 to inhibit inflammatory responses
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    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The class of long non-coding RNAs (lncRNAs) consists of RNA molecules, which lack protein coding potential and regulate a wide variety of cellular processes. At the molecular level, lncRNAs act as regulators of gene expression by interacting with chromatin, other types of RNA or proteins. Transforming growth factor β (TGFβ) plays pivotal roles in diverse biological processes, such as cell growth arrest, embryonic development and regulation of the immune system. In this study, we describe the long intergenic non-protein coding RNA 707 (LINC00707) as a TGFβ responsive gene. By combining transcriptomic data from human keratinocytes and glioblastoma cancer stem cells, we observed that TGFβ signaling down-regulates the expression of LINC00707. RNA sequencing revealed that in keratinocytes knockdown of LINC00707 or stimulation by TGFβ, affected expression of genes involved in inflammatory responses and interferon-γ-mediated signaling. In summary, we suggest that the immune suppressive actions of TGFβ involve suppression of the pro-inflammatory LINC00707.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-363898 (URN)
    Available from: 2018-10-19 Created: 2018-10-19 Last updated: 2018-12-03
    3. The non-coding MIR100HG RNA mediates cytostatic responses of epithelial cells to transforming growth factor β
    Open this publication in new window or tab >>The non-coding MIR100HG RNA mediates cytostatic responses of epithelial cells to transforming growth factor β
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Transforming growth factor β (TGFβ) stimulation modulates the expression of many epithelial genes involved in cell growth arrest, epithelial-to-mesenchymal transition and development. Many recent reports provide evidence that TGFβ signaling regulates the expression of long non-coding RNAs (lncRNAs), i.e. RNAs lacking protein coding potential. After screening for lncRNAs whose expression is regulated by TGFβ signaling, we observed that TGFβ induced the expression of the mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG), a genetic locus which gives rise to multiple lncRNAs (MIR100HG splice variants), as well as the micro-RNA clusters miR-100, let-7a-2 and miR-125b-1. In addition, TGFβ stimulation led to increased levels of mature let-7a-2-3p, miR-125b-5p and miR-125b-1-3p miRNAs. MIR100HG depletion attenuated the TGFβ/Smad-mediated transcriptional responses, the expression of the TGFβ-target genes SERPINE1 (PAI-1) and fibronectin 1 (FN1), and TGFβ-mediated cell growth arrest. Moreover, overexpressing let-7a-2-3p, but not miR-125b-5p or miR-125b-1-3p miRNAs, mimicked enhanced TGFβ/Smad-mediated transcription and inhibited cell proliferation, while inhibition of let-7a-2-3p slightly reduced PAI-1 and fibronectin expression. Thus, we identified MIR100HG and the miRNA clusters generated by its locus as TGFβ-target non-coding RNAs, and ascribed to them a potential role in mediating cytostatic responses by modulating the magnitude of TGFβ signaling.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-363899 (URN)
    Available from: 2018-10-19 Created: 2018-10-19 Last updated: 2018-10-23
    4. Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
    Open this publication in new window or tab >>Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
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    2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 8, p. e103651-Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND:

    Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.

    METHODS:

    A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.

    RESULTS:

    TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.

    CONCLUSION:

    Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.

    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:uu:diva-231920 (URN)10.1371/journal.pone.0103651 (DOI)000341302700014 ()25133494 (PubMedID)
    Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2018-10-23Bibliographically approved
    5. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation
    Open this publication in new window or tab >>Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation
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    2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 24, p. 12706-12723Article in journal (Refereed) Published
    Abstract [en]

    We previously established a mechanism of negative regulation of transforming growth factor beta signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-299724 (URN)10.1074/jbc.M116.729699 (DOI)000378119900024 ()27129221 (PubMedID)
    Funder
    Swedish Research Council, K2010-67X-14936-07-3 K2013-66X-14936-10-5
    Available from: 2016-07-26 Created: 2016-07-26 Last updated: 2018-10-23Bibliographically approved
  • Public defence: 2018-12-17 13:15 Hörsal 2, 753 13 Uppsala
    Brandén, Gunnar
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Social Sciences, Department of Economics.
    Understanding Intergenerational Mobility: Inequality, Student Aid and Nature-Nurture Interactions2018Doctoral thesis, monograph (Other academic)
    Abstract [en]

    Essay I: A body of evidence has emerged in the literature on intergenerational mobility documenting that unequal countries experience less social mobility: a relationship known as the Great Gatsby Curve. In this paper I estimate the Great Gatsby Curve within Sweden across 125 commuting zones and 20 cohorts, exploiting both cross-sectional and longitudinal variation. I find that children who were exposed to higher levels of inequality during childhood experienced less social mobility as adults, thereby confirming the existence of a Great Gatsby Curve in Sweden. I also present new evidence on the underlying mechanisms of the Great Gatsby Curve. By decomposing intergenerational mobility into separate transmission channels, I find that the Great Gatsby Curve is exclusively driven by the mediating effect that children's educational attainment and development of cognitive and non-cognitive skills has on the persistence of income across generations. Hence, the results suggest that adverse effects of inequality on mobility can be alleviated by policies that target children's educational attainment and development of cognitive and non-cognitive skills.

  • Public defence: 2018-12-19 09:00 Polhemsalen, Uppsala
    Carstensen, Hauke
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Physics.
    Self-assembly of magnetic particles2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Self-assembly is the spontaneous formation of larger structures from small building blocks. This process is driven and determined by the interactions between the constituents. Examples of self assembly are found almost everywhere and, in particular, biological systems in general rely on a hierarchical formation of structures over a range of length scales. Technologically, self-assembly can be used to form mesoscopic structures and artificial crystals. In the case of particles with micrometer size suspended in a liquid phase, it is possible to use optical microscopy for the the investigation of self-assembly.

    In this thesis, the self-assembly of microbeads with tunable magnetic interactions is studied, based on the statistic analysis of microscope images and computer simulations. Magnetic and non-magnetic microbeads are suspended in a ferrofluid, which is a dispersion of magnetic nanoparticles in water. As a result, the magnetic properties of the microbeads in the ferrofluid are altered and can be described by effective magnetic susceptibilities and magnetic dipole moments, which can be tuned continuously. The liquid is confined between glass slides and effectively the microbeads are studied in a 2D geometry under a magnetic field, applied either in- or out-of-plane. The resulting structures are detected by image analysis algorithms, analyzed and correlated to the dipolar interaction between the beads, as well as to macroscopic quantities, like the particle density and ratio. For the in-plane field a phase transition from square to hexagonal lattice is observed. This phase transition is explained by the change in dipole interaction between the microbeads as the moments change from anti-parallel to parallel alignment.  For the out-of-plane field the situation becomes diverse and more phases appear. It turns out that the phase formation in this case is strongly dependent on the bead ratio, density and interactions.

    We identify regions in the phase diagram, where isolated beads, percolated structures, and crystals dominate. To cover a wide parameter range the experiments are complemented by computer simulations. The tools developed in this thesis enable us to construct phase diagrams extracted from direct imaging and dependence on the extracted relevant parameters.

    List of papers
    1. Phase formation in colloidal systems with tunable interaction
    Open this publication in new window or tab >>Phase formation in colloidal systems with tunable interaction
    2015 (English)In: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics, ISSN 1539-3755, E-ISSN 1550-2376, Vol. 92, no 1, article id 012303Article in journal (Refereed) Published
    Abstract [en]

    Self-assembly is one of the most fascinating phenomena in nature and is one key component in the formation of hierarchical structures. The formation of structures depends critically on the interaction between the different constituents, and therefore the link between these interactions and the resulting structure is fundamental for the understanding of materials. We have realized a two-dimensional system of colloidal particles with tunable magnetic dipole forces. The phase formation is studied by transmission optical microscopy and a phase diagram is constructed. We report a phase transition from hexagonal to random and square arrangements when the magnetic interaction between the individual particles is tuned from antiferromagnetic to ferrimagnetic.

    National Category
    Physical Sciences
    Identifiers
    urn:nbn:se:uu:diva-259093 (URN)10.1103/PhysRevE.92.012303 (DOI)000357262700007 ()
    Funder
    Swedish Research Council, A0505501Carl Tryggers foundation , CT 13:513
    Available from: 2015-07-28 Created: 2015-07-27 Last updated: 2018-11-12Bibliographically approved
    2. Statistical analysis of phase formation in 2D colloidal systems
    Open this publication in new window or tab >>Statistical analysis of phase formation in 2D colloidal systems
    2018 (English)In: The European Physical Journal E Soft matter, ISSN 1292-8941, E-ISSN 1292-895X, Vol. 41, no 1, article id 9Article in journal (Refereed) Published
    Abstract [en]

    Colloidal systems offer unique opportunities for the study of phase formation and structure since their characteristic length scales are accessible to visible light. As a model system the two-dimensional assembly of colloidal magnetic and non-magnetic particles dispersed in a ferrofluid (FF) matrix is studied by transmission optical microscopy. We present a method to statistically evaluate images with thousands of particles and map phases by extraction of local variables. Different lattice structures and long-range connected branching chains are observed, when tuning the effective magnetic interaction and varying particle ratios.

    Place, publisher, year, edition, pages
    SPRINGER, 2018
    National Category
    Physical Sciences
    Identifiers
    urn:nbn:se:uu:diva-346233 (URN)10.1140/epje/i2018-11615-x (DOI)000423452000001 ()29353322 (PubMedID)
    Funder
    The Swedish Foundation for International Cooperation in Research and Higher Education (STINT), IG2011-2067Swedish Research Council, A0505501Carl Tryggers foundation , CT 13:513
    Available from: 2018-03-16 Created: 2018-03-16 Last updated: 2018-11-12Bibliographically approved
    3. Tunable self assembly of crystals and branching chain networks
    Open this publication in new window or tab >>Tunable self assembly of crystals and branching chain networks
    (English)Manuscript (preprint) (Other academic)
    National Category
    Condensed Matter Physics
    Research subject
    Physics
    Identifiers
    urn:nbn:se:uu:diva-365206 (URN)
    Available from: 2018-11-11 Created: 2018-11-11 Last updated: 2018-11-12
  • Public defence: 2018-12-20 09:15 A1:111a, BMC, Uppsala
    Ahnfelt, Emelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    In vitro evaluation of formulations used in the treatment of hepatocellular carcinoma2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Hepatocellular carcinoma (HCC) causes ~ 600,000 deaths annually, making it the second most deadly cancer form. HCC is classified into five stages and for the intermediate HCC treatment, the two most commonly used drug delivery systems (DDSs) are lipiodol-based emulsions and drug-eluting beads. The aims of this thesis were to develop in vitro methods suitable for studying these DDSs. It is important to investigate the release mechanisms and release rates with relevant in vitro methods, as this can improve the understanding of the in vivo performance. Miniaturized in vitro methods with sample reservoirs separated from the release medium by a diffusion barrier were developed and shown to be suitable for studying drug release from particle DDSs (Paper I). In Paper II these methods were further developed and used to study the release of doxorubicin (DOX) from the clinically used drug-eluting beads. DOX release rates were affected by the method set-up and the characteristics of the release medium. The choice of method and volume of release medium could improve the in vivo-likeness of the in vitro release profiles. Applied theoretical models suggested a film-controlled type of DOX release mechanism from the beads when self-aggregation, DOX-bead interaction, and DOX deprotonation were taken into account.

    A micropipette-assisted microscopy method was used to further improve the understanding of the release mechanism of amphiphilic molecules from the beads (Paper III). A detailed analysis suggested an internal depletion-layer model dependent on molecular self-aggregation for the release. It was further suggested that a simple ion-exchange mechanism is unrealistic in physiological conditions.

    The important pharmaceutical factors for the emulsion-based formulations were investigated in Paper IV. DOX solubility, lipid phase distribution, and emulsion stability increased when the contrast agent iohexol was added. Also, an increase in release half-life (h) was observed from emulsions with iohexol.

    The in vitro methods and theoretical models presented in this thesis can be used during development and optimization of future DDSs.

    List of papers
    1. A miniaturized in vitro release method for investigating drug-release mechanisms
    Open this publication in new window or tab >>A miniaturized in vitro release method for investigating drug-release mechanisms
    2015 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 486, no 1-2, p. 339-349Article in journal (Refereed) Published
    Abstract [en]

    We have evaluated a miniaturized in vitro method, based on the mDISS Profiler (TM) technique that enables on-line monitoring of drug release from a 21 mu l sample with 10 ml of release medium. Four model drugs in eight clinically used formulations, including both solid and non-solid drug delivery systems, were investigated. The acquired data were compared with historical in vitro release data from the same formulations. Use of the Weibull function to describe the in vitro drug-release profiles allowed discrimination between the selected formulations with respect to the drug-release mechanisms. Comparison of the release data from the same formulation in different in vitro set-ups showed that the methodology used can affect the mechanism of in vitro release. We also evaluated the ability of the in vitro methods to predict in vivo activity by comparing simulated plasma concentration-time profiles acquired from the application of the biopharmaceutical software GI-Sim to the in vitro observations. In summary, the simulations based on the miniaturized-method release data predicted the plasma profiles as well as or more accurately than simulations based on the historical release data in 71% of the cases and this miniaturized in vitro method appears to be applicable for both solid and non-solid formulations.

    Keywords
    In vitro release methods, Release mechanisms, Weibull function, GI-Sim, In vivo prediction
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-255064 (URN)10.1016/j.ijpharm.2015.03.076 (DOI)000353999100037 ()25843760 (PubMedID)
    Available from: 2015-06-22 Created: 2015-06-12 Last updated: 2018-10-30Bibliographically approved
    2. In Vitro Release Mechanisms of Doxorubicin From a Clinical Bead Drug-Delivery System
    Open this publication in new window or tab >>In Vitro Release Mechanisms of Doxorubicin From a Clinical Bead Drug-Delivery System
    2016 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 105, no 11, p. 3387-3398Article in journal (Refereed) Published
    Abstract [en]

    The release rate of doxorubicin (DOX) from the drug-delivery system (DDS), DC Bead, was studied by 2 miniaturized in vitro methods: free-flowing and sample reservoir. The dependencies of the release mechanisms on in vitro system conditions were investigated experimentally and by theoretical modeling. An inverse relationship was found between release rates and bead size, most likely due to the greater total surface area. The release rates correlated positively with temperature, release medium volume, and buffer strength, although the release medium volume had larger effect than the buffer strength. The sample reservoir method generated slower release rates, which described the in vivo release profile more accurately than the free-flowing method. There was no difference between a pH of 6.3 or 7.4 on the release rate, implying that the slightly acidic tumor microenvironment is less importance for drug release. A positive correlation between stirring rate and release rate for all DDS sizes was observed, which suggests film controlled release. Theoretical modeling highlighted the influence of local equilibrium of protonation, self-aggregation, and bead material interactions of DOX. The theoretical release model might describe the observed larger sensitivity of the release rate to the volume of the release medium compared to buffer strength. A combination of miniaturized in vitro methods and theoretical modeling are useful to identify the important parameters and processes for DOX release from a micro gel-based DDS.

    Keywords
    controlled release, diffusion, dissolution, dissolution rate, drug-delivery systems, in vitro models, mathematical model, microspheres
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-311211 (URN)10.1016/j.xphs.2016.08.011 (DOI)000388268200018 ()27663384 (PubMedID)
    Funder
    Swedish Research Council, 521-2011-373
    Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2018-10-30Bibliographically approved
    3. Single bead investigation of a clinical drug delivery system – a novel release mechanism
    Open this publication in new window or tab >>Single bead investigation of a clinical drug delivery system – a novel release mechanism
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    2018 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 292, p. 235-247Article in journal (Other academic) Published
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-360988 (URN)10.1016/j.jconrel.2018.11.011 (DOI)
    Available from: 2018-09-20 Created: 2018-09-20 Last updated: 2018-11-19
    4. In vitro evaluation of lipiodol-based emulsions in clinical use
    Open this publication in new window or tab >>In vitro evaluation of lipiodol-based emulsions in clinical use
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-360987 (URN)
    Available from: 2018-09-20 Created: 2018-09-20 Last updated: 2018-10-30
  • Public defence: 2018-12-20 13:15 Rosénsalen, Uppsala
    Georgantzi, Kleopatra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Research group (Dept. of women´s and children´s health), Neuropediatrics/Paediatric oncology.
    On the Diagnostics of Neuroblastoma: Clinical and Experimental Studies2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Neuroblastoma (NB) is one of the most common childhood cancers. Patients with low stage tumor have high survival rate, while those with advanced stage and/or unfavorable molecular biology have poor prognosis. A correct histopathological diagnosis, clinical stage, and identified genetic aberrations are crucial for treatment stratification according to current protocol. The tumor sample is obtained either by fine needle aspiration, cutting needle biopsy or open biopsy. NB exhibits neuroendocrine differentiation by showing immunoreactivity for chromogranin A (CgA), synaptophysin (syn), and neuron specific enolase (NSE) and 90% of the patients have increased levels of urine catecholamine metabolites.

    Experimental and clinical NB tumor samples were immunostained for somatostatin receptors (SSTRs) 1-5, somatostatin and CgA. Clinical tumor samples were also immunostained for syn, synaptic vesicle protein 2 (SV2), and vesicle monoamine transporter 1 (VMAT1) and 2 (VMAT 2). Blood samples from 92 patients were analyzed for level of CgA, NSE, and chromogranin B and compared with control groups. The urinary excretion of catecholamine metabolites was analyzed in samples collected at diagnosis. Clinical and laboratory data were extracted from patient records, including information on the diagnostic accuracy of ultrasound guided cutting needle biopsies (UCNB) and potential complications.

    We found that NB expressed the different SSTRs and that receptor 2 was the most frequently expressed before chemotherapy. Furthermore, NB tumors showed immunoreactivity for SV2, VMAT 1 and VMAT2 alongside CgA and syn. The immunoreactivity of SV2 was comparable to CgA and superior to syn. Patients with NB had higher blood concentrations of CgA and NSE compared with controls. Patients with advanced stage disease, MYCN amplification and 1 p deletion had higher concentrations of both CgA and NSE while only NSE was correlated to outcome with higher concentrations in the deceased patients.

    A high urinary excretion of homovanillic acid and dopamine were correlated to inferior outcome. UCNB were found to be safe and may provide all necessary diagnostic requirements for adequate therapy stratification according to current treatment protocols.

    List of papers
    1. Differentiated expression of somatostatin receptor subtypes in experimental models and clinical neuroblastoma
    Open this publication in new window or tab >>Differentiated expression of somatostatin receptor subtypes in experimental models and clinical neuroblastoma
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    2011 (English)In: Pediatric Blood & Cancer, ISSN 1545-5009, E-ISSN 1545-5017, Vol. 56, no 4, p. 584-589Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND:

    Neuroblastoma (NB) is a solid tumor of childhood originating from the adrenal medulla or sympathetic nervous system. Somatostatin (SS) is an important regulator of neural and neuroendocrine function, its actions being mediated through five specific membrane receptors. The aim of this study was to investigate the expression of the different somatostatin receptors (SSTRs) in NB tumor cells that may form targets for future therapeutic development.

    PROCEDURE:

    Tumor specimens from 11 children with stage II-IV disease were collected before and/or after chemotherapy. Experimental tumors derived from five human NB cell lines were grown subcutaneously in nude mice. Expression of SSRTs, the neuroendocrine marker chromogranin A (CgA) and SS was detected by immunohistochemistry using specific antibodies.

    RESULTS:

    SSTR2 was detected in 90%, SSTR5 in 79%, SSTR1 in 74%, SSTR3 in 68% whereas SSTR4 was expressed in 21% of the clinical tumors. The experimental tumors expressed SSTRs in a high but variable frequency. All clinical tumors showed immunoreactivity for CgA but not for SS.

    CONCLUSION:

    The frequent expression of SSTRs indicates that treatment with unlabeled or radiolabeled SS analogs should be further explored in NB.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-140509 (URN)10.1002/pbc.22913 (DOI)000287986700013 ()21120894 (PubMedID)
    Available from: 2011-01-05 Created: 2011-01-05 Last updated: 2018-10-31
    2. Chromogranin A and neuron-specific enolase in neuroblastoma: Correlation to stage and prognostic factors.
    Open this publication in new window or tab >>Chromogranin A and neuron-specific enolase in neuroblastoma: Correlation to stage and prognostic factors.
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    2018 (English)In: Pediatric Hematology & Oncology, ISSN 0888-0018, E-ISSN 1521-0669, Vol. 35, no 2, p. 156-165Article in journal (Refereed) Published
    Abstract [en]

    Chromogranin A (CgA) and neuron specific enolase (NSE) are important markers in adult neuroendocrine tumors (NET). Neuroblastoma (NB) has certain neuroendocrine properties. The aim of this study was to correlate blood concentrations of CgA, chromogranin B (CgB), and NSE to prognostic factors and outcome in children with NB. Blood samples from 92 patients with NB, 12 patients with benign ganglioneuroma (GN), 21 patients with non-NB solid tumors, 10 patients with acute leukemias, and 69 healthy children, were analyzed. CgA concentrations were higher in neonates vs. children older than one month in the control group (p < 0.0001), and in neonates with NB vs. the control group (p < 0.01). CgA and NSE concentrations were higher in patients with stages 3 and 4 disease (p < 0.05 and p < 0.05), in patients having tumors with amplification of MYCN (p < 0.05 and p < 0.001), or chromosome 1 p deletion (p < 0.05 and p < 0.05). NSE correlated to the tumor size at diagnosis (p < 0.001) and to tumor related death (p < 0.01) in NB. CgA and NSE concentrations were elevated in patients with NB and especially in those with advanced disease. Both CgA and NSE correlated to genetic markers, while only NSE correlated to primary tumor size and outcome in NB. We found that CgA and NSE are clinically valuable tumor markers in NB and they merit prospective clinical evaluations as such.

    Keywords
    Chromogranin A, neuroblastoma, neuron-specific enolase, prognosis, tumor markers
    National Category
    Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-360970 (URN)10.1080/08880018.2018.1464087 (DOI)000446356300007 ()29737901 (PubMedID)
    Funder
    Swedish Childhood Cancer Foundation
    Available from: 2018-09-20 Created: 2018-09-20 Last updated: 2018-12-07Bibliographically approved
    3. Synaptic Vesicle Protein 2 and Vesicular Monoamine Transporter 1 and 2 are expressed in Neuroblastoma
    Open this publication in new window or tab >>Synaptic Vesicle Protein 2 and Vesicular Monoamine Transporter 1 and 2 are expressed in Neuroblastoma
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    neuroblastoma, neuroendocrine, immunohistochemistry, urine-dopamine, urine-HVA, urine-VMA
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-364674 (URN)
    Available from: 2018-10-31 Created: 2018-10-31 Last updated: 2018-10-31
    4. Diagnostic Ultrasound-Guided Cutting Needle Biopsies in Neuroblastoma: a safe and efficient procedure
    Open this publication in new window or tab >>Diagnostic Ultrasound-Guided Cutting Needle Biopsies in Neuroblastoma: a safe and efficient procedure
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    ultrasound, biopsy, neuroblastoma, complications, child
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-364673 (URN)
    Available from: 2018-10-31 Created: 2018-10-31 Last updated: 2018-10-31
  • Public defence: 2018-12-21 13:15 Polhemsalen, Ångströmlaboratoriet, Uppsala
    Blomqvist, Björn Rune Helmer
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Mathematics, Applied Mathematics and Statistics.
    Gaussian process models of social change2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Social systems produce complex and nonlinear relationships in the indicator variables that describe them. Traditional statistical regression techniques are commonly used in the social sciences to study such systems. These techniques, such as standard linear regression, can prevent the discovery of the complex underlying mechanisms and rely too much on the expertise and prior beliefs of the data analyst. In this thesis, we present two methodologies that are designed to allow the data to inform us about these complex relations and provide us with interpretable models of the dynamics.

    The first methodology is a Bayesian approach to analysing the relationship between indicator variables by finding the parametric functions that best describe their interactions. The parametric functions with the highest model evidence are found by fitting a large number of potential models to the data using Bayesian linear regression and comparing their respective model evidence. The methodology is computationally fast due to the use of conjugate priors, and this allows for inference on large sets of models. The second methodology is based on a Gaussian processes framework and is designed to overcome the limitations of the first modelling approach. This approach balances the interpretability of more traditional parametric statistical methods with the predictability and flexibility of non-parametric Gaussian processes.

    This thesis contains four papers where we apply the methodologies to both real-life problems in the social sciences as well as on synthetic data sets. In paper I, the first methodology (Bayesian linear regression) is applied to the classic problem of how democracy and economic development interact. In paper II and IV, we apply the second methodology (Gaussian processes) to study changes in the political landscape and demographic shifts in Sweden in the last decades. In paper III, we apply the second methodology on a synthetic data set to perform parameter estimation on complex dynamical systems.

    List of papers
    1. Using Bayesian dynamical systems, model averaging and neural networks to determine interactions between socio-economic indicators
    Open this publication in new window or tab >>Using Bayesian dynamical systems, model averaging and neural networks to determine interactions between socio-economic indicators
    2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 5, article id e0196355Article in journal (Refereed) Published
    Abstract [en]

    Social and economic systems produce complex and nonlinear relationships in the indicator variables that describe them. We present a Bayesian methodology to analyze the dynamical relationships between indicator variables by identifying the nonlinear functions that best describe their interactions. We search for the 'best' explicit functions by fitting data using Bayesian linear regression on a vast number of models and then comparing their Bayes factors. The model with the highest Bayes factor, having the best trade-off between explanatory power and interpretability, is chosen as the 'best' model. To be able to compare a vast number of models, we use conjugate priors, resulting in fast computation times. We check the robustness of our approach by comparison with more prediction oriented approaches such as model averaging and neural networks. Our modelling approach is illustrated using the classical example of how democracy and economic growth relate to each other. We find that the best dynamical model for democracy suggests that long term democratic increase is only possible if the economic situation gets better. No robust model explaining economic development using these two variables was found.

    Place, publisher, year, edition, pages
    PUBLIC LIBRARY SCIENCE, 2018
    National Category
    Probability Theory and Statistics
    Identifiers
    urn:nbn:se:uu:diva-359665 (URN)10.1371/journal.pone.0196355 (DOI)000431757400027 ()29742126 (PubMedID)
    Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2018-10-30Bibliographically approved
    2. Explaining and predicting the rise of a radical right-wing party using Gaussian processes
    Open this publication in new window or tab >>Explaining and predicting the rise of a radical right-wing party using Gaussian processes
    (English)In: Article in journal (Other academic) Submitted
    National Category
    Mathematics
    Identifiers
    urn:nbn:se:uu:diva-364653 (URN)
    Available from: 2018-10-30 Created: 2018-10-30 Last updated: 2018-10-30
    3. Model selection and parameter estimation of complex dynamical systems using semi-parametric Gaussian processes
    Open this publication in new window or tab >>Model selection and parameter estimation of complex dynamical systems using semi-parametric Gaussian processes
    (English)Manuscript (preprint) (Other academic)
    National Category
    Mathematics
    Research subject
    Mathematics with specialization in Applied Mathematics
    Identifiers
    urn:nbn:se:uu:diva-364652 (URN)
    Available from: 2018-10-30 Created: 2018-10-30 Last updated: 2018-10-30
    4. Last night in Sweden? Using Gaussian processes to study changing demographics at the level of municipalities
    Open this publication in new window or tab >>Last night in Sweden? Using Gaussian processes to study changing demographics at the level of municipalities
    (English)In: Article in journal (Other academic) Submitted
    National Category
    Mathematics
    Identifiers
    urn:nbn:se:uu:diva-364654 (URN)
    Available from: 2018-10-30 Created: 2018-10-30 Last updated: 2018-10-30
  • Public defence: 2018-12-21 13:15 Humanistiska teatern, Uppsala
    Schumann, Åsa
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Theology, Department of Theology.
    Vilken mening!?: En blandad metodstudie i religionspsykologi av meningsskapandets betydelse för skolungdomar2018Doctoral thesis, monograph (Other academic)
    Abstract [en]

    The purpose of the study was to explore the role of religion in the development of a meaning system among Swedish adolescents by examining the interactions of their: sense of coherence (SOC), identity process (U-MICS), moral development (SRM-SF), and views on existential and religious questions.

    The study used a Mixed Methods Design, with a Sequential Explanatory Strategy consisting of quantitative and qualitative parts. In the quantitative part of the study, 90 students in 8th grade, 50 girls and 40 boys, participated. The qualitative part of the study consisted of 24 semi-structured interviews drawn from the original group.

    According to the results, there was a relation between SOC and the following variables in the identity process (U-MICS): commitment to school and reconsideration of commitment to friends, and commitment to school and perception of the importance of religion. The SOC value among girls was significantly lower than among boys. The results did not indicate a significant relationship between moral development, SOC values, and the religious variables.

    In the qualitative interview results, the participants in the commitment phase relating to school expressed more satisfaction with their school situations and relationships than those who were predominantly in the reconsideration of commitment phase. Those with a higher level of commitment to school were also more likely to express the view that religion was important in life. Interview material showed that those young people who expressed the belief that religion was important in life today or believed that religion constituted a potential future source of strength dominated the group with higher SOC. The results served to reinforce theoretical perspectives on the meaning-making function of religion. The study results suggest that safe and trustful relationships with peers and adults related closely to SOC values among these adolescents and were singled out as their most important sources of strength and support in life.

  • Public defence: 2019-01-11 13:00 B41, Uppsala
    Söderholm, Annika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    The Importance of Being Promiscuous: Understanding enzyme function, specificity, and evolution through structure2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzymes are known to be amazingly specific and efficient catalysts. However, many enzymes also have so-called promiscuous functions, i.e., they are able to catalyze other reactions than their main one. The promiscuous activities are often low, serendipitous, and under neutral selection but if conditions arise that make them beneficial, they can play an important role in the evolution of new enzymes. In this thesis, I present three studies where we have characterized different enzyme families by structural and biochemical methods. The studies demonstrate the occurrence of enzyme promiscuity and its potential role in evolution and organismal adaptation.

    In the first study, I describe the characterization of wild type and mutant HisA enzymes from Salmonella enterica. In the first part of this study, we could clarify the mechanistic cycle of HisA by solving crystal structures that showed different conformations of wild type HisA in complex with its labile substrate ProFAR (N´-[(5´-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide). In the second part of this study, structures of mutant enzymes from a real-time evolution study provided us with an atomic-level description of how HisA had evolved a new function. The HisA mutants had acquired TrpF activity, either in addition to (bifunctional generalists) or instead of (TrpF specialists) their HisA activity. In the second study, I present the crystal structure and demonstrate promiscuous activity of the TrpC enzyme from Pseudomonas aeruginosa. The activity data demonstrates that the enzyme can turn over a substrate that lacks a substituent that was previously considered essential for catalysis. In the third study, I present the structural and functional characterization of SAM (S-Adenosyl methionine) hydrolases from bacteriophages. These enzymes were discovered because of their ability to rescue auxotrophic bacteria by inducing expression of a promiscuous bacterial enzyme.  

    List of papers
    1. Structure of a phage-encoded SAM hydrolase enzyme provides insights in substrate binding and catalysis
    Open this publication in new window or tab >>Structure of a phage-encoded SAM hydrolase enzyme provides insights in substrate binding and catalysis
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    SAM hydrolase, phage enzyme
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-366689 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-22
    2. Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
    Open this publication in new window or tab >>Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
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    2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 41, p. 24657-24668Article in journal (Refereed) Published
    Abstract [en]

    HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of ProFAR to PRFAR in the histidine biosynthesis pathway and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp7 acts as the catalytic base and Asp176 as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure, and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen-bond network including His47, Asp129, Thr171 and Ser202, all shown to be functionally important. The closed-conformation structure is highly similar to the bi-functional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-260701 (URN)10.1074/jbc.M115.678086 (DOI)000362598300003 ()26294764 (PubMedID)
    Funder
    Swedish Research CouncilSwedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 283570
    Available from: 2015-08-23 Created: 2015-08-23 Last updated: 2018-11-22
    3. Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes
    Open this publication in new window or tab >>Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes
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    2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 8, p. 4727-4732Article in journal (Refereed) Published
    Abstract [en]

    New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα)8 barrel as an inherently evolvable protein scaffold.

    Keywords
    HisA, TrpF, adaptive evolution, enzyme performance threshold
    National Category
    Evolutionary Biology Structural Biology
    Research subject
    Biology with specialization in Structural Biology; Biochemistry; Biology with specialization in Molecular Evolution
    Identifiers
    urn:nbn:se:uu:diva-320223 (URN)10.1073/pnas.1618552114 (DOI)000400358000052 ()
    Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2018-11-22Bibliographically approved
    4. A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function
    Open this publication in new window or tab >>A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function
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    2018 (English)In: Nature Ecology & Evolution, E-ISSN 2397-334X, Vol. 2, no 8, p. 1321-1330Article in journal (Refereed) Published
    Abstract [en]

    One key concept in the evolution of new functions is the ability of enzymes to perform promiscuous side-reactions that serve as a source of novelty that may become beneficial under certain conditions. Here, we identify a mechanism where a bacteriophage-encoded enzyme introduces novelty by inducing expression of a promiscuous bacterial enzyme. By screening for bacteriophage DNA that rescued an auxotrophic Escherichia coli mutant carrying a deletion of the ilvA gene, we show that bacteriophage-encoded S-adenosylmethionine (SAM) hydrolases reduce SAM levels. Through this perturbation of bacterial metabolism, expression of the promiscuous bacterial enzyme MetB is increased, which in turn complements the absence of IlvA. These results demonstrate how foreign DNA can increase the metabolic capacity of bacteria, not only by transfer of bona fide new genes, but also by bringing cryptic bacterial functions to light via perturbations of cellular physiology.

    Place, publisher, year, edition, pages
    Nature Publishing Group, 2018
    National Category
    Evolutionary Biology Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-355286 (URN)10.1038/s41559-018-0568-5 (DOI)000439505600024 ()
    Funder
    Swedish Research CouncilKnut and Alice Wallenberg Foundation
    Available from: 2018-06-27 Created: 2018-06-27 Last updated: 2018-11-22Bibliographically approved
    5. Structure and substrate ambiguity of TrpC from Pseudomonas aeruginosa
    Open this publication in new window or tab >>Structure and substrate ambiguity of TrpC from Pseudomonas aeruginosa
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The enzyme TrpC catalyzes the formation of Indole-3-glycerol phosphate (IGP) from 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate as part of the tryptophan biosynthesis pathway. The reaction mechanism follows a series of condensation, decarboxylation, and dehydration. The decarboxylation has been assumed to constitute an essential step of the mechanism since no activity with decarboxylated substrate was observed in an early study on the TrpC:TrpF fusion protein from Escherichia coli (Smith 1962). Here, we refute this assumption by demonstrating IGP formation catalyzed by both TrpC from Pseudomonas aeruginosa and from E.coli. We show that P. aeruginosa TrpC is more active on decarboxylated substrate than E.coli TrpC and, by solving the crystal structure of P. aeruginosa TrpC, we provide structure-based hypotheses on their difference in promiscuous activity.

    Keywords
    TrpC, IGPS
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-366660 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-30
  • Public defence: 2019-01-18 09:15 Room B21, Uppsala
    Jiang, Wangshu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Of spiders, bugs, and men: Structural and functional studies of proteins involved in assembly2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Protein assembly enables complex machineries while being economical with genetic information. However, protein assembly also constitutes a potential threat to the host, and needs to be carefully regulated.

    Sulfate is a common source of sulfur for cysteine synthesis in bacteria. A putative sulfate permease CysZ from Escherichia coli appears much larger than its apparent molecular mass when analyzed by chromatography and native gel. Clearly CysZ undergoes homo-oligomerization. Using isothermal titration calorimetry, we confirmed that CysZ binds to its putative substrate sulfate, and also sulfite with higher affinity. CysZ-mediated sulfate transport—in both E. coli whole cells and proteoliposomes—was inhibited in the presence of sulfite, indicating a feedback inhibition mechanism.

    Proteus mirabilis is a Gram-negative bacterium causing urinary tract infections. Its simultaneous expression of multiple fimbriae enables colonization and biofilm formation. Fimbriae are surface appendages assembled from protein subunits, with distal adhesins specifically recognizing host-cell receptors. We present the first three structures of P. mirabilis fimbrial adhesins. While UcaD and AtfE adopt the canonical immunoglobulin-like fold, MrpH has a previously unknown fold. The coordination of Zn or Cu ion by three conserved histidine residues in MrpH is required for MrpH-dependent biofilm formation.

    Spider silk is an assembly of large proteins called spidroins. The N-terminal domain (NT) of spidroins senses the pH decrease along the silk spinning gland, and transits from monomer to dimer. A locked NT dimer interlinks spidroin molecules into polymers. We identified a new asymmetric dimer form of NT by x-ray crystallography. With additional evidence from small angle x-ray scattering (SAXS), we propose the asymmetric dimer as a common intermediate of NT in silk formation.

    Alzheimer’s disease is a life-threatening dementia, where aggregation-prone Aβ peptides self-assemble into amyloid fibrils. Bri2 BRICHOS is a molecular chaperone that efficiently delays Aβ fibrillation, and protects the region of its pro-protein with high β-propensity from aggregation. Combining SAXS and microscale thermophoresis data, we confirmed binding between Bri2 BRICHOS and its native client peptide. Using site-directed mutagenesis, we showed that three conserved tyrosine residues in Bri2 BRICHOS are important for its anti-Aβ fibrillation activity.

    List of papers
    1. The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
    Open this publication in new window or tab >>The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
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    2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 7, p. 1809-1816Article in journal (Refereed) Published
    Abstract [en]

    The Escherichia coli inner membrane protein CysZ mediates the sulfate uptake subsequently utilized for the synthesis of sulfur-containing compounds in cells. Here we report the purification and functional characterization of CysZ. Using Isothermal Titration Calorimetry, we have observed interactions between CysZ and its putative substrate sulfate. Additional sulfur-containing compounds from the cysteine synthesis pathway have also been analyzed for their abilities to interact with CysZ. Our results suggest that CysZ is dedicated to a specific pathway that assimilates sulfate for the synthesis of cysteine. Sulfate uptake via CysZ into E. coil whole cells and proteoliposome offers direct evidence of CysZ being able to mediate sulfate uptake. In addition, the cysteine synthesis pathway intermediate sulfite can interact directly with CysZ with higher affinity than sulfate. The sulfate transport activity is inhibited in the presence of sulfite, suggesting the existence of a feedback inhibition mechanism in which sulfite regulates sulfate uptake by CysZ. Sulfate uptake assays performed at different extracellular pH and in the presence of a proton uncoupler indicate that this uptake is driven by the proton gradient. (C) 2014 Elsevier B.V. All rights reserved.

    Keywords
    CysZ, Sulfate, Transport, Membrane protein, Inhibition
    National Category
    Biochemistry and Molecular Biology Biophysics
    Identifiers
    urn:nbn:se:uu:diva-227987 (URN)10.1016/j.bbamem.2014.03.003 (DOI)000336695300014 ()
    Available from: 2014-07-04 Created: 2014-07-02 Last updated: 2018-11-23Bibliographically approved
    2. Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis.
    Open this publication in new window or tab >>Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis.
    2018 (English)In: Acta crystallographica. Section D, Structural biology, ISSN 2059-7983, Vol. 74, no Pt 11, p. 1053-1062Article in journal (Refereed) Published
    Abstract [en]

    The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Å resolution) and AtfE (1.58 Å resolution) from two P. mirabilis fimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding of P. mirabilis fimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.

    Keywords
    Proteus mirabilis, adhesins, fimbriae, urinary-tract infection
    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-366693 (URN)10.1107/S2059798318012391 (DOI)30387764 (PubMedID)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
    3. Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
    Open this publication in new window or tab >>Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
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    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Proteus mirabilis is a Gram-negative uropathogen and the major causative agent in catheter-associated  (CAUTI) and complicated UTIs. Mannose resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonisation. Here, the X-ray structure of the MR/P tip-located MrpH adhesin is reported. The structure has an unusual fold not previously observed, and contains a transition metal centre with Cu2+ or Zn2+ ligated by three conserved histidine residues and a ligand. Using metal complementation biofilm assays and site directed mutagenesis of the three histidines we show that an intact metal binding site occupied by zinc or copper is essential for MR/P-mediated biofilm formation. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and will serve as a starting point for identifying the physiological MR/P receptor(s).

    Keywords
    fimbriae; adhesins; biofilm; urinary tract infection; Proteus mirabilis
    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-366696 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
    4. Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
    Open this publication in new window or tab >>Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-366699 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
    5. Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
    Open this publication in new window or tab >>Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    Bri2, BRICHOS, amyloid, Alzheimer's disease, molecular chaperone
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-366698 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23