| 1. |
- Bratengeier, Cornelia, 1983-
(författare)
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Mechanisms of mechanically induced Osteoclastogenesis : in a novel in vitro model for bone implant loosening
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Total joint arthroplasty is the primary intervention in the treatment of end-stage osteoarthritis. Despite the high success rate, in some patients, the replacement will fail during their lifetime requiring a revision of the implant. These revisions are strenuous for the patient and costly for health care. Joint replacement at a younger age, in combination with a more active lifestyle, increases the need for an early revision of the joint prosthesis. The main reason for revision surgeries is aseptic loosening, a condition where the prosthesis is loosening due to bone degradation at the peri-prosthetic interface in the absence of infections. The most well-established pathological mechanism for aseptic loosening is related to wear particles, generated from different parts of the prosthesis that will trigger bone degradation and bone loss. In addition, early micromotions of the prosthesis and resulting local pressurized fluid flow in the peri-prosthetic interface (supraphysiological loading) have also been identified as a cause for aseptic loosening. However, it remains unknown what cells are the primary responders to supraphysiological loading, and what underlying physical, cellular and molecular mechanism that triggers osteoclast differentiation and osteolysis.In this thesis, we intended to shed light on three currently unknown aspects of mechanical loading-induced peri-prosthetic osteolysis, leading to aseptic loosening of orthopedic prostheses: (1)Which cells are the primary responder to supraphysiological loading? (2)What characteristics of the mechanical stimulus induce an osteo-protective or osteo-destructive response? (3)Which cellular mechano-sensing mechanisms are involved in an osteo-destructive response?We successfully implemented supraphysiological mechanical loading, mimicking the periprosthetic pressurized fluid flow around a loosening implant, in an in vitro model for bone implant loosening. Using this model, we uncovered the involvement of mesenchymal stem cells and myeloid progenitor cells (monocytes) in mechanical loading-induced peri-prosthetic osteolysis. Applying supraphysiological loading on cells from patients undergoing primary hip arthroplasty, successfully validated the in vitro model for the use of cells of human origin. We further identified in murine myeloid progenitor cells that a combination of high loading amplitude (3.0±0.2Pa), prolonged active loading duration per cycle (duty cycle 22%-50%), and rapid alterations in minimum/maximum values of the loading profile (square wave) is necessary to induce an osteo-destructive response. Further, the loading-induced ATP release and subsequent activation of the P2X7 receptor was essential for the release of soluble factors modulating osteoclastogenesis.In conclusion, we expect that the proposed new in vitro model is a helpful tool to further advance the knowledge in aseptic loosening, by uncovering the mechanoresponsive cellular mechanism to supraphysiological mechanical loading. The identification of the respondent cells in mechanical loading-induced prosthetic loosening gives the opportunity to deliver targeted treatment strategies. Furthermore, identifying the physical parameters that define the shift towards an osteo-destructive response emphasizes the importance of the prosthetic design and surgical technique to reduce mechanical loading-induced bone degradation around a prosthesis.
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| 2. |
- Eliasson, Pernilla, 1979-
(författare)
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Live and Let Die : Critical regulation of survival in normal and malignant hematopoietic stem and progenitor cells
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3.
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| 3. |
- Halvarsson, Camilla, 1985-
(författare)
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Hypoxia inducible factor 1 alpha : dependent and independent regulation of hematopoietic stem cells and leukemia
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis has studied the role of low oxygen levels, or hypoxia, in hematopoietic stem cells (HSCs) and how, at the molecular level, it regulates stem cell maintenance and protects against oxidative stress induced by reactive oxygen species (ROS). HSCs reside within the bone marrow in specific niches created by a unique vascularized environment, which is suggested to be hypoxic and crucial for HSCs by maintaining a quiescent state of cell cycle and by redirecting metabolism away from the mitochondria to glycolysis. The niches are also believed to limit the production of ROS, which could damage DNA and disrupt the stem cell features. The hypoxia-responsive protein hypoxia-inducible factor 1 alpha (HIF-1α) is a major regulator of the hypoxic cell response in HSCs as well as in leukemic stem cells. Both these cells are thought to reside in the bone marrow where they are protected from stress and chemotherapy by niche cells and hypoxia.The thesis demonstrates that pyruvate dehydrogenase kinase 1 regulates a metabolic shift to glycolysis, and maintains the engraftment potential of both HSCs and multipotent progenitors upon transplantation. Furthermore, we wanted to determine whether HIF-1α or other signaling pathways are involved in protecting HSCs from ROS-induced cell death. Overexpression, silencing or a knockout mouse model of Hif-1α could not identify HIF-1α as important for protecting HSCs from oxidative stress-induced cell death through inhibition of synthesis of the antioxidant glutathione. Gene expression analysis instead identified the transcription factor nuclear factor kappa B (NF-κB) as induced by hypoxia. By studying NF- κB signaling we found increased NF-κB activity in cells cultured in hypoxia compared to normoxia. Suppression of inhibitor of kappa B indicated a putative role of NF-κB signaling in hypoxia-induced protection against oxidative stress. The findings show that hypoxia-induced protection to elevated levels of ROS upon glutathione depletion seems to be attributed to activation of the NF-κB signaling pathway independently of HIF-1α.To address the question whether hypoxic in vitro cultures support maintenance and promote HSC expansion we performed a limited dilution-transplantation assay. Our data indicate that hypoxic cultures maintain more long-term-reconstituting HSCs than normoxia, but this could not be confirmed statistically. Finally, we wanted to study the mechanisms by which hypoxia protect against chemotherapy. We could demonstrate that hypoxic culture protects leukemic cell lines against apoptosis induced by chemotherapy or inhibitors used for treatment of leukemia. This multidrug resistance seems to be mediated by ATP-binding cassette transporter genes, which are upregulated by hypoxia and whose inhibition has been shown to increase chemosensitivity. In addition, HIF-1α was upregulated in the leukemic cell lines in hypoxia and its inhibition increased the sensitivity to chemotherapy, indicating a role in inducing chemotherapy resistance.Conclusively, the results presented in this thesis stress the importance of hypoxia in regulating metabolism, oxidative-stress response and maintenance of both HSCs as well as leukemic cells, especially through the critical transcription factors HIF-1α and NF-κB and their target genes.
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| 4. |
- Kuruvilla, Jacob
(författare)
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Proteomics as a multifaceted tool in medicine and environmental assessment
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteomics is evolving as a multi-faceted tool for addressing various biochemical and biomedical queries in the field of scientific research. This involves various stages, ranging from sample preparation to data analysis and biological interpretation. Sample preparation involves isolating proteins from the sample source, purifying and digesting them to initiate shotgun proteomics. Shotgun proteomics identifies proteins by bottom-up proteomic approaches where proteins are identified from the fragmentation spectra of their own peptides.Paper I: deals with the simplification of functional characterization for nanoparticles intended for use in biomedicine. Proteomics was constructive in differentiating and semi-quantifying the surface of protein corona. This could be beneficial in predicting the interactions between nanoparticles and a biological entity like the cell or a receptor protein and provide initial valuable information related to targeting, uptake and safety.Paper II: deals with understanding effects of TiO2 nanoparticles on endothelial cells. A combinatorial approach, involving transcriptomics and proteomics was used to identify aberrations in the permeability and integrity of endothelial cells and tissues. Our study also investigated the correlation of size and how they motivated a differential cellular response. In case of intravenous entry for nanoparticles in targeted drug delivery systems, endothelial cells are the first barrier encountered by these drug carriers. This evaluation involving endothelial cell response could be very instrumental during the designing of NP based drug delivery systems.Paper III: Pharmaceuticals and its metabolites could be very hazardous, especially if its disposal is not managed properly. Since water bodies are the ultimate sink, these chemicals could end up there, culminating in toxicity and other ‘mixture effects’ in combination with other factors. To evaluate the effects of the pharmaceutical, propranolol and climatic factors like low salinity conditions, a microcosm exposure was designed and shotgun proteomics helped understand its impact on mussel gills. In this study too, a combination of transcriptomics and proteomics unveiled molecular mechanisms altered in response to stressors, both individually and in combination.Paper IV: An interplay of various factors like EBF1 and PAX5 determines B-cell lineage and commitment. This might have been materialized by direct and transient proteinprotein interactions. A unique method called BioID helped screen relevant interactions in living cells by the application of a promiscuous biotin ligase enzyme capable of tagging proteins through biotinylation based on a proximity radius. Biotinylation of endogenous proteins enabled their selective isolation by exploiting the high affinity of biotin and streptavidin on streptavidin coated agarose beads, leading to their identification by mass spectrometry. The biotinylated proteins were potential candidate interactors of EBF1 and PAX5, which were later confirmed by sequencing techniques like ChIP-Seq, ATAC seq, and visualization techniques like proximity ligation assay (PLA).
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| 5. |
- Stjernberg (Zetterblad), Jenny
(författare)
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Knock Knock Knock, Who is there? - Cell Crosstalk within the Bone Marrow
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is focused on the subject of cell-cell interaction. Our body is composed of cells, most of them are integrated in a network with other cells that together forms tissues and organs. Every cell type in these complex organs has its special task and location. This is true whether we are doing research on humans or, as we have been, investigating mice. Mice are excellent models for studies of blood cell development since this process in mice resembles human blood cell generation in many regards.Cells communicate with each other by sending out small molecules or by directly binding to surrounding cells; to cells of the same kind as well as to cells with different origins and tasks. A cell is surrounded by hundreds of different signal-carrying entities; soluble, bound to the extra cellular matrix or bound to its surface. Every cell has to distinguish and respond to the environment according to its own specific nature.In the first article interleukin 7 (IL-7) a growth factor expressed by the stroma cells was studied. Results show that IL-7 is crucial for the immature progenitor cell in its development towards antibody producing B-lymphocytes. The second article is about stroma cells and their ability to support the development of B-cells. It is a comparative study on two different cell lines, where we focus on transcription factors and their regulation of protein induction of factors supporting B-cells. This study increased our knowledge of stroma cells. In the third paper we combined our knowledge from the first two papers in regard to stroma cells as well as B-cell development by testing if there is a possibility to theoretically find new factors of importance for the maturing B-cell. We achieved this by the development of GCINT, a database investigating possible receptorligand interactions between two cells, verifying these results in vitro with cell lines as well as primary cells. This revealed a two way communication between blood cells and stroma cells, highlighting the complexity of the bone marrow environment. In the last article we continued this work with primary FACS sorted stroma cells investing the potential connections between each of the stroma cell populations with primary blood cells in different stages of development. This work supports a model where hematopoietic cells can interact with stroma cells in a stage-specific manner and that the exchange between cells is of importance for their maturation.
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| 6. |
- Zandi, Sasan
(författare)
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What’s in a name? : Sub-fractionation of common lymphoid progenitors
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The hematopoietic system is a highly dynamic organ developed in many multi-cellular organisms to provide oxygen, prevent bleeding and to protect against microorganisms. The blood consist of many different specialized cells that all derive from rare hematopoietic stem cells (HSCs) located in the bone marrow in mice and humans. Blood cell production from HSCs occurs in a stepwise manner through development of intermediate progenitors that gradually loose lineage potentials. This is a tightly regulated process with complex regulatory mechanisms and many checkpoints that ensure a high and balanced production of blood cells. One of the fundamental questions in hematopoiesis relates to how the maturation of the cells is controlled and driven towards defined cell fates. The understanding of these processes is largely facilitated by isolation of intermediate populations of cells at defined stages of development.This thesis is focused on the regulatory mechanisms that regulate the maturation of B-lymphocytes constituting an important part of adaptive immunity by being responsible for the production of antibodies. It has been suggested that all the lymphoid cells have a common lineage restricted ancestor defined as a Lin-KitloSca1loFlt3+IL7R+ common lymphoid progenitor (CLP). These cells are believed retain the combined potentials for B, T and NK cells and it has been presumed that commitment of CLPs to B lineage is associated with expression of CD19 and B220 on progenitor B-cells.The aim of this thesis has been to identify the point of no return in B-cell development in order to allow for a better understanding of lineage restriction events in early lymphopoesis.To this end, we have used reporter transgenic mice where marker gene expression has been controlled by the transcription regulatory elements from one early lymphoid marker (Rag1) and one B-lymphoid restricted gene (λ5, Igll1). This allowed us to identify three functionally distinct sub-populations within the conventional CLP compartment. The cells were identified as CLPRaglowλ5- cells retaining B, T, Nk and a limited myeloid potential while up-regulation of Rag1 to generate CLPRaghighλ5- cells, was associated with loss of Nk potential as well as of the residual myeloid potential. Ultimately expression of λ5 in the CLPRag1highλ5+ compartment identifies the first committed B cells. Hence, our data suggest that the point of no return in B-cell development can be found within the CD19- CLP compartment. Using this new model for B-cell development, we investigated the instructive vs. permissive role of IL7 signaling in B cell commitment. Our results show that in absence of IL7, CLP maturation is impaired and generation of the earliest committed B-lineage cells is severely impaired. CLP maturation could not be rescued by ectopic expression of the anti-apoptotic Bcl2 protein even though the cells were able to generate normal B lineage cells after restoration of the IL7 signal. These findings suggest that Il7 is crucial for the maturation of lineage restricted CLPs and provide support for an instructive role of IL7 in early Bcell development.This thesis highlights the importance of precise identification of the point of commitment in B cell development and provides insight to the hematopoietic hierarchical model with the potential to serve as a map to better understand the mechanisms of hematopoietic disorders.
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| 7. |
- Åhsberg, Josefine
(författare)
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Molecular mechanisms in lymphoid restriction : securing the B lineage fate
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With the work in this thesis I have aimed to deepen the understanding of the mechanisms behind the development of different blood cell lineages with a specific focus on B cell development.To understand the interplay between extracellular signaling and transcription factor networks in early lymphoid development we investigated the functional collaborations of FLT3 and IL7R. We found that signaling via FLT3 and IL7R act in powerful synergy on proliferation of common lymphoid progenitors (CLPs). In addition to a role in expansion of progenitor cells we provided evidence for that IL7R signaling play a crucial role in B-cell commitment. IL7 deficient mice display a dramatic block in development before functional lineage restriction in the Ly6D+ CLP-compartment. The few Ly6D+CLPs that do develop have reduced mRNA levels of transcription factor EBF1, a protein with crucial functions in lineage restriction and activation of the B-lymphoid program. One crucial function of EBF1 is to activate Pax5. Even though Pax5 deficient fetal liver cells upon transplantation to congenic hosts will generate an abundance of cells with an activated B-lineage transcriptional program, the pro-B cells have disrupted regulation of non-B-lineage transcripts and a propensity to develop into T- and NK-cells in vitro. Both the activation of the B-lineage program and lineage restriction was dependent on the dose of transcription factors. Mice carrying a heterozygous mutation for the transcription factor E2A had slightly reduced relative frequency of progenitor cells and an impaired B-lineage specification in CLPs. Loss of one allele of Ebf1 resulted in reduced surface expression of IL2Rα and pre-B cell receptor (BCR), reduced IL7-response in vitro, and disrupted cell cycle dynamics in pro- and pre-B cells. While heterozygous loss of Pax5 did not result in any dramatic phenotype, the combined loss of one allele of Pax5 and one allele of Ebf1 (Pax5+/-Ebf1+/-) had a dramatic effect on lineage plasticity in B-cell progenitors compared to the single heterozygotes. Furthermore, these Pax5+/-Ebf1+/- mice developed spontaneous, transplantable pro-B cell tumors and had a significantly reduced probability to survive over time. The transformed cells show high in vitro plasticity and tumor cells with induced overexpression of intracellular Notch1 can transform into T-lineage cell in vivo.The data presented in this thesis add important pieces of information to the field of developmental hematopoiesis. By increasing the analytical depth of development in normal circumstances, and by understanding the consequence of genetic mutations in relation to cell type, we hope to contribute to the understanding of hematopoietic development in health and disease.
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