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- El-Habta, Roine, 1988-
(author)
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Cell therapy for denervated tissue
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Background: Peripheral nerve injury results in denervation of tendons and muscles. The biology of denervated muscle has been well studied but little is known about the associated tendons. Denervation of muscle leads to atrophy which includes muscle fiber shrinkage and cell death, a process that is influenced by the lack of acetylcholine (ACh) signaling to the muscle cells. Recovery of long-term denervated muscle function is often poor. This thesis describes how a cell therapy approach using adipose tissue-derived stromal vascular fraction (SVF) may be used to protect and regenerate denervated muscle. Previous studies have shown how adipose tissue-dervied stem cells (ASCs), commonly expanded from the SVF, have pro-regenerative effects on the injured peripheral nervous system, and how ASCs differentiated towards a “Schwann cell-like phenotype” (dASCs) reduce muscle atrophy. In this thesis work, we studied the possible mechanisms underlying the regenerative potential of both SVF and culture expanded dASCs.Hypotheses: We hypothesized that: 1) denervated tendon displays morphological and biochemical properties that resemble the chronic degenerative tendon condition known as tendinosis; 2) denervated muscle up-regulates expression of muscarinic acetylcholine (ACh) receptors and apoptosis-associated signaling mechanisms; 3) dASCs enhance the proliferation of myoblasts in vitro through secretion of ACh; 4) SVF influences the proliferation, differentiation, and survival of myoblasts in vitro via secretion of growth factors; and 5) SVF can preserve denervated muscle tissue. To test our hypotheses, two model systems were used: an in vitro model based on indirect co-culture, and an in vivo rat sciatic nerve transection model.Results: Denervated tendon displayed morphological changes similar to tendinosis, including hypercellularity, disfigurement of cells, and disorganized collagen architecture, along with an increased expression of type I and type III collagen. In addition, levels of neurokinin 1 receptor (NK-1R) were upregulated in the tendon cells. In denervated muscle, there was an increased expression of muscarinic ACh receptors, as well as of genes associated with apoptosis, such as caspases, cytokines (e.g., tumor necrosis factor-alpha; TNF-a), and death domain receptors. We subsequently used TNF-aas an inducer of apoptosis in an in vitrorat primary myoblast culture model. TNF-aactivated/cleaved caspase 7 and increased poly ADP-ribose polymerase (PARP) levels. Moreover, Annexin V and TUNEL were increased after TNF-atreatment. Indirect co-culture with SVF significantly reduced all these measures of apoptosis. Proliferation studies showed that both dASCs and SVF enhanced growth of myoblasts in vitro. With dASCs, the effect was partially explained by secretion of ACh, and for SVF by released growth factors, such as hepatocyte growth factor (HGF). In both cases, the signal was mediated via phosphorylation of ERK1/2 (MAPK). HGF also had an inhibitory effect on the differentiation of myoblasts into myotubes. Finally, the protective effects of SVF were confirmed in vivo: injections of SVF into denervated muscle significantly increased the mean fiber area and diameter, as well as reduced the expression of apoptotic genes and TUNEL reactivity.Conclusions: Denervated tendons undergo severe degenerative changes similar to tendinosis. Furthermore, SVF has the ability to reduce muscle atrophy in vivo. Using in vitro systems, we showed that this might occur through secretion of growth factors which activate MAPK signaling and anti-apoptotic pathways. In conclusion, SVF offers a promising approach for future clinical application in the treatment of denervated muscle.
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| 2. |
- Lindholm, Karl-Johan, 1970-
(author)
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Wells of Experience : A pastoral land-use history of Omaheke, Namibia.
- 2006
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Doctoral thesis (other academic/artistic)abstract
- The conventional view on the Kalahari in southern Africa expresses that the area is unsuitable for livestock herding. For this reason, it is argued that livestock herders avoided the Kalahari in the past and were only able to establish themselves in the later half of the twentieth century, when deep-reaching boreholes were introduced in the area. An effect of this concept was that the archaeological record of pastoralists in the Kalahari either was perceived as non-existent or received little attention from scientific enquiry.Based on an archaeological survey in the Kalahari of the northeastern part of Namibia, the purpose of this study is to construct an alternative approach to the archaeology of livestock herding. The aim is to contribute to a better understanding of the areas unrecorded land-use history. I depart from the notion that the main ecological constraint for dryland pastoralism is the availability of dry season water and fodder resources. For this reason, the fundamental basis for a pastoral land-use system is places that contain dry season resources. By reviewing recent ecological research, historical and anthropological accounts and previous archaeological research, I establish a link between livestock herders’ procurement of dry season key resources and the practice of digging wells. The link can be motivated from the pastoral ambition of accumulating livestock and high water requirements in the restrained dry season. On this basis, I suggest that artificial wells are useful indicators of pastoral land use in the Kalahari. The most crucial task for the study is to address the archaeological visibility of pastoral well sites. By a research approach integrating the theoretical understanding of pastoralism and a methodology including ecology, archaeology, history and the knowledge of the people who keep livestock in the region today, the archaeological survey revealed 40 well sites, including nearly 200 well structures that have all been used for watering livestock. However, it would be unfortunate if a study of pastoral wells would solely address the ecological foundation and the archaeological visibility of pastoralism. I suggest that the wells signify the labour of peoples with common or separate histories, with or without own herds, but probably talked about in relation to herds. I will also argue that the wells can be used for tracking and reconstructing a pastoral land-use system that predated the colonial era. Furthermore, the wells can be used to identify changes of the land-use that took place during the twentieth century, which involved that livestock herding was more or less abandoned in large parts of northwestern Kalahari. The study surmises that the critical historical perspective is valuable for development projects and conservationist interventions active in the region, especially in the light of the recent trends in the dryland ecology, which shows a larger appreciation for the indigenous understanding of the management of dryland ecosystems. With modifications, the developed approach can be applicable for land-use historical research elsewhere in southern Africa.
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| 3. |
- Paul, Elisabeth R., 1991-
(author)
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Immunological Changes and Brain Function over a Psychotic-Depressive Spectrum
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Psychotic and depressive disorders are severe psychiatric disorders that contribute significantly to the global burden of disease. They are distinct disorders with different symptom profiles according to both the Diagnostic and Statistical Manual and the International Classification of Diseases. However, these disorders share commonalities in various aspects, such as high comorbidity, prevalence of subclinical symptoms, and shared genetics. Furthermore, both disorders have been associated with a dysregulated immune system functioning.In this thesis, we aimed to identify common biological dimensions of both depression and psychosis by first investigating proteins related to immune system activation in depression and psychosis separately, and then identifying biological underpinnings of psychotic-like symptoms in depression.Specifically, we first assessed in major depressive disorder central nervous system levels of metabolites along the kynurenine pathway, a pathway that is regulated by the immune system and implicated in depressive and psychotic disorders (paper I). We found an imbalance between neuroprotective versus neurotoxic metabolites in blood and decreased levels of a neuroprotective metabolite in cerebrospinal fluid of patients with depression.Next, we assessed patterns of proteins implicated in immune-system function that distinguish first episode psychosis and healthy controls (paper II). Results indicate prominent changes in patients compared to controls, partially replicating previous findings and partially highlighting proteins that have not previously been assessed in psychosis.Lastly, we investigated psychotic-like symptoms in patients with major depressive disorder, finding a relation to immune system markers (paper III) and changes in connectivity between brain structures that integrate information about the physical body and autobiographic information into a sense of self (paper IV).In summary, the results from this thesis suggest that both in major depressive disorder and first episode psychosis there might be a dysregulation of the immune system and closely related systems such as the kynurenine pathway. These commonalities could further underlie the prevalence of subclinical psychotic-like symptoms in major depressive disorder. Ultimately, a better understanding of the underlying biological mechanisms of psychiatric disorders and, transdiagnostically, their symptoms will help formulate empiricallyinformed frameworks to guide clinical diagnostic processes and treatments.
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| 6. |
- Kolar, Mallappa K., 1981-
(author)
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Transplantation of mesenchymal stem cells and injections of microRNA as therapeutics for nervous system repair
- 2016
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Doctoral thesis (other academic/artistic)abstract
- Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.
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| 7. |
- Larsen, Kasper J.
(author)
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Maximal Unitarity at Two Loops : A New Method for Computing Two-Loop Scattering Amplitudes
- 2012
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Doctoral thesis (other academic/artistic)abstract
- The study of scattering amplitudes beyond one loop is necessary for precision phenomenology for the Large Hadron Collider and may also provide deeper insights into the theoretical foundations of quantum field theory. In this thesis we develop a new method for computing two-loop amplitudes, based on unitarity rather than Feynman diagrams. In this approach, the two-loop amplitude is first expanded in a linearly independent basis of integrals. The process dependence thereby resides in the coefficients of the integrals. These expansion coefficients are then the object of calculation.Our main results include explicit formulas for a subset of the integral coefficients, expressing them as products of tree-level amplitudes integrated over specific contours in the complex plane. We give a general selection principle for determining these contours. This principle is then applied to obtain the coefficients of integrals with the topology of a double box. We show that, for four-particle scattering, each double-box integral in the two-loop basis is associated with a uniquely defined complex contour, referred to as its master contour. We provide a classification of the solutions to setting all propagators of the general double-box integral on-shell. Depending on the number of external momenta at the vertices of the graph, these solutions are given as a chain of pointwise intersecting Riemann spheres, or a torus. This classification is needed to define master contours for amplitudes with arbitrary multiplicities.We point out that a basis of two-loop integrals with as many infrared finite elements as possible allows substantial technical simplications, in terms of obtaining the coefficients of the integrals, as well as for the analytic evaluation of the integrals themselves. We compute two such integrals at four points, obtaining remarkably compact expressions. Finally, we provide a check on a recently developed recursion relation for the all-loop integrand of the amplitudes of N=4 supersymmetric Yang-Mills theory, examining the two-loop six-gluon MHV amplitude and finding agreement. The validity of the approach to two-loop amplitudes developed in this thesis extends to all four-dimensional gauge theories, in particular QCD. The approach is suited for obtaining compact analytical expressions as well as for numerical implementations.
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| 8. |
- Lauvrud, Anne Therese, 1975-
(author)
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Optimizing stem cells for reconstructive surgery
- 2024
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Doctoral thesis (other academic/artistic)abstract
- Fat grafting has become an established method in plastic surgery for treating soft tissue defects. The results for survival of the fat being transplanted is unpredictable and supplementation of the graft with the Stromal Vascular Fraction (SVF) or cultures Adipose tissue-derived stem cells (ASCs) can enhance graft viability. The ASCs are a heterogenous group of cells with various cell membrane markers, and differing growth promoting and differentiation characteristics of the stem cells derived from the fat. It is of high importance when expanding cells prior to the transplantation of the cells into patients, that the culture conditions are well defined and ideally are xenofree, avoiding use of animal-derived products. Furthermore, the procedures must be safe and not increase risk for recurrence of cancer after reconstructive surgeries. This thesis explores the phenotypic properties of a selected population of ASCs, with a view to determining their suitability for transplantation into fat grafts. ASCs were isolated from SVF of human abdominal fat and CD146+ cells were selected using immunomagnetic beads. The proliferation, angiogenic and adipogenic properties were significantly higher in the CD146+ cells. Stem cells were also isolated from lipoaspirate obtained using two different liposuction methods. Waterjet lipoaspirates yielded the greatest number of CD146+ cells with high adipogenic potential and angiogenic activity. The cells could also be successfully isolated using a closed processing system. Cells were expanded in either foetal bovine serum, platelet lysate or a chemically defined xenofree (XV) medium. Cultures in XV medium proliferated the fastest, expressed the highest number of CD146+ cells, and showed the best adipogenic and angiogenic properties. To test possible ASCs interactions with cancer cells, co-cultures with MCF-7 breast cancer cells were established. Conditioned medium from co-cultures significantly increased the migration of the cancer cells but not their proliferation, and there was increased expression of Tenascin-C in these cultures. The research in this thesis work has shown more optimal ways to isolate and expand ASCs, potentially offering new therapeutic reconstructive treatment options for a variety of medical conditions.
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| 9. |
- Löf, Liza
(author)
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Applications of in situ proximity ligation assays for cancer research and diagnostics
- 2016
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Doctoral thesis (other academic/artistic)abstract
- In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system. Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt. A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry.
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| 10. |
- Oliveros Anerillas, Luis, 1995-
(author)
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Development and optimization of a 3D in vitro model for osteogenic biomaterial evaluation
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Despite the innate regenerative capabilities of bone tissue, self-repair is impaired when an injury exceeds the critical size threshold because of trauma, congenital, or pathological conditions. Autologous transplantation is the gold standard to reconstruct large bone defects. However, this method has drawbacks such as limited amount of graftable material, limited accessibility, and donor site morbidity. For these reasons, alternative regenerative medicine and tissue engineering approaches are being developed, including implantable scaffolds.The use of in vitro-made scaffolds containing biomaterials that mimic the functional characteristics and composition of extracellular bone matrix has been favored in 3D vs 2D in vitro culture systems. Adult stem cells such as mesenchymal stem cells (MSCs), that give rise to bone building cells, have been used in combination with various biomaterials. The development of an implantable scaffold with or without cells requires extensive in vitro validation and optimization prior to its testing in vivo. Thus, the primary aim of this thesis was to develop a 3D model for the optimization of MSC differentiation. Further aims were to utilize this 3D model to evaluate the MSC response to a novel osteogenic biomaterial. To achieve these objectives human bone marrow MSCs (BMSCs) were utilized in various hydrogels in combination with chemical differentiation factors or biomaterials. Moreover, the osteogenic capability of the tested biomaterials and their induced inflammatory/angiogenic responses were investigated, and the culture conditions were optimized for clinical application. In this thesis, a comparison between 2D and 3D (hydrogels) in vitro culture models was developed with the purpose of studying osteogenic differentiation in BSMCs. Testing various hydrogels revealedt he superiority of type I collagen hydrogels for the osteogenic 3D in vitro culture system. Further, cell culture conditions were improved for the expansion and differentiation of BMSCs to fulfill clinically approved standards according to Good Manufacturing Practice (GMP) conditions. Comparisons between fetal bovine serum (FBS) and human platelet lysate (PLT) showed superior cellular differentiation in FBS, while PLT enhanced cell proliferation. Based on the developed 3D model, the osteogenic properties of a novel nanoporous silica calcium phosphate (nSCP) material were investigated. The results indicated that nSCP was osteoinductive, involving different pathways compared with the traditional chemical differentiation protocols or other tested osteogenic biomaterials. Finally, the inflammatory and angiogenic responses from human BMSCs and an immortalized monocyte cell line (THP-1) exposed to nSCP in the established 3D model were assessed.The results indicated limited inflammatory effect of nSCP, while inducing the secretion of pro-angiogenic cytokines. The bioactivity of these released factors was confirmed in an assay using human endothelial cells.Taken together, this thesis presents a 3D in vitro model for studying osteogenic differentiation in MSCs, which can be utilized to evaluate, validate, and optimize biomaterial candidates for bone regeneration applications.
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