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- Danielsson, Marcus, 1985-
(författare)
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Mosaic loss of chromosome Y : methods for detection and consequences for affected leukocytes and men
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- It has been known for centuries that men live shorter lives than women, but until recently, the biological mechanisms driving this sex bias has been poorly understood. Mosaic loss of chromosome Y (mLOY) refers to chromosome Y aneuploidy, a male specific and the most common somatic mutation in human blood cells. Known risk factors include age, smoking and genetic predisposition. Men with mLOY carry a fraction of blood cells without the Y chromosome, due to its loss from hematopoietic progenitor cells during life. Hence, in a single cell LOY is a binary event causing the absence of almost 2% of the male haploid nuclear genome. When measured in bulk samples, it is present as a continuous mosaicism affecting a fraction of cells. A paper published in 1963 demonstrated that mLOY is frequent in cells of the hematopoietic system in aging men, but it was long viewed as a neutral event. In contrast, recent discoveries demonstrate that mLOY in blood is associated with various forms of cancer, autoimmune conditions, Alzheimer’s disease, cardiovascular events, diabetes as well as age-related macular degeneration. Studies support the hypothesis that mLOY in leukocytes may exacerbate disease processes in other organs. Thus, given the associations with several common diseases, mLOY in blood cells could help explain reduced male longevity.A main aim and a long term goal of the work presented in this thesis is the development of novel methods for improved mLOY detection. Focus here is exploration of analytes such as DNA, RNA and proteins, including studies of bulk samples as well as single cell approaches. Among the evaluated methods are SNP-arrays, ddPCR, WGS, RNA-seq and CITE-seq. Furthermore, a novel method called SPARC was developed for co-detection of the transcriptome and a panel of 92 proteins in single cells. Future methods with clinical utility for mLOY should, in addition to robust detection, be able to simultaneously discriminate mLOY in different types of immune cells. The latter builds on recent results showing cell type specificity with regard to disease associations. To meet these needs, single cell analyses using mLOY associated cell surface proteins have been pursued and proof-of-concept established. Implementation of mLOY screening in general populations has the potential to identify men with increased risk for various disease. It could be envisioned that further medical examination of men affected with mLOY would enable earlier diagnoses of ongoing disease processes as well as serving to guide targeted interventions.
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| 2. |
- Carlberg, Konstantin
(författare)
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Spatial Tissue Mapping on Joint Biopsies from Arthritis Patients
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that mainly affects joints, causing discomfort and pain that severely reduces the life quality of affected individuals. Its etiology is largely unknown, but some pathophysiological mechanisms have been identified. These include formation of anti-citrullinated protein antibodies (ACPAs) and rheumatic factors (RFs), local proliferation of mesenchymal cells, and recruitment of T- and B cells to the affected synovium. Lymphocyte infiltration results in elevated levels of cytokines such as Tumor Necrosis Factor alpha (TNF-α) and interleukin signaling, which in turn triggers protease activation that gradually degrades the synovium and underlying bone. In many cases RA can be effectively managed by early diagnosis followed by treatment with disease-modifying anti-rheumatic drugs (DMARDs). However, this is not true for all patients and there is currently no cure for RA. Synovial lesions in RA patients exhibit complex histopathological manifestations involving the formation of lymphoid follicles with highly organized Ectopic Lymphoid Structures (ELS). These have been extensively studied using immunostaining and other cytological methods, either by targeting a few specific molecular markers in tissue sections or by examining homogenized suspensions of complex samples, which causes a loss of local and spatial tissue information. This thesis reports the use of Spatial Transcriptomics (ST) to study gene expression in tissue samples from RA patients while preserving spatial information. The method was applied to RA biopsies from early onset and untreated RA to late-stage established disease with edema, providing comprehensive coverage of the spatio-temporal dynamics of the inflamed joints. Paper I introduces sRIN, a novel method of assessing the quality of RNA in tissue sections that is similar to RNA Integrity Number (RIN) analysis for bulk RNA but with single-cell resolution. The aim was to find ways of analyzing clinically rare samples for further processing with ST. Paper II uses ST to study tissue samples from RA joints with long-standing disease, using Spondyloarthritis (SpA) as a disease control. The resulting comprehensive transcriptomic data were used to perform in silico immune cell prediction and revealed how immune cell infiltration in RA differs from that in SpA in more detail than was previously possible using traditional pathological methods. As a follow up, Paper III investigates inflamed RA joints in even greater detail by using several adjacent tissue sections to build a three-dimensional atlas of assumed ELS areas. Finally, Paper IV uses four distinct technologies to study untreated early onset RA patients. Spatial tissue analysis with ST was combined with single cell RNA sequencing (scRNA-Seq) of fluorescence-activated cell sorted (FACS) B cells. These two methods were complemented with immunohistochemistry (IHC) for validation and sRIN to assess the quality of the clinical samples. B cells are known to play a key role in RA by producing self-reactive antibodies. This work showed that B cell maturation and ELS formation are detectable even in early onset RA, and revealed mechanisms supporting survival niches in hyperplastic joints. Overall, these studies shed new light on the complex nature of Rheumatoid arthritis, characterize the site of infection with greater granularity than was previously possible, and reveal novel disease patterns with clinical implications that warrant further study.
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| 3. |
- Marklund, Maja
(författare)
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Spatial transcriptome and epigenome analysis with focus on prostate cancer
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Each cancer is unique, and therefore the use of general treatments are often suboptimal. If we can understand the mechanisms of cancer development, we might be able to develop effective treatments tailored to each patient. Our bodies are complex three-dimensional structures and how things are organized correlate with proper functioning. Technologies for biological research have escalated enormously in the last years. Going from bulk analysis of tissues to the advent of single cell sequencing and spatially resolved transcriptomics has initiated a new era in biological research. The technology Spatial Transcriptomics (ST) combines histology with next-generation sequencing, making it possible to map which genes that are active at thousands of sub-areas in a tissue section. In Paper I, ST was combined with an in-house developed artificial intelligence method to explore the landscape of prostate cancer tissue. We identified a gene expression-based tumor signature in healthy tissue areas not possible to recognize through visual assessment, indicating that the genotype changes before phenotype. A gradient of the tumor microenvironment was also identified. In Paper II, prostate cancer tissue from three patients were investigated before and after androgen deprivation therapy using ST. All patients treated with this therapy long enough will reach a clinically defined stage called castration-resistant prostate cancer. We could see that only a set of cancer cells across the tissue responded to the treatment, which allowed comparison of gene expression program in responding versus non-responding cells. By understanding the underlying mechanisms to resistance, it might be possible to target these cells and decrease relapse risk. In Paper III, we inferred copy number variation from ST data allowing for the generation of genome integrity maps in cancerous tissue of prostate, breast, brain, and skin, and in a lymph node. This allowed us to identify tumor clones not recognizable histologically, indicating how genomic instability can be initiated and spread before visible for the naked eye. In Paper IV, we developed a method for spatial ATAC-seq by fusing the ST-technology with ATAC-seq, enabling the analyses of accessible chromatin while preserving histological information. The Visium platform by 10x Genomics was used and we demonstrate a similar capture efficiency to single-cell ATACseq.
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