| 1. |
- Ceder, Mikaela, 1991-
(author)
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Characterization of Novel Solute Carriers in Humans, Mice and Flies : Solute Carriers in a Broad and Narrow Perspective
- 2020
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Doctoral thesis (other academic/artistic)abstract
- The solute carrier family is the largest family of membrane-bound transporters in humans, with 430 members divided into 65 subfamilies. They transport various substrates across lipid barriers and are vital for absorption, distribution, metabolism and excretion in all cell types in the body. Despite being involved in vital functions, and their effect on both physiology and pathophysiology, many transporters are not characterized. The aim of this thesis was to study newly identified putative solute carriers of which little is known. In Paper I, the relationship of solute carriers in humans and fruit flies was studied. The study revealed that 54 of the 65 subfamilies in humans have one or more orthologues in fruit flies, and a total of 381 orthologues were identified in fruit flies. In Paper II, a comprehensive study of the putative solute carriers and their response to different sugar concentrations were performed. Several, but not all, putative solute carriers were altered in cell cultures maintained in media containing low or no glucose, and the expression normalized upon refeeding with glucose. Similar results were observed in fruit flies subjected to complete starvation or diets with varying sugar concentrations. Last, in Paper III and IV, characterization of one putative solute carrier, UNC93A, was performed. The studies revealed that UNC93A was a conserved protein with an abundant expression in the body of mice but with a restricted expression in fruit flies. The protein was found to possibly be expressed at, or close to, the plasma membrane of cells and to co-localize with Twik-Acid sensitive potassium channels. UNC93A was found to be important for the renal function in fruit flies and to affect survival and membrane potentials in cells. The findings of this thesis establish a high conservation of several putative solute carriers and that they have a highly dynamic regulation during fluctuating energy and glucose availability. Further, while several clear biological aspects of UNC93A was identified, the exact function of transporter proteins is cumbersome to find and more research about these transporters is needed to fully understand their mechanistic role and their association and/or involvement in health and sickness.
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| 2. |
- Nätt, Daniel, et al.
(author)
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Male reproductive health and intergenerational metabolic responses from a small RNA perspective
- 2020
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In: Journal of Internal Medicine. - : WILEY. - 0954-6820 .- 1365-2796. ; 288:3, s. 305-320
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Research review (peer-reviewed)abstract
- The world has recently experienced a decline in male reproductive (e.g. sperm counts and motility) and metabolic (e.g. obesity and diabetes) health. Accumulated evidence from animal models also shows that the metabolic health of the father may influence the metabolic health in his offspring. Vectors for such paternal intergenerational metabolic responses (IGMRs) involve small noncoding RNAs (sncRNAs) that often increase in spermatozoa during the last days of maturation in the epididymis. We and others have shown that the metabolic state - depending on factors such as diet, obesity and physical exercise - may affect sperm quality and sperm sncRNA. Together, this suggests that there are overlapping aetiologies between the male metabolic syndrome, male factor infertility and intergenerational responses. In this review, we present a theoretical framework for an overlap of these aetiologies by exploring the advances in our understanding of the roles of sncRNA in spermatogenesis and offspring development. A special focus will lie on novel findings about tRNA-derived small RNA (tsRNA), rRNA-derived small RNA (rsRNA) and small mitochondrial RNA (mitoRNA), and their emerging roles in intergenerational metabolic and reproductive health.
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| 3. |
- Ramesh, Rashmi, 1984-, et al.
(author)
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Dietary Sugar Shifts Mitochondrial Metabolism and Small RNA Biogenesis in Sperm
- 2023
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In: Antioxidants and Redox Signaling. - : MARY ANN LIEBERT, INC. - 1523-0864 .- 1557-7716. ; 38:16-18, s. 1167-1183
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Journal article (peer-reviewed)abstract
- Aims: Increasing concentrations of dietary sugar results in a linear accumulation of triglycerides in male Drosophila, while inducing a U-shaped obesity response in their offspring. Here, using a combination of proteomics and small RNA (sRNA) sequencing, we aimed at understanding the molecular underpinning in sperm for such plasticity.Results: Proteomic analysis of seminal vesicles revealed that increasing concentrations of dietary sugar resulted in a bell-shaped induction of proteins involved in metabolic/redox regulation. Using stains and in vivo redox reporter flies, this pattern could be explained by changes in sperm production of reactive oxygen species (ROS), more exactly mitochondria-derived H2O2. By quenching ROS with the antioxidant N-acetyl cysteine and performing sRNA-seq on sperm, we found that sperm miRNA is increased in response to ROS. Moreover, we found sperm mitosRNA to be increased in high-sugar diet conditions (independent of ROS). Reanalyzing our previously published data revealed a similar global upregulation of human sperm mitosRNA in response to a high-sugar diet, suggesting evolutionary conserved mechanisms.Innovation: This work highlights a fast response to dietary sugar in mitochondria-produced H2O2 in Drosophila sperm and identifies redox-sensitive miRNA downstream of this event.Conclusions: Our data support a model where changes in the sperm mitochondria in response to dietary sugar are the primary event, and changes in redox homoeostasis are secondary to mitochondrial ROS production. These data provide multiple candidates for paternal intergenerational metabolic responses as well as potential biomarkers for human male fertility.
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| 4. |
- Skog, Signe, et al.
(author)
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Seqpac: a framework for sRNA-seq analysis in R using sequence-based counts
- 2023
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In: Bioinformatics. - : OXFORD UNIV PRESS. - 1367-4803 .- 1367-4811. ; 39:4
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Journal article (peer-reviewed)abstract
- Motivation: Feature-based counting is commonly used in RNA-sequencing (RNA-seq) analyses. Here, sequences must align to target features (like genes or non-coding RNAs) and related sequences with different compositions are counted into the same feature. Consequently, sequence integrity is lost, making results less traceable against raw data.Small RNA (sRNA) often maps to multiple features and shows an incredible diversity in form and function. Therefore, applying feature-based strategies may increase the risk of misinterpretation. We present a strategy for sRNA-seq analysis that preserves the integrity of the raw sequence making the data lineage fully traceable. We have consolidated this strategy into Seqpac: An R package that makes a complete sRNA analysis available on multiple platforms. Using published biological data, we show that Seqpac reveals hidden bias and adds new insights to studies that were previously analyzed using feature-based counting.We have identified limitations in the concurrent analysis of RNA-seq data. We call it the traceability dilemma in alignment-based sequencing strategies. By building a flexible framework that preserves the integrity of the read sequence throughout the analysis, we demonstrate better interpretability in sRNA-seq experiments, which are particularly vulnerable to this problem. Applying similar strategies to other transcriptomic workflows may aid in resolving the replication crisis experienced by many fields that depend on transcriptome analyses.
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| 5. |
- Toia, Beatrice, 1993-
(author)
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Molecular characterization of the interplay between DNA-PK and TRF2 in telomere protection
- 2024
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Doctoral thesis (other academic/artistic)abstract
- The genetic material of mammalian cells is organized as linear chromosomes inside their nucleus. In order to preserve genomic stability, mammalian cells have developed several mechanisms to detect and repair DNA lesions that can occasionally occur in the genome. Nevertheless, in certain circumstances, these pathways can promote aberrant repair that can result in genomic instability and tumorigenesis. One such case are the ends of linear chromosomes, which are particularly susceptible since they can be mistakenly recognized as DNA lesions by the DNA repair machinery, which seeks to repair the damage by joining the two DNA ends together and thus causing rearrangements and genome instability. Therefore, the linear ends of chromosomes need to be protected from being sensed as sites of damage, and this is promoted by the presence of specialized structures known as telomeres, with the aim of preventing determinantal consequences of having exposed DNA ends. Telomeres are formed by a repetitive DNA sequence that is bound by a protein complex named shelterin, which forms a protective structure at the end of chromosomes. Among the shelterin subunits, TRF2 plays a crucial role. Other factors that have been found essential for the protection of telomeres are Apollo and DNA-PK proteins which, strikingly, are also involved in DNA repair mechanisms.Understanding the underlying mechanism of telomere protection and the interplay between factors involved is of relevance since defects in telomere protection activate aberrant repair, which is associated with genome instability, a prime mechanism shaping cancer.In Paper I, we focused on how Apollo and the catalytic subunit of DNA-PK (DNA-PKcs) cooperate in preserving telomere homeostasis, and we shed light on the mechanistic function of DNA-PKcs in granting access to Apollo to the telomeric ends. We found that this function requires the kinase activity of DNA-PKcs to promote autophosphorylation, and the binding of Apollo to DNA-PKcs for optimal positioning at the DNA ends. This study shows an analogous mechanism of function of DNA-PK at sites of DNA lesions and at telomeres.In Paper II, we found that the localization of DNA-PK at telomeres post-replication is essential to block the nucleolytic erosion of the telomeric ends by any other nuclease than Apollo. This protective function is also independently fulfilled by the iDDR domain of TRF2, which specifically inhibits the endonucleolytic activity of MRN protein complex. This study reveals the high pressure for cells to keep tight control of resection at telomeres.In Paper III, we expanded our studies by investigating the role of Apollo and DNA-PKcs in ALT cancerous cells, which utilize a recombination-mediated mechanism to elongate telomeres. Here, we show that DNA-PKcs and Apollo have a conserved role in promoting G-overhang formation, but in the absence of Apollo and/or DNA-PKcs kinase activity, telomeres are insensitive to fusion. Moreover, we found that Apollo promotes telomere recombination events at ALT telomeres. This study opens the possibility for different mechanisms of telomere maintenance in ALT cells.
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| 6. |
- Örkenby, Lovisa, 1992-
(author)
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Small non-coding RNA in early fly development : plasticity, interactions and improved bioinformatic tools
- 2023
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Doctoral thesis (other academic/artistic)abstract
- At fertilization, the male and female pronuclei undergo a transformation from germline to pluripotency as they fuse, marking the beginning of Drosophila embryogenesis. As the parental contributions decrease, the zygote takes control of its genome in a process called the maternal-to-zygotic transition (MZT). Several small non-coding RNAs (sncRNAs), a very large and diverse group of RNAs, have regulatory roles during this transition. This includes for example microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs). Regulation by miRNAs mainly occurs through mediating maternal mRNA degradation, while piRNAs operate by repressing transposable elements (TEs) and regulating the nanos-induced embryonic body axis determination.In this thesis, the complex and dynamic field of early Drosophila embryogenesis and sncRNAs are put in relation to the included papers. In Paper I, I explored the most stress-sensitive embryonic period and found that stress before the midblastula transition retains maternal miRNAs. These miRNAs impact zygotic gene activation by modulating the boundary factor Elba1, leading to compromised transcription control. Paper III examines the piRNA population during MZT. I find differences of unique piRNA sequences in embryos of different ages but not in target preferences, potentially highlighting the importance of constant repression of certain TEs. Paper II addresses specific difficulties with sncRNA seq data analysis and presents a bioinformatic framework to improve these analyses using a sequence-based strategy.This thesis highlights the intricate interplay of sncRNAs in the critical period of early Drosophila embryogenesis and offers insights into their regulatory roles.
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| 7. |
- Örkenby, Lovisa, et al.
(author)
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Stress-sensitive dynamics of miRNAs and Elba1 in Drosophila embryogenesis
- 2023
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In: Molecular Systems Biology. - : WILEY. - 1744-4292 .- 1744-4292. ; 19:5
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Journal article (peer-reviewed)abstract
- Early-life stress can result in life-long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. Here, we show that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher-order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer-1 mutant, we show that the stress-induced decrease in one of these genes, the insulator-binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV w(m4h) reporter. We propose that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.
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| 8. |
- Örtegren Kugelberg, Unn, et al.
(author)
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5 XP sRNA-seq: efficient identification of transcripts with and without 5 phosphorylation reveals evolutionary conserved small RNA
- 2021
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In: RNA Biology. - : TAYLOR & FRANCIS INC. - 1547-6286 .- 1555-8584. ; 18:11, s. 1588-1599
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Journal article (peer-reviewed)abstract
- Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5 nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that captures a more complete picture of sRNA. By tagging 5 P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5MODIFIER LETTER PRIME-terminal modifications (5 X), with a selective approach for 5-phosphorylated sRNA (5 P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5 insensitive library strategies with 5 sensitive tagging, we have successfully tackled an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.
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