Dissecting immune interactions in health and disease with multiomics and spatial technologies

Cells are the basic building blocks of life, forming the enormous plethora of tissues and living organisms on Earth. They have a high diversity of phenotypes and functions in different environments. The high-throughput tools to profile different modalities from a single cell have grown exponentially in recent years. This now allows us to draw a complete picture of how cells function in different environments for the first time. In the work of my thesis, I use high-throughput multiomics and spatial technologies to create comprehensive cell atlases. I focus on studying the immune cell communication among themselves and with other cells in the context of disease and development. Chapter 1 starts with an outline of the background on cell biology and the impact that genomic technologies have on how we can study cellular processes. I then discuss the experimental methodology of high-throughput multiomics and spatial techniques, and computational tools for the analysis of such data. Following is the introduction to the two projects comprising the work of my thesis: (i) a multiomics study of Common Variable Immunodeficiency (CVID) and, (ii) a spatial multiomics map of the Maternal-Fetal Interface (MFI) in early pregnancy in humans. Chapter 2 outlines materials and methods used in this work, showcasing the workflow for each project. Chapter 3 details the multiomics atlas of Common Variable Immunodeficiency (CVID). This condition is characterised by defects in the function of B cells, a type of adaptive immune cells capable of producing antibodies to fight infections. I analyse gene expression and chromatin accessibility data of B cells from a pair of monozygotic CVID-discordant twins. I uncover potential defects in the epigenome of the affected twin’s B cells. Next, after in vitro stimulation of these twins’ PBMCs, I observe CVID-associated transcriptional dysregulation in immune subsets additional to those in B cells. I discover defects in the immune cell crosstalk between B cells and other immune compartments of the CVID twin. With an expanded cohort of CVID patients and healthy individuals, I go on to further validate these findings. These results show that, in addition to B-cell-intrinsic alterations, defects in cell-cell communication between B cells and other immune compartments may be compromising the correct immune response in CVID. Chapter 4 presents the work on creating a comprehensive spatial multiomics atlas of the maternal-fetal interface in early pregnancy. Firstly, I characterise the signatures and differentiation trajectories of trophoblast cells - the building blocks of placenta. I then focus on the crosstalk between invading trophoblast and maternal immune cells. I predict putative cell-cell communication events and validate in situ the selected molecules mediating these interactions. I propose a model of arterial transformation facilitated by fetal trophoblast and their communication with maternal cells. This work expands our knowledge about the cellular and molecular players in the maternal-fetal dialog in the first trimester of pregnancy, definitive of its success. Chapter 5 describes the work on modelling the dialog between decidual natural killer (dNK) cells, a type of innate immune cell most abundant in pregnant decidua, and the invading trophoblast at the maternal-fetal interface using primary trophoblast organoids (PTO). I benchmark the PTO system against the in vivo trophoblast atlas I described in chapter 4. After defining trophoblast cell states in vitro, I perform comparative analysis of PTOs stimulated with a cocktail of chemokines that in vivo are secreted by dNK cells and unstimulated PTOs as control. I propose a putative effect of the signals from dNK cells on trophoblast invasion in the first trimester of pregnancy. Lastly, Chapter 6 provides an overview of all the described work, as well as a discussion of how the novel high-throughput multiomic and spatial technologies together with in vitro models shape our current view of fundamental biology, and how they will impact future directions of research.Wellcome Trust 4-Year PhD Studentshi

"I think of crime when I´m in a N.Y state of mind" : Hur hiphopmusik utrycker det amerikanska samhället.

This bachelor thesis examine how the early hip-hop music mirrors the society in the US ghettos in the 80s and 90s in order to explore how the artists' individual experiences take the expression in artistic form of coherent discourse on public issues. It uses structural textual analysis thatencode and analyze lyrics from ten hip-hop songs. The material was studied with the help of Elias and Scotsons theories established and outsiders, Bourdieu's capital types, field and habitus, and postcolonial theory that Eriksson, Eriksson Baaz and Thorn highlights in there book GlobaliseringensKulturer. The survey shows that the songs we analyzed tells of similar problems, which we divided into five themes: crime, poverty, ideals, discrimination and depression. In this way, we concluded that there are common patterns in the songs we chose. In conclusion, the study shows that the analyzed songs express the artists’, both individual problems such as depression, poverty, criminality in an artistic form. But also other issues such as discrimination and racism in a larger level.Den här studien undersöker hur tidig hiphop speglar samhället i USAs förorter på 80- och 90 talet i syfte att utforska hur artisternas individuella upplevelser tar sig utryck i konstnärlig form i sammanhängande diskurser om allmänna samhällsproblem.    Metoden består utav strukturalistisk textanalys, där texter från hiphopmusik kodas och analyseras. Teorier som använts består utav Elias och Scotsons teorier kring etablerade och outsiders, Bourdieus kapital typer, fält och habitus samt postkolonial teori som Eriksson, Eriksson Baaz och Thörn behandlar i Globaliseringens Kulturer. Undersökningen visar att artisterna berättar om liknade problem och teman i sina låtar som beskrivs efter: kriminalitet, fattigdom, ideal, diskriminering och depression. Slutsatserna som studien drar består av hur låttexterna blir utryck i konstnärlig from sådant som artisternas individuella problem som depression, fattigdom, kriminalitet samt tar upp saker som diskriminering och rasism på ett större makro nivå

Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice

<div><h3>Background</h3><p>Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types <em>in vivo</em>. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.</p> <h3>Methodology/Principal Findings</h3><p><em>Bcr</em> and <em>abr</em> null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia <em>bcr−/−</em> and <em>abr−/−</em> macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.</p> <h3>Conclusions</h3><p>Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.</p> </div

Expression of <it>cassini</it>, a murine gamma-satellite sequence conserved in evolution, is regulated in normal and malignant hematopoietic cells

Abstract Background Acute lymphoblastic leukemia (ALL) cells treated with drugs can become drug-tolerant if co-cultured with protective stromal mouse embryonic fibroblasts (MEFs). Results We performed transcriptional profiling on these stromal fibroblasts to investigate if they were affected by the presence of drug-treated ALL cells. These mitotically inactivated MEFs showed few changes in gene expression, but a family of sequences of which transcription is significantly increased was identified. A sequence related to this family, which we named cassini, was selected for further characterization. We found that cassini was highly upregulated in drug-treated ALL cells. Analysis of RNAs from different normal mouse tissues showed that cassini expression is highest in spleen and thymus, and can be further enhanced in these organs by exposure of mice to bacterial endotoxin. Heat shock, but not other types of stress, significantly induced the transcription of this locus in ALL cells. Transient overexpression of cassini in human 293 embryonic kidney cells did not increase the cytotoxic or cytostatic effects of chemotherapeutic drugs but provided some protection. Database searches revealed that sequences highly homologous to cassini are present in rodents, apicomplexans, flatworms and primates, indicating that they are conserved in evolution. Moreover, CASSINI RNA was induced in human ALL cells treated with vincristine. Surprisingly, cassini belongs to the previously reported murine family of γ-satellite/major satellite DNA sequences, which were not known to be present in other species. Conclusions Our results show that the transcription of at least one member of these sequences is regulated, suggesting that this has a function in normal and transformed immune cells. Expression of these sequences may protect cells when they are exposed to specific stress stimuli.</p

Chain-End Functionalization of Poly(&epsilon;-caprolactone) for Chemical Binding with Gelatin: Binary Electrospun Scaffolds with Improved Physico-Mechanical Characteristics and Cell Adhesive Properties

Composite biocompatible scaffolds, obtained using the electrospinning (ES) technique, are highly promising for biomedical application thanks to their high surface area, porosity, adjustable fiber diameter, and permeability. However, the combination of synthetic biodegradable (such as poly(&epsilon;-caprolactone) PCL) and natural (such as gelatin Gt) polymers is complicated by the problem of low compatibility of the components. Previously, this problem was solved by PCL grafting and/or Gt cross-linking after ES molding. In the present study, composite fibrous scaffolds consisting of PCL and Gt were fabricated by the electrospinning (ES) method using non-functionalized PCL1 or NHS-functionalized PCL2 and hexafluoroisopropanol as a solvent. To provide covalent binding between PCL2 and Gt macromolecules, NHS-functionalized methyl glutarate was synthesized and studied in model reactions with components of spinning solution. It was found that selective formation of amide bonds, which provide complete covalent bonding of Gt in PCL/Gt composite, requires the presence of weak acid. With the use of the optimized ES method, fibrous mats with different PCL/Gt ratios were prepared. The sample morphology (SEM), hydrolytic resistance (FT-IR), cell adhesion and viability (MTT assay), cell penetration (fluorescent microscopy), and mechanical characteristics of the samples were studied. PCL2-based films with a Gt content of 20 wt% have demonstrated the best set of properties

Loss of Bcr or Abr promotes hypoxia-induced Rac1 activation <i>in vitro</i> and <i>in vivo</i>. A,

<p>Real-time RT-PCR analysis for quantification of <i>rac1</i>, <i>rac2</i> and <i>rac3</i> mRNA in PASMCs. <b>B,</b> Representative gel electrophoresis of RT-PCR products showing absence of <i>rac2</i> in PASMCs. Samples loaded are indicated above the lanes; spleen, positive control; RNA (−), no RNA, negative control. <b>C–F,</b> Assay for Rac1 activation. <b>C–D,</b> Analysis of activation of Rac1 <i>in vivo</i> in the lungs of <i>bcr−/−</i>, <i>abr−/−</i> and <i>wt</i> mice after normoxia or hypoxia exposure. <b>E–F,</b> Analysis of Rac1 activation in PASMC under normoxia or hypoxia. <b>C</b> and <b>E,</b> representative Western blots; <b>D</b> and <b>F,</b> quantification. Three independent samples of each genotype were tested and the entire experiment was repeated independently. To quantitate results, Western blots were scanned and the ratio of GTP-Rac/total Rac was determined (panels <b>D, F</b>). * p<0.05 when compared with the results of the same genotype under normoxia. # p<0.05 when compared with WT exposed to the same condition.</p

Hypoxia-treated <i>bcr−/−</i> and <i>abr−/−</i> mice have higher RVSP and more severe right ventricular hypertrophy. A,

<p>RVSP from <i>bcr−/−, abr−/−</i> and <i>wt</i> mice with exposure to normoxia or hypoxia. <b>B,</b> Ratio of RV/LV+S calculated using the weight of RV, LV+S from the hearts of normoxic and hypoxic <i>bcr−/−, abr−/−</i> and <i>wt</i> mice. *p<0.05 compared to the values of mice with the same genotype at normoxia. # p<0.05 when compared to <i>wt</i> mice in the same exposure condition. Bars represent mean ±SD; n  = 6 mice per group.</p

Hypoxia-induced pulmonary vascular remodeling in <i>bcr−/−</i> and <i>abr−/−</i> mice.

<p><b>A,</b> Hematoxylin and eosin staining on representative lung specimens from <i>bcr−/−, abr−/−</i> and <i>wt</i> mice under normoxia or hypoxia. Note that the walls of the pulmonary arteries of the <i>bcr−/−</i> and <i>abr−/−</i> mice are remarkably thicker than those of the <i>wt</i> mice after hypoxia. Magnification, 200×. <b>B,</b> Quantification of changes in the pulmonary artery walls. Percent wall thickness was determined on H&E stained sections as described in Methods on nine vessels of comparable size per mouse, with 6 mice per genotype per condition. *p<0.05 compared with the same genotype at normoxia. # p<0.05 compared with WT mice in the same exposure condition. Bars, mean ± SD. <b>C,</b> Immunostaining with α-SMA antibodies on pulmonary vessels from representative normoxia or hypoxia-treated mice. <b>D,</b> Quantification of areas for α-SMA-positive cells. Areas of α-SMA-positive cells were calculated using ImageJ software as described in the Materials and Methods.</p