Self-Assembly of DNA Graphs and Postman Tours

DNA graph structures can self-assemble from branched junction molecules to yield solutions to computational problems. Self-assembly of graphs have previously been shown to give polynomial time solutions to hard computational problems such as 3-SAT and k-colorability problems. Jonoska et al. have proposed studying self-assembly of graphs topologically, considering the boundary components of their thickened graphs, which allows for reading the solutions to computational problems through reporter strands. We discuss weighting algorithms and consider applications of self-assembly of graphs and the boundary components of their thickened graphs to problems involving minimal weight Eulerian walks such as the Chinese Postman Problem and the Windy Postman Problem

Unraveling the pathology of different disease severities in human cerebral organoid models of LIS1-lissencephaly

The human neocortex is greatly expanded and exhibits a highly organized and extensively folded (gyrencephalic) structure. Model systems gave a fundamental understanding about how the cortex is generated although the applied models often involve species with a smooth (lissencephalic) brain surface, such as mice. Thus, key cellular events that impact human-specific brain expansion and our understanding of how disease-linked mutations disrupt human cortical development remains elusive. Lissencephaly is a malformation of cortical development which is characterized by a smooth brain and a disorganized cortex. Heterozygous deletions or mutations in the LIS1 gene, encoding a microtubule-associated protein in humans, were identified to cause lissencephaly with diverse clinical phenotypic variations ranging from mild pachygyria (broad gyri) to severe agyria (no gyri) resulting in epilepsy and intellectual disabilities. While the clinical severity generally correlates with the degree of agyria, the location and type of mutation in the LIS1 gene does not. From LIS1 mouse models we know that LIS1 regulates the microtubule motor cytoplasmic dynein and by that dynein-dependent processes such as neuronal migration, nucleokinesis, interkinetic nuclear migration and mitotic spindle orientation. Even though the observed LIS1-deficiency-associated phenotypes appeared drastically milder in murine systems compared to humans these studies suggest that LIS1 gene dosage is relevant for the phenotypic severities. However, why a specific mutation within the LIS1 gene as identified in LIS1-lissencephalic patients (LIS1-patients) leads to different disease severities and whether human-specific processes during cortical development are differentially affected by the specific mutations could, due to a lack of adequate model systems, so far not been investigated. Here, I explore the ability to recapitulate different disease severities of LIS1-lissencephaly using LIS1-patient-specific iPS cells and thereof derived forebrain-type cerebral organoids. To do so, I selected from a LIS1-patient cohort comprising 63 cases 7 patients who cover the whole spectrum of gyrification alterations of LIS1-lissencephaly ranging from Dobyns grade 5 (mild) to 1 (severe). Each patient harbors a different molecular characterized heterozygous mutation in the LIS1 gene. To analyze the consequences of each LIS1 mutation on human brain development a 3D cell culture forebrain-organoid protocol was developed. Following reprogramming of patient-derived somatic cells and basic characterization (2 clones each) the iPS cells were applied to the organoid protocol. Organoids reproduced, in correlation with the patient’s severity, alterations in organoid cytoarchitecture and premature neurogenesis. To assess the direct consequences of the patient-specific mutations on LIS1 microtubule stabilizing function I investigated the stability of the cytoskeleton of apical (a) RG cells within the cortical ventricular-like zone (VZ) structures and found a progressive collapse of tubulin strand stability with increasing patient disease severity leading to a disruption of cellular organization. These phenotypic alterations could in part be reversed by stabilizing the microtubule array using the FDA-approved drug EpothiloneD. In addition, organoids from individuals with severe but not mild disease showed a non-random aRG cell division switch from proliferative to neurogenic division. As an underlying molecular cause, WNT-signaling alterations were identified, most prominently in severe conditions. To test to what extend perturbed WNT-signaling contributes to the observed patient-specific alterations, organoids were exposed to the GSK3ß inhibitor CHIR99021 leading to a significant rescue of non-random aRG cell division switch in severe organoids and to enlarged VZ diameters as well as reduced neurogenesis in all patient derived organoids. The here demonstrated research underlines the capability of cerebral organoids to sensitively model individual disease severities, a so far not addressed major challenge of the system. My data show that different patient-specific mutations in the LIS1 gene have divergent direct impact on microtubule stability, which directly and/or indirectly lead to perturbed human corticogenesis providing the missing link between the patient-specific LIS1 mutation and the clinical severity grade. Future applications analyzing individual diseases have the potential to advance personalize medicine and improve the understanding of individual pathology for personalized therapy

Dissecting regional heterogeneity and modeling transcriptional cascades in brain organoids

Over the past decade, there has been a rapid expansion in the development and utilization of brain organoid models, enabling three-dimensional in vivo-like views of fundamental neurodevelopmental features of corticogenesis in health and disease. Nonetheless, the methods used for generating cortical organoid fates exhibit widespread heterogeneity across different cell lines. Here, we show that a combination of dual SMAD and WNT inhibition (Triple-i protocol) establishes a robust cortical identity in brain organoids, while other widely used derivation protocols are inconsistent with respect to regional specification. In order to measure this heterogeneity, we employ single-cell RNA-sequencing (scRNA-Seq), enabling the sampling of the gene expression profiles of thousands of cells in an individual sample. However, in order to draw meaningful conclusions from scRNA-Seq data, technical artifacts must be identified and removed. In this thesis, we present a method to detect one such artifact, empty droplets that do not contain a cell and consist mainly of free-floating mRNA in the sample. Furthermore, from their expression profiles, cells can be ordered along a developmental trajectory which recapitulates the progression of cells as they differentiate. Based on this ordering, we model gene expression using a Bayesian inference approach in order to measure transcriptional dynamics within differentiating cells. This enables the ordering of genes along transcriptional cascades, statistical testing for differences in gene expression changes, and measuring potential regulatory gene interactions. We apply this approach to differentiating cortical neural stem cells into cortical neurons via an intermediate progenitor cell type in brain organoids to provide a detailed characterization of the endogenous molecular processes underlying neurogenesis.Im letzten Jahrzent hat die Entwicklung und Nutzung von Organoidmodellen des Gehirns stark zugenommen. Diese Modelle erlauben dreidimensionale, in-vivo ähnliche Einblicke in fundamentale Aspekte der neurologischen Entwicklung des Hirnkortex in Gesundheit und Krankheit. Jedoch weisen die Methoden, um die Entwicklung kortikaler Organoide zu verfolgen, starke Heterogenität zwischen verschiedenen Zelllinien auf. Hier weisen wir nach, dass eine Kombination dualer SMAD und WNT Hemmung (Triple-i Protokoll) eine konstante kortikale Zuordnung in Hirnorganoiden erzeugt, während andere, weit verbreitete und genutzte Protokolle in Bezug auf kortikale Spezifizierung keine konstanten Ergebnisse liefern. Um die Heterogenität zu messen, haben wir Einzelzell-RNA Sequenzierung (scRNA-Seq) benutzt, wodurch die Erfassung der Genexpression von Tausenden von Zellen in einer Probe möglich ist. Um jedoch sinnvolle Schlüsse aus diesen scRNA-Seq Daten zu ziehen, müssen technische Artifakte identifiziert und aus den Daten entfernt werden. In dieser Dissertation stellen wir eine Methode vor, um eines solcher Artifakte zu erkennen: leere Tröpfchen (ohne Zellen), die hauptsächlich aus freischwebender mRNA in der Probe bestehen. Weiterhin können Zellen anhand ihrer Genexpressionsprofile entlang einer Entwicklungsschiene angeordnet werden, die die Entwicklung der Zellen während ihrer Differenzierung rekapituliert. Auf der Grundlage dieser Entwicklungsreihenfolge modellieren wir die Genexpression mit einem Bayes’schen Inferenzansatz, um die Dynamik der Transkription in sich differenzierenden Zellen zu messen. Dies ermöglicht das Anordnen von Genen entlang einer Transkriptionskaskade, sowie statistische Untersuchungen in Hinblick auf Unterschiede in der Veränderung von Genexpression, und das Messen des Einflusses möglicher Regulationsgene. Wir wenden diese Methode an, um kortikale neuronale Stammzellen zu untersuchen, die sich über einen intermediären Vorläuferzelltyp in kortikale Neuronen in Hirnorganoiden differenzieren, und um eine detaillierte Charakterisierung der molekularen Prozesse zu liefern, die der Neurogenese zugrunde liegen

The role of primary cilia and sonic hedgehog signalling in adrenal development function.

PhDPrimary cilia are sensory organelles found on most vertebrate cells during interphase. They play key roles in development, cell signalling and cancer, and are involved in signal transduction pathways such as Hh and Wnt signalling. The adrenal cortex produces steroid hormones essential for controlling homeostasis and mediating the stress response. Signalling pathways involved in the process of its development and differentiation are still being identified but include Hh and Wnt, and adrenal development is thus likely to require cilia. I have demonstrated that inhibiting cilia formation, using siRNA targeted to different ciliary components, results in reduced differentiation of the human adrenal carcinoma cell line H295R towards a zona glomerulosa (zG)-like phenotype. These data suggest that primary cilia play a key role in adrenal differentiation, but which signalling pathways are involved still remains unclear. I have also discovered that adrenals from Bardet-Biedl syndrome (BBS) mice, the most prominently studied ciliopathy, have thin capsules, the proposed adrenal stem cell niche, and abnormal histology, while zebrafish embryos injected with morpholinos targeting BBS genes show delayed and reduced expression of ff1b, a marker of interrenal tissue. These data further suggest a role for primary cilia in adrenal development and maintenance. These studies are the foundation for elucidating the role of primary cilia in the development and function of the adrenal gland, and furthering our understanding of adrenocortical development. This promises to lead to improved management of adrenal dysfunction, and demonstrating that adrenal defects are a characteristic of ciliopathies will potentially inform new strategies for patient care.Medical Research Counci

Decoding the heterogeneity of skin in homeostasis and regeneration at single-cell resolution

The skin plays a critical role in securing homeostasis in the mammalian body. Its epidermis forms a tight barrier, which separates the internal from the external environment, thereby shielding the body from physical and chemical insult. Due to the exposed position of skin as the outermost organ of the body, skin cells need to be replaced continuously. Cellular maintenance and regeneration of the skin and its associated hair follicles is orchestrated by a variety of stem cell populations. Because of its regenerative properties, the mouse skin is one of the most important model organs in stem cell research and regenerative medicine. The skin is a complex multicellular system composed of a large variety of molecularly and functionally distinct cell populations. The physiology of the skin is a result of the intricate interplay of these diverse cell types. Accordingly, knowledge about the cellular composition of the skin is an essential step in understanding its biology. For a long time, cell populations in the skin were defined based on the expression of individual molecular markers, thus making a comprehensive analysis of cellular heterogeneity impossible. In this thesis, I describe how we used single-cell transcriptomics to create systematic cell type maps of the skin in order to analyze complex molecular processes at single-cell resolution. In the first part of this thesis, I provide an overview of the morphology, function and cellular heterogeneity of the skin. I put particular emphasis on the skin as a self-maintaining tissue and model organ for stem cell research, describing regenerative process such as skin barrier maintenance, cyclical regeneration of hair follicles and cutaneous wound healing in great detail. Then, I introduce single-cell RNA-sequencing as a technique, which has revolutionized the way we analyze and conceptualize cellular heterogeneity in complex tissues. Next, I portray how we championed the application of single-cell transcriptomics in skin biology with three key papers. In Paper I, we used single-cell RNA-sequencing to analyze the mouse epidermis including hair follicles during its resting stage (telogen). We discovered previously unknown cellular heterogeneity in the epidermis and demonstrated that the complexity of this tissue is the result of just two vectors of variation: differentiation stage and spatial position. In Paper II, we analyzed the complete mouse skin, including both epidermal and stromal cells, during hair growth (anagen) and rest (telogen). In addition to describing novel cell types in the stromal part of the skin, we model cellular differentiation and lineage specification in the growing hair follicle at unprecedented resolution. In Paper III, we use single-cell transcriptomics to track molecular changes in different stem cell populations during wound healing and answer several key questions related to stem cell identity and plasticity during regenerative processes. In the last section of this thesis, I demonstrate that our studies have not just allowed us to analyze the cellular heterogeneity of the mouse skin at unprecedented detail, but have also enabled us to address a variety of critical questions such as how stem cell identity is shaped and how regenerative processes are orchestrated in the skin. I thus outline how our endeavors mark the first step towards a systems biology of the skin

Zebrafish and mouse models for studying deubiquitinating enzyme genes as candidates for retinal dystrophies

[eng] The retina consists of several structured layers of highly specialized neurons that capture and process light stimuli enabling vision. Such a fine architecture turns retinal differentiation into an extremely complex event that must be accurately regulated. The ubiquitin-proteasome system (UPS) is considered one of the most dynamic and versatile mechanisms of protein regulation in eukaryotic cells. As ubiquitination is reversible, deubiquitinating enzymes (DUBs) play a major regulatory role in the UPS. Despite the importance of proteostasis and the UPS in health and disease, a more comprehensive in-depth analysis of DUB expression and function on particular tissues or organs, such as the retina, is still missing. Combining expression quantification, mRNA localization assays and functional analyses in animal and cellular models, we analyzed the function of several DUB genes in the retina to identify DUBs that regulate important retinal cell mechanisms, explore their relevance in retinal function in health and disease, and finally, posit them as new potential candidate genes for retinal dystrophies. Taking into consideration our results in the expression levels and pattern of DUBs in the retina, we first selected USP45 to perform functional assays in animal models in order to define its role and function in the retina. By morpholino-knockdown of usp45 in zebrafish embryos, our results showed moderate to severe eye morphological defects, eye size reduction, small body size with small tail or without tail, and disruption in notochord formation. There is also defective lamination and formation of the retinal structures, with no distinguishable layers and smaller retinas. Overall, our results supported the relevance of USP45 in the normal development and formation of the vertebrate retina, and we proposed this gene as a good candidate for causing hereditary retinal dystrophies, as later confirmed by other authors in several families. We also selected ATXN3, a DUB gene that causes the dominant polyQ disease Spinocerebellar ataxia type 3 (SCA3), and we aimed to analyze its function in the retina. We showed that depletion of Atxn3 in zebrafish and mice caused retinal morphological and functional alterations with photoreceptor outer segment elongation, cone opsin mislocalization, and cone hyperexcitation upon light stimuli. A pool of ATXN3 resides at the basal body and axoneme of the photoreceptor cilium, where it controls the levels and recruitment of the regulatory proteins KEAP1 and HDAC6. Abrogation of Atxn3 expression causes delayed phagosome maturation in the retinal pigment epithelium. We propose that ATXN3 regulates two relevant biological processes in the retina, ciliogenesis and phagocytosis, by modulating microtubule polymerization and microtubule-dependent retrograde transport, and propose ATXN3 as a causative or modifier gene in retinal/macular dystrophies. We further aimed to explore whether the SCA3 humanized mouse model showed specific retinal phenotype traits. We showed that polyQ-expanded ATXN3 protein formed a high number of progressive pathogenic aggregates in the retinal layers of transgenic Atxn3Q84 mice, and caused a decrease in the number of cone photoreceptors. Optical coherence tomography revealed a general decrease in the thickness of the retinal layers whereas retinal electrophysiological analyses showed a strong decrease in photoreceptor response to light, thus supporting severe retinal dysfunction in Atxn3Q84 mice. Similar analyses in human patients detected a correlation of retinal alterations with the number of CAG repeats and the age of onset of SCA3 symptoms. We propose that retinal alterations detected by non-invasive eye examination and electroretinography tests in SCA3 patients could serve as a valuable early-onset symptom and a biological marker of disease progression. As a conclusion, our work posits several DUB genes as candidates for inherited retinal dystrophies, but further investigation is needed to dissect the function of DUBs in retinal cell differentiation, photoreceptor function, and retinal homeostasis

The role of neutrophils in telomere induced senescence via bystander effects

PhD ThesisSenescence is classically defined as a state of permanent cell-cycle arrest. Senescence can occur in response to various stresses, which have been shown to act mainly through the activation of a DNA damage response (DDR). Senescence is characterised not solely by a cell-cycle arrest but also by increased production of Reactive Oxygen Species (ROS) and the development of a Senescence-Associated Secretory Phenotype (SASP). SASP components include growth factors, cytokines, chemokines and immune modulators and have been shown to contribute to senescence in an autocrine manner but also impact on the tissue microenvironment trough paracrine effects. Several studies have linked the SASP with immune surveillance suggesting that Natural Killer cells, monocytes and T lymphocytes CD4+ can effectively eliminate senescent cells. However, the interaction between neutrophils (the first innate immune responders to infection or injury) and cellular senescence has not yet been investigated. In this thesis, I have shown that neutrophils induce premature senescence in human fibroblasts in a telomere-dependent manner. My data indicates that hydrogen peroxide released by neutrophils damages telomeric DNA, thereby accelerating the rate of telomere shortening and contributing to the early onset of senescence. Consistently, pre-treatment with the antioxidant enzyme catalase, prevents neutrophil-induced telomere shortening and premature senescence. In addition, overexpression of the catalytic subunit of telomerase (hTERT), which maintains telomere length in cultured fibroblasts, is able to bypass neutrophil-induced premature senescence. In accordance with my in vitro results, I have shown that following acute liver injury (using CCl4) which is characterised by neutrophil infiltration, mouse hepatocytes show increased markers of telomere dysfunction, which can be prevented by neutralisation of neutrophils. Importantly, I have found that during the ageing process or after injection with lipopolysaccharide (LPS), mouse livers experience increased neutrophil infiltrations which positively correlate with markers of telomere-dysfunction. Finally, I have shown that senescent cells secrete factors which act as a neutrophil chemoattractant and that neutrophils preferentially induce cell-death in senescent cells but not young cells. These data suggest for the first time that neutrophils play an important role in the immune clearance of senescent cells. viii Altogether, my data propose that neutrophils act as a double-edged sword: on one hand, they can induce senescence by accelerating telomere shortening; on the other hand, they can be recruited to sites where senescent cells are present and accelerate their specific clearance

MSI-based mapping strategies in tumour-heterogeneity

Since the early 2000s, considerable innovations in MS technology and associated gene sequencing systems have enabled the "-omics" revolution. The data collected from multiple omics research can be combined to gain a better understanding of cancer's biological activity. Breast and ovarian cancer are among the most common cancers worldwide in women. Despite significant advances in diagnosis, treatment, and subtype identification, breast cancer remains the world's second leading cause of cancer-related deaths in women, with ovarian cancer ranking fifth. Tumour heterogeneity is a significant hurdle in cancer patient prognosis, response to therapy, and metastasis. As such, heterogeneity is one of the most significant and clinically relevant areas of cancer research nowadays. Metabolic reprogramming is a hallmark of malignancy that has been widely acknowledged in recent literature. Metabolic heterogeneity in tumours poses a challenge in developing therapies that exploit metabolic vulnerabilities. Consequently, it is crucial to approach tumour heterogeneity with an unlabeled yet spatially specific read-out of metabolic and genetic information. The advantage of DESI-MSI technology originates from its untargeted nature, which allows for the investigation of thousands of component distributions, at a micrometre scale, in a single experiment. Most notably, using a DESI-MSI clustering approach could potentially offer novel insights into metabolism, providing a method to characterise metabolically distinct sub-regions and subsequently delineate the underlying genetic drivers through genomic analyses. Hence, in this study, we aim to map the inter-and intra-tumour metabolic heterogeneity in breast and ovarian cancer by integrating multimodal MSI-based mapping strategies, comprising DESI and MALDI, with IMC (Imaging Mass Cytometry) analysis of the tumour section, using CyTOF, and high- throughput genetic characterisation of metabolically-distinct regions by transcriptomics. The multimodal analysis workflow was initially performed using sequential breast cancer Patient-Derived Xenografts (PDX) models and was expanded on primary tumour sections. Moreover, a newly developed DESI-MSI friendly, hydroxypropyl-methylcellulose and polyvinylpyrrolidone (HPMC/PVP) hydrogel-based embedding was successfully established to allow simultaneous preparation and analysis of numerous fresh frozen core-size biopsies in the same Tissue Microarray (TMA) block for the investigation of tumour heterogeneity. Additionally, a single section strategy was combined with DESI-MSI coupled to Laser Capture Microdissection (LCM) application to integrate gene expression analysis and Liquid Chromatography-Mass Spectrometry (LC-MS) on the same tissue segment. The developed single section methodology was then tested with multi-region collected ovarian tumours. DESI-MSI-guided spatial transcriptomics was performed for co-registration of different omics datasets on the same regions of interest (ROIs). This co-registration of various omics could unravel possible interactions between distinct metabolic profiles and specific genetic drivers that can lead to intra-tumour heterogeneity. Linking all these findings from MSI-based or guided various strategies allows for a transition from a qualitative approach to a conceptual understanding of the architecture of multiple molecular networks responsible for cellular metabolism in tumour heterogeneity.Open Acces

Demonstration of Tissue Factor expression using a Tissue Factor – Green Fluorescent Protein Reporter Model

Blood coagulation is initiated when circulating factor (F) VII comes into contact with membrane bound tissue factor (TF). Subsequently a series of enzymatic cleavages results in the formation of insoluble fibrin. The coagulation network is of fundamental importance in maintaining the integrity of the circulation and is closely integrated with other processes such as inflammation. Both under- and over-activity of the network have significant pathological consequences and thus the network is tightly regulated. The first point of regulation is in the formation of the TF•FVIIa initiating complex. Circulating FVII is kept separate from membrane-bound TF by an intact vascular wall. The initiation of coagulation is thus dependant on the site of TF expression. In addition abnormal TF expression has been shown to be important in various pathological processes such as atherosclerosis and cancer metastasis. The accurate demonstration of TF activity is of critical importance in the understanding of these processes. This thesis describes the development of a TF-Enhanced Green Fluorescent Protein (EGFP) reporter model for analysis of TF expression. A targeting vector containing the cDNA for EGFP in-frame with and flanked by regions homologous to the mouse TF gene was generated. The vector was designed such that EGFP would be knocked in to the TF locus by homologous recombination to produce a fusion protein with EGFP tagged onto the intact cytoplasmic domain of TF. Two strategies were used for generating transgenic mice. A targeting vector was electroporated into mouse embryonic stem (ES) cells and correctly targeted ES cells were then used in blastocyst injections. Alternatively a bacterial artificial chromosome (BAC) containing the mouse TF gene and flanking regions was targeted in a bacterial host using a similar vector. The targeted BAC was used in pro-nuclear injections of fertilized mouse ova resulting in random integration into the mouse genome. The function of the TF-EGFP reporter model was investigated by analysis of transgene expression in whole tissues and specific cell types taken from transgenic mice