5,092 research outputs found

    Undergraduate Catalog of Studies, 2023-2024

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    Exploring missing heritability in neurodevelopmental disorders:Learning from regulatory elements

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    In this thesis, I aimed to solve part of the missing heritability in neurodevelopmental disorders, using computational approaches. Next to the investigations of a novel epilepsy syndrome and investigations aiming to elucidate the regulation of the gene involved, I investigated and prioritized genomic sequences that have implications in gene regulation during the developmental stages of human brain, with the goal to create an atlas of high confidence non-coding regulatory elements that future studies can assess for genetic variants in genetically unexplained individuals suffering from neurodevelopmental disorders that are of suspected genetic origin

    Undergraduate Catalog of Studies, 2023-2024

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    Exploring missing heritability in neurodevelopmental disorders:Learning from regulatory elements

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    Identifying alterations in adipose tissue-derived islet GPCR peptide ligand mRNAs in obesity: implications for islet function

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    In addition to acting as an energy reservoir, white adipose tissue is a vital endocrine organ involved in the modulation of cellular function and the maintenance of metabolic homeostasis through the synthesis and secretion of peptides, known as adipokines. It is known that some of these secretory peptides play important regulatory roles in glycaemic control by acting directly on islet β-cells or on insulin-sensitive tissues. Excess adiposity causes alterations in the circulating levels of some adipokines which, depending on their mode of action, can have pro-inflammatory, pro-diabetic or anti-inflammatory, anti-diabetic properties. Some adipokines that are known to act at β-cells have actions that are transduced by binding to G protein- coupled receptors (GPCRs). This large family of receptors represents ~35% of all current drug targets for the treatment of a wide range of diseases, including type 2 diabetes (T2D). Islets express ~300 GPCRs, yet only one islet GPCR is currently directly targeted for T2D treatment. This deficit represents a therapeutic gap that could be filled by the identification of adipose tissue-derived islet GPCR peptide ligands that increase insulin secretion and overall β-cell function. Thus, by defining their mechanisms of action, there is potential for the development of new pharmacotherapies for T2D. Therefore, this thesis describes experiments which aimed to compare the expression profiles of adipose tissue-derived islet GPCR peptide ligand mRNAs under lean and obese conditions, and to characterise the functional effects of a selected candidate of interest on islet cells. Visceral fat depots were retrieved from high-fat diet-induced and genetically obese mouse models, and from human participants. Fat pads were either processed as whole tissue, or mature adipocyte cells were separated from the stromal vascular fraction (SVF) which contains several other cell populations, including preadipocytes and macrophages. The expression levels of 155 islet GPCR peptide ligand mRNAs in whole adipose tissue or in isolated mature adipocytes were quantified using optimised RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols. Comparisons between lean and obese states in mice models and humans revealed significant modifications in the expression levels of several adipokine mRNAs. As expected, mRNAs encoding the positive control genes, Lep and AdipoQ were quantifiable, with the expression of Lep mRNA increasing and that of AdipoQ mRNA decreasing in obesity. Expression of Ccl4 mRNA, encoding chemokine (C-C motif) ligand 4, was significantly upregulated in whole adipose tissue across all models of obesity compared to their lean counterparts. This coincided with elevated circulating Ccl4 peptide levels. This increase was not replicated in isolated mature adipocytes, indicating that the source of upregulated Ccl4 expression in obesity was the SVF of adipose tissue. Based on this significant increase in Ccl4 mRNA expression within visceral fat and its undetermined effects on β-cell function, Ccl4 was selected for further investigation in MIN6 β-cells and mouse islets. PRESTO-Tango β-arrestin reporter assays were performed to determine which GPCRs were activated by exogenous Ccl4. Experiments using HTLA cells expressing a protease-tagged β- arrestin and transfected with GPCR plasmids of interest indicated that 100ng/mL Ccl4 significantly activated Cxcr1 and Cxcr5, but it was not an agonist at the previously identified Ccl4-target GPCRs Ccr1, Ccr2, Ccr5, Ccr9 and Ackr2. RNA extraction and RT-qPCR experiments using MIN6 β-cells and primary islets from lean mice revealed the expression of Cxcr5 mRNA in mouse islets, but it was absent in MIN6 β-cells. The remaining putative Ccl4 receptors (Ccr1, Ccr2, Ccr5, Ccr9, Cxcr1 and Ackr2) were either absent or present at trace levels in mouse islets and MIN6 β-cells. Recombinant mouse Ccl4 protein was used for functional experiments at concentrations of 5, 10, 50 and 100ng/mL, based on previous reports of biological activities at these concentrations. Trypan blue exclusion testing was initially performed to assess the effect of exogenous Ccl4 on MIN6 β-cell viability and these experiments indicated that all concentrations (5-100ng/mL) were well-tolerated. Since β-cells have a low basal rate of apoptosis, cell death was induced by exposure to the saturated free fatty acid, palmitate, or by a cocktail of pro-inflammatory cytokines (interleukin-1β, tumour necrosis factor-α and interferon-γ). In MIN6 β-cells, Ccl4 demonstrated concentration-dependent protective effects against palmitate-induced and cytokine-induced apoptosis. Conversely, while palmitate and cytokines also increased apoptosis of mouse islets, Ccl4 did not protect islets from either inducer. Quantification of bromodeoxyuridine (BrdU) incorporation into β-cell DNA indicated that Ccl4 caused a concentration-dependent reduction in proliferation of MIN6 β-cells in response to 10% fetal bovine serum (FBS). In contrast, immunohistochemical quantification of Ki67-positive mouse islet β-cells showed no differences in β-cell proliferation between control- and Ccl4-treated islets. Whilst the number of β-cells and δ-cells were unaffected, α- cells were significantly depleted by Ccl4 treatment. Exogenous Ccl4 had no effect on nutrient- stimulated insulin secretion from both MIN6 β-cells and primary mouse islets. The 3T3-L1 preadipocyte cell line was used to assess potential Ccl4-mediated paracrine and/or autocrine signalling within adipose tissue. Ccl4 did not alter the mRNA expression of Pparγ, a master regulator of adipocyte differentiation, but did significantly downregulate the mRNA expression of the crucial adipogenic gene, adiponectin. Oil Red O staining and Western blotting were performed to assess lipid accumulation, and insulin and lipolytic signalling, respectively, and these experiments indicated that the observed Ccl4-induced decrease in adiponectin expression failed to correlate with any changes in adipocyte function. In summary, these data demonstrated anti-apoptotic and anti-proliferative actions of the adipokine, Ccl4, on MIN6 β-cells that were not replicated in mouse islets. The absence of any anti-apoptotic, insulin secretory and/or pro-proliferative effects of Ccl4 in islet β-cells suggests that it is unlikely to play a role in regulating β-cell function via crosstalk between adipose tissue and islets. The divergent functional effects highlight that whilst MIN6 cells are a useful primary β-cell surrogate for some studies, primary islets should always be used to confirm physiological relevance. On the other hand, significant α-cell depletion following Ccl4 treatment suggests a cell-specific function within the islets. Furthermore, Ccl4 impaired adiponectin mRNA expression in adipocytes, although, how adipocyte function is affected as a result requires further investigation. Collectively, these data have contributed increased understanding of the role of obesity in modifying the expression of adipose tissue-derived islet GPCR peptide ligands

    Dissecting structural and biochemical features of DNA methyltransferase 1

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    DNA methylation is an epigenetic modification found in every branch of life. An essential enzyme for the maintenance of DNA methylation patterns in mammals is DNA methyltransferase 1 (DNMT1). Its recruitment is regulated through its large N-terminus, which contains six annotated domains. Although most of these have been assigned a function, we are still lacking a holistic understanding of the enzyme's spatio-temporal regulation. Interestingly, a large segment of the N-terminus is devoid of any known domain and appears to be disordered in its sequence. Over the past years, such disordered sequences have increasingly gained attention, due to their role in forming biomolecular condensates through liquid-liquid phase separation (LLPS). These liquid compartments offer specific environmental conditions distinct from the surrounding that can enhance protein recruitment and function. In this work, we explore a potential role for the intrinsically disordered domain (IDR) in the recruitment of DNMT1. Taking an evolutionary approach, we uncover that structural features of the region that are key for IDR function are highly conserved. Moreover, we find conserved biochemical signatures compatible with a role in LLPS. Using a reconstitution assay and an opto-genetic approach in cells, we for the first time show that the DNMT1 IDR is capable of undergoing LLPS in vitro and in vivo. In addition, we define a novel region of interest (ROI) of about 120 amino acids in the IDR that appears to have been inserted in the ancestor of eutherian mammals. Although the ROI has a distinct biochemical signature, we find no effect on the LLPS behavior of the IDR. Therefore, we discuss other potential roles of the ROI related to DNA methylation, for example, imprinting. Finally, we lay the foundation for investigating a biological function of the IDR and establish a system for screening DNMT1 mutant phenotypes in mouse embryonic stem cells. Swift depletion of the endogenous protein is enabled by degron-mediated degradation, while our optimized construct design and efficient derivation strategy ensure the robust expression of the large transgenes. In combination with different methods for DNA methylation read-out, this system can now be used to study the role of the IDR and ROI in maintaining the steady-state level of DNA methylation against mechanisms of passive and active demethylation, but also for studying phenotypes affecting the efficiency of DNMT1 recruitment in the future

    Investigating the early interaction between Mycobacterium avium ssp paratuberculosis and the host using a bovine enteroid system

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    Mycobacterium avium ssp paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), a chronic granulomatous enteritis of ruminant’s prevalent world-wide. Infection of calves occurs through the faecal oral route, typically in animals <6 months old. Animals are asymptomatic for 2-5 years before clinical signs begin to show, which typically present as emaciation and chronic diarrhoea. In the subclinical phase, animals will have decreased milk yield, increased susceptibility to other diseases and decreased feed conversion. This has a severe impact on the farming economy and animal welfare, as affected animals are often prematurely culled. Infected subclinical animals are extremely difficult to identify but can still act as a source of transmission for the rest of the herd by shedding MAP in their faeces. There is no treatment for JD, and the current diagnostic tests are ineffective. By investigating the initial interaction between MAP and the host at the intestinal lining, a greater understanding of MAP pathogenesis can be gained and better diagnostic and therapeutic targets can be identified. In this work, proteins expressed on the surface of MAP were assessed for their ability to aid attachment, invasion and intracellular survival in epithelial and phagocytic cells when expressed on the membrane of a non-invasive E. coli host strain. The proteins investigated were encoded by mammalian cell entry (mce) genes, mce1A, mce1D, mce3C and mce4A, which have been implicated in attachment and invasion of epithelial cells by other mycobacteria. Interestingly, E. coli expressing Mce1A had enhanced uptake by phagocytic cells and E. coli expressing Mce1D had enhanced attachment and invasion of epithelial cells, but neither protein conferred this phenotype in both eukaryotic cell types investigated. To identify key intestinal cell types involved in MAP pathogenesis, bovine intestinal organoids (enteroids) were assessed for their ability to model a MAP infection in a physiologically representative system. Baso-out 3D enteroids, apical-out 3D enteroids and 2D monolayers were created, and the cell types present were compared to bovine intestinal tissue samples using RT-PCR and immunofluorescence microscopy. The models contained the mature epithelial cell types of the intestine including goblet cells, enteroendocrine cells, Paneth cells and enterocytes. 3D baso-out enteroids and 2D monolayers also contained proliferative cells, but the 3D apical-out enteroids did not and so could not be maintained past 2 weeks of culture. The models were infected with two strains of MAP over the course of 72 hours, the reference strain K10, and a recent clinical isolate C49. MAP C49 was shown to be present in all three intestinal models in consistently higher numbers than MAP K10, quantified using qPCR of the genomic DNA. This indicates that MAP C49 was better able to infect these models than K10, which may suggest a loss of virulence in MAP K10. Overall, the data presented has increased our understanding of MAP pathogenesis by emphasising the need for multicellular models which accurately represent the pathogen target cell type/s in vivo and the confirmation of the role of two hypothetical MAP proteins in cellular interactions

    Dissecting Extracellular Matrix Internalisation Mechanisms using Functional Genomics

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    Breast and ovarian malignancies account for one third of female cancers. The role of the stroma in supporting invasive growth in breast cancer has become clear. Breast cancer cells interact and respond to the cues from the surrounding extracellular matrix (ECM). Integrins are main cell adhesion receptors and key players in invasive migration by linking the ECM to the actin cytoskeleton. In addition, integrins mediate distinctive biochemical and biomechanical signals to support cancer invasion. The role of matrix proteases in promoting ECM degradation and cancer dissemination has been extensively studied; however, cancer cells possess additional means to support those processes, such as integrin-mediated ECM endocytosis and consequent degradation in the lysosomes. Internalisation of the extracellular matrix is upregulated in invasive breast cancer. Nonetheless, the mechanisms by which cancer cells regulate this process are poorly understood. We developed a high throughput pH sensitive system to detect ECM uptake. Here, we show that MDA-MB-231 breast cancer cells converge in macropinocytosis to internalise diverse ECM components and we confirm that this process is modulated by PAK1. To unravel which ECM components breast cancer cells internalise in a complex environment (namely, cell derived matrices), we performed mass spectrometry. Proteomic analysis identified Annexin A6, Collagen VI, Tenascin C and fibronectin, among other matrisome proteins, to be internalised by invasive breast cancer cells. Following ECM endocytosis, ECM is targeted for lysosomal degradation. To unravel the molecular mechanisms behind this process, we performed a trafficking screen and identified the AP3 complex, VAMP7, Arf1 and ARFGEF2. Our results suggest that the AP3 complex may regulate ECM-integrin delivery to lysosomes. To gain more insight on the signalling pathways governing macropinocytosis in breast cancer cells, we performed a kinase and phosphatase screen that unravelled MAP3K1 and PPP2R1A, a subunit of protein phosphatase 2A (PP2A) as relevant regulators of ECM endocytosis. Furthermore, our data suggests that p38 mitogen-activated protein kinase (MAPK) activation upon binding to the ECM is required for ECM macropinocytosis. Outstandingly, inhibiting p38 MAPK led to profound changes in the ability of breast cancer cells to migrate in cell derived matrices. Previous work from the Rainero lab focused on characterising the receptors involved in ECM internalisation; α2β1 integrin was identified as the main regulator of ECM uptake in MDA-MB-231 cells. In particular, α2β1 integrin has been shown to activate p38 MAPK pathway. Taken together, we hypothesise that binding of ECM to α2β1 integrin results in the activation of PAK1 and MAP3K1, which in turn leads to ECM endocytosis. p38 MAPK activity may induce changes in actin polymerisation via PPP2R1A and/or focal adhesion turnover, which consequently promotes ECM macropinocytosis and invasive migration

    Role of ethylene and other signals in the regulation of responses to Iron, Phosphorus or Sulfur deficiencies

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    Para su correcto crecimiento y desarrollo, las plantas necesitan diversos nutrientes minerales, entre los que se encuentran los elementos objeto de estudio de esta Tesis: hierro (Fe), fósforo (P) y azufre (S). Estos elementos son esenciales para las plantas y, aunque suelen ser abundantes en la mayoría de los suelos, su disponibilidad para las plantas puede ser baja, sobre todo en determinados suelos, como los calcáreos. Para facilitar su adquisición, las plantas han desarrollado diferentes mecanismos. En el caso del hierro, y dependiendo del tipo de mecanismos, las plantas se dividen en plantas de Estrategia I (todas las plantas excepto las gramíneas) y plantas de Estrategia II (gramíneas). En condiciones de deficiencia de alguno de estos nutrientes, las plantas dicotiledóneas (con Estrategia I y objeto de esta Tesis) ponen en marcha una serie de respuestas, principalmente en sus raíces, para mejorar la movilización y transporte del nutriente en cuestión. Entre estas respuestas, encontramos cambios morfológicos de la raíz, como el desarrollo de pelos radicales subapicales y raíces proteoides (en respuesta a las deficiencias de hierro o fósforo), o un incremento del desarrollo de raíces laterales (en respuesta a las deficiencias de hierro, fósforo o azufre). También ocurren respuestas fisiológicas, como el incremento del número de transportadores, el incremento de la síntesis y liberación de compuestos que solubilizan los nutrientes en el suelo, y el incremento de algunas actividades enzimáticas, como la actividad de la reductasa férrica (en respuesta a la deficiencia de hierro) o de la fosfatasa ácida (en respuesta a la deficiencia de fósforo). Una vez el nutriente se ha adquirido en suficiente cantidad, estas respuestas deben ser inhibidas para minimizar el coste energético y evitar la toxicidad por exceso, lo que implica que estas respuestas deben estar sujetas a un control muy estricto. En la regulación de estas respuestas, el etileno juega un papel fundamental. Por ello, uno de los objetivos principales de esta Tesis ha sido profundizar en el papel de la proteína EIN2, clave en la ruta de señalización del etileno, en la regulación de las respuestas a la deficiencia de hierro. Este objetivo se abordó en el primer trabajo de esta Tesis “Comparative study of several Fe deficiency responses in the Arabidopsis thaliana ethylene insensitive mutants ein2-1 and ein2-5”, publicado en la revista Plants. Aunque las respuestas a cada deficiencia se inducen de manera específica, es frecuente que ocurra un cierto cruce de respuestas, de manera que, a veces, la deficiencia de un nutriente puede inducir respuestas relacionadas con las deficiencias de otros nutrientes. En la regulación de las respuestas a las deficiencias de hierro, fósforo o azufre, se ha implicado al etileno. Esa común implicación podría explicar parcialmente la inducción cruzada de respuestas a las distintas deficiencias. Para conferir especificidad, el etileno podría actuar junto con otras señales y/o a través de diferentes rutas de señalización para la regulación de las distintas respuestas a las diferentes deficiencias. En este sentido, uno de los principales objetivos de esta Tesis ha sido tratar de esclarecer la participación de algunos de los componentes clave de la ruta de señalización del etileno en la regulación de las respuestas a las deficiencias de hierro, fósforo o azufre. Para ello, se realizaron ensayos con distintos mutantes de Arabidopsis thaliana, afectados en alguno de los componentes de la ruta de señalización del etileno, y se sometieron a deficiencia de hierro, fósforo o azufre. A continuación, se estudiaron respuestas específicas a cada una de las tres deficiencias, así como respuestas a las otras deficiencias, en cada uno de estos mutantes. Estos estudios nos permitieron conocer qué elementos de la ruta de señalización del etileno son claves para cada una de las respuestas y a qué nivel se produce la interacción entre ellas Los resultados obtenidos se han publicado en Frontiers in Plant Science “Influence of ethylene signaling in the crosstalk between Fe, S, and P deficiency responses in Arabidopsis thaliana”. Por último, el tercer gran objetivo de esta Tesis ha sido tratar de dilucidar la posible relación existente entre distintas señales reguladoras de las respuestas a la deficiencia de hierro ya conocidas, como es el caso del etileno y de LODIS (‘LOng Distance Iron Signal’; señal represiva de las respuestas relacionada con el contenido interno de hierro), y unos pequeños péptidos (IMA1, 2 y 3), recientemente identificados en Arabidopsis y que actúan como activadores de las respuestas a la deficiencia de hierro. Nuestro objetivo ha sido tratar de encajar los péptidos IMA en el modelo de regulación existente. Los resultados obtenidos relacionados con este objetivo se han publicado también en Frontiers in Plant Science “A shoot derived long distance iron signal may act upstream of the IMA peptides in the regulation of Fe deficiency responses in Arabidopsis thaliana roots”.For their correct growth and development, plants need various mineral nutrients, like the elements studied in this Thesis: iron (Fe), phosphorus (P) and sulfur (S). These elements are essential for plants and, although they are usually abundant in most soils, their availability for plants can be low, especially in certain soils, such as the calcareous ones. To facilitate their acquisition, plants have developed different mechanisms. In the case of iron, based on the acquisition mechanisms, plants are divided into Strategy I plants (all plants except grasses) and Strategy II plants (grasses). Under nutrient deficiency conditions, dicotyledonous plants (with Strategy I and object of this Thesis) induce several responses, mainly in their roots, to improve the mobilization and transport of the deficient nutrient. Among these responses, there are morphological changes of the root, such as the development of subapical root hairs and proteoid roots (in response to iron or phosphorous deficiencies), or an increase in lateral root development (in response to iron, phosphorus or sulfur deficiencies). Physiological responses also occur, such as an increase of nutrient transporters, an increase in the synthesis and release of several compounds that solubilize nutrients in the soil, and an increase in some enzymatic activities, such as ferric reductase activity (in response to iron deficiency) or acid phosphatase (in response to phosphorus deficiency). Once the nutrient has been acquired in sufficient quantity, these responses must be inhibited to minimize energy cost and to avoid toxicity by excess. This implies that the responses must be subjected to very strict control. In the regulation of these responses, ethylene plays a key role. For this reason, one of the main objectives of this Thesis has been to look further into the role of the EIN2 protein, a key component of the ethylene signaling pathway, in the regulation of the iron deficiency responses. This objective has been addressed in the first work of this Thesis "Comparative study of several Fe deficiency responses in the Arabidopsis thaliana ethylene insensitive mutants ein2-1 and ein2-5", published in the journal Plants. Although responses to each deficiency are induced in a specific manner, some crosstalk among the responses to different nutrient deficiencies often occur. This implies that, sometimes, the typical responses to a nutrient deficiency can be induced by other nutrient deficiencies. Ethylene has been involved in the regulation of the iron, phosphorous or sulfur deficiency responses. This common implication could partially explain the crosstalk of the responses to these different deficiencies. To confer specificity, ethylene could act in conjunction with other signals, and/or through different signaling pathways, to regulate different responses to different deficiencies. In this sense, one of the main objectives of this Thesis has been to clarify the participation of some of the key components of the ethylene signaling pathway in the regulation of the responses to iron, phosphorus or sulfur deficiencies. For this, several experiments were carried out with Arabidopsis thaliana mutants, affected in some components of the ethylene signaling pathway, and subjected to iron, phosphorus or sulfur deficiencies. Next, specific responses to each deficiency, as well as responses to the other deficiencies, in each of these mutants were studied. These studies allowed us to find out which elements of the ethylene signaling pathway play key roles in each response and the interactions between them. The results obtained have been published in Frontiers in Plant Science "Influence of ethylene signaling in the crosstalk between Fe, S, and P deficiency responses in Arabidopsis thaliana”. Finally, the third major objective of this Thesis has been to elucidate the possible connection between different regulatory signals of the iron deficiency responses already known, such as ethylene and LODIS ('LOng Distance Iron Signal'; repressive signal of responses related to internal iron content), and some small peptides (IMA1, 2 and 3), recently identified in Arabidopsis that act as activators of the iron deficiency responses. Our objective has been to fit the IMA peptides into the existing regulatory model. The results obtained related to this objective have also been published in Frontiers in Plant Science “A shoot derived long distance iron signal may act upstream of the IMA peptides in the regulation of Fe deficiency responses in Arabidopsis thaliana roots”

    The brown algal genus Fucus : A unique insight into reproduction and the evolution of sex-biased genes

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    Doctoral thesis (PhD) - Nord University, 2023publishedVersio
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