5,438 research outputs found

    Undergraduate Catalog of Studies, 2023-2024

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    Structure of the Borrelia burgdorferi ATP-dependent metalloprotease FtsH in its functionally relevant hexameric form

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    Publisher Copyright: © 2023 The AuthorsATP-dependent proteases FtsH are conserved in bacteria, mitochondria, and chloroplasts, where they play an essential role in degradation of misfolded/unneeded membrane and cytosolic proteins. It has also been demonstrated that the FtsH homologous protein BB0789 is crucial for mouse and tick infectivity and in vitro growth of the Lyme disease-causing agent Borrelia burgdorferi. This is not surprising, considering B. burgdorferi complex life cycle, residing in both in mammals and ticks, which requires a wide range of membrane proteins and short-lived cytosolic regulatory proteins to invade and persist in the host organism. In the current study, we have solved the crystal structure of the cytosolic BB0789 166 - 614, lacking both N-terminal transmembrane α-helices and the small periplasmic domain. The structure revealed the arrangement of the AAA+ ATPase and the zinc-dependent metalloprotease domains in a hexamer ring, which is essential for ATPase and proteolytic activity. The AAA+ domain was found in an ADP-bound state, while the protease domain showed coordination of a zinc ion by two histidine residues and one aspartic acid residue. The loop region that forms the central pore in the oligomer was poorly defined in the crystal structure and therefore predicted by AlphaFold to complement the missing structural details, providing a complete picture of the functionally relevant hexameric form of BB0789. We confirmed that BB0789 is functionally active, possessing both protease and ATPase activities, thus providing novel structural-functional insights into the protein, which is known to be absolutely necessary for B. burgdorferi to survive and cause Lyme disease.Peer reviewe

    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

    Phylogenetic conservation of Trop-2 across species—rodent and primate genomics model anti-Trop-2 therapy for pre-clinical benchmarks

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    A phylogenetic conservation analysis of Trop-2 across vertebrate species showed a high degree of sequence conservation, permitting to explore multiple models as pre-clinical benchmarks. Sequence divergence and incomplete conservation of expression patterns were observed in mouse and rat. Primate Trop-2 sequences were found to be 95%–100% identical to the human sequence. Comparative three-dimension primate Trop-2 structures were obtained with AlphaFold and homology modeling. This revealed high structure conservation of Trop-2 (0.66 ProMod3 GMQE, 0.80–0.86 ± 0.05 QMEANDisCo scores), with conservative amino acid changes at variant sites. Primate TACSTD2/TROP2 cDNAs were cloned and transfectants for individual ORF were shown to be efficiently recognized by humanized anti-Trop-2 monoclonal antibodies (Hu2G10, Hu2EF). Immunohistochemistry analysis of Macaca mulatta (rhesus monkey) tissues showed Trop-2 expression patterns that closely followed those in human tissues. This led us to test Trop-2 targeting in vivo in Macaca fascicularis (cynomolgus monkey). Intravenously injected Hu2G10 and Hu2EF were well tolerated from 5 to 10 mg/kg. Neither neurological, respiratory, digestive, urinary symptoms, nor biochemical or hematological toxicities were detected during 28-day observation. Blood serum pharmacokinetic (PK) studies were conducted utilizing anti-idiotypic antibodies in capture-ELISA assays. Hu2G10 (t1/2 = 6.5 days) and Hu2EF (t1/2 = 5.5 days) were stable in plasma, and were detectable in the circulation up to 3 weeks after the infusion. These findings validate primates as reliable models for Hu2G10 and Hu2EF toxicity and PK, and support the use of these antibodies as next-generation anti-Trop-2 immunotherapy tools

    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

    Investigation of the metabolism of rare nucleotides in plants

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    Nucleotides are metabolites involved in primary metabolism, and specialized metabolism and have a regulatory role in various biochemical reactions in all forms of life. While in other organisms, the nucleotide metabolome was characterized extensively, comparatively little is known about the cellular concentrations of nucleotides in plants. The aim of this dissertation was to investigate the nucleotide metabolome and enzymes influencing the composition and quantities of nucleotides in plants. For this purpose, a method for the analysis of nucleotides and nucleosides in plants and algae was developed (Chapter 2.1), which comprises efficient quenching of enzymatic activity, liquid-liquid extraction and solid phase extraction employing a weak-anionexchange resin. This method allowed the analysis of the nucleotide metabolome of plants in great depth including the quantification of low abundant deoxyribonucleotides and deoxyribonucleosides. The details of the method were summarized in an article, serving as a laboratory protocol (Chapter 2.2). Furthermore, we contributed a review article (Chapter 2.3) that summarizes the literature about nucleotide analysis and recent technological advances with a focus on plants and factors influencing and hindering the analysis of nucleotides in plants, i.e., a complex metabolic matrix, highly stable phosphatases and physicochemical properties of nucleotides. To analyze the sub-cellular concentrations of metabolites, a protocol for the rapid isolation of highly pure mitochondria utilizing affinity chromatography was developed (Chapter 2.4). The method for the purification of nucleotides furthermore contributed to the comprehensive analysis of the nucleotide metabolome in germinating seeds and in establishing seedlings of A. thaliana, with a focus on genes involved in the synthesis of thymidilates (Chapter 2.5) and the characterization of a novel enzyme of purine nucleotide degradation, the XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Chapter 2.6). Protein homology analysis comparing A. thaliana, S. cerevisiae, and H. sapiens led to the identification and characterization of an enzyme involved in the metabolite damage repair system of plants, the INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Chapter 2.7). It was shown that this enzyme dephosphorylates deaminated purine nucleotide triphosphates and thus prevents their incorporation into nucleic acids. Lossof-function mutants senesce early and have a constitutively increased content of salicylic acid. Also, the source of deaminated purine nucleotides in plants was investigated and it was shown that abiotic factors contribute to nucleotide damage.Nukleotide sind Metaboliten, die am Primärstoffwechsel und an spezialisierten Stoffwechselvorgängen beteiligt sind und eine regulierende Rolle bei verschiedenen biochemischen Reaktionen in allen Lebensformen spielen. Während bei anderen Organismen das Nukleotidmetabolom umfassend charakterisiert wurde, ist in Pflanzen vergleichsweise wenig über die zellulären Konzentrationen von Nukleotiden bekannt. Ziel dieser Dissertation war es, das Nukleotidmetabolom und die Enzyme zu untersuchen, die die Zusammensetzung und Menge der Nukleotide in Pflanzen beeinflussen. Zu diesem Zweck wurde eine Methode zur Analyse von Nukleotiden und Nukleosiden in Pflanzen und Algen entwickelt (Kapitel 2.1), die ein effizientes Stoppen enzymatischer Aktivität, eine Flüssig-Flüssig-Extraktion und eine Festphasenextraktion unter Verwendung eines schwachen Ionenaustauschers umfasst. Mit dieser Methode konnte das Nukleotidmetabolom von Pflanzen eingehend analysiert werden, einschließlich der Quantifizierung von Desoxyribonukleotiden und Desoxyribonukleosiden mit geringer Abundanz. Die Einzelheiten der Methode wurden in einem Artikel zusammengefasst, der als Laborprotokoll dient (Kapitel 2.2). Darüber hinaus wurde ein Übersichtsartikel (Kapitel 2.3) verfasst, der die Literatur über die Analyse von Nukleotiden und die jüngsten technologischen Fortschritte zusammenfasst. Der Schwerpunkt lag hierbei auf Pflanzen und Faktoren, die die Analyse von Nukleotiden in Pflanzen beeinflussen oder behindern, d. h. eine komplexe Matrix, hochstabile Phosphatasen und physikalisch-chemische Eigenschaften von Nukleotiden. Um die subzellulären Konzentrationen von Metaboliten zu analysieren, wurde ein Protokoll für die schnelle Isolierung hochreiner Mitochondrien unter Verwendung einer Affinitätschromatographie entwickelt (Kapitel 2.4). Die Methode zur Analyse von Nukleotiden trug außerdem zu einer umfassenden Analyse des Nukleotidmetaboloms in keimenden Samen und in sich etablierenden Keimlingen von A. thaliana bei, wobei der Schwerpunkt auf Genen lag, die an der Synthese von Thymidilaten beteiligt sind (Kapitel 2.5), sowie zu der Charakterisierung eines neuen Enzyms des Purinnukleotidabbaus, der XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Kapitel 2.6). Eine Proteinhomologieanalyse, die A. thaliana, S. cerevisiae und H. sapiens miteinander verglich führte zur Identifizierung und Charakterisierung eines Enzyms, das an der Reparatur von geschädigten Metaboliten in Pflanzen beteiligt ist, der INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Kapitel 2.7). Es konnte gezeigt werden, dass dieses Enzym desaminierte Purinnukleotidtriphosphate dephosphoryliert und so deren Einbau in Nukleinsäuren verhindert. Funktionsverlustmutanten altern früh und weisen einen konstitutiv erhöhten Gehalt an Salicylsäure auf. Außerdem wurde die Quelle der desaminierten Purinnukleotide in Pflanzen untersucht, und es wurde gezeigt, dass abiotische Faktoren zur Nukleotidschädigung beitragen

    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

    2023-2024 Catalog

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    The 2023-2024 Governors State University Undergraduate and Graduate Catalog is a comprehensive listing of current information regarding:Degree RequirementsCourse OfferingsUndergraduate and Graduate Rules and Regulation

    Undergraduate Catalog of Studies, 2022-2023

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