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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
101

Mécanismes cellulaires et moléculaires des fonctions tolérogèniques et immunogéniques des cellules dendritiques dans les réponses auto-immunes

Zerif, Echarki 05 1900 (has links)
La contribution des DCs dans l’initiation et la perpétuation des maladies auto-immunes est bien établie. Chez la souris Non Obèse Diabétique (NOD), modèle spontané du diabète de type 1 (DT1), plusieurs travaux ont rapportés des anomalies phénotypiques et fonctionnelles des DCs. Les DCs sont parmi les premières cellules qui infiltrent les ilots pancréatiques, produisent des quantités excessives de cytokines pro-inflammatoires et contribuent à l’activation des lymphocytes T auto-réactifs (Teff). Cette capacité accrue des DCs à activer les Teffs est régulée par plusieurs voies de signalisation intracellulaire. STAT5 est parmi les facteurs de transcription critiques dans la régulation des gènes associés au développement, la maturation et les fonctions des DCs. La prédisposition au DT1 chez la NOD est déterminée par plusieurs régions de susceptibilités au diabète (idd1-20). De façon intéressante, le gène Stat5b est localisé dans la région de susceptibilité idd4 chez la souris NOD suggérant son implication dans le développement du diabète. En effet des études récentes ont identifiés un dysfonctionnement dans la voie de signalisation JAK-STAT5 chez les souris NOD, y compris la présence d’une mutation (L327M) au niveau du domaine de liaison à l’ADN de Stat5b qui altère sa liaison à l’ADN. Par ailleurs, les études réalisées dans notre laboratoire ont montré que le conditionnement des DCs au GM-CSF ou à la TSLP, qui activent la voie de signalisation Jak-Stat5, constitue une voie potentielle d’immunothérapie chez la souris NOD. Ces données suggèrent un rôle central de Stat5b dans la régulation des fonctions tolérogènes des cellules du système immunitaire. Nous avons généré un modèle de souris NOD transgéniques (NOD.CD11cStat5b-CA) exprimant de façon constitutive la forme active de STAT5B de la souris C57BL/6 spécifiquement dans les DCs. Nos résultats ont montré que ces souris transgéniques ont développées une protection totale contre le diabète auto-immun. Cette résistance au diabète à long terme est associe à l’acquisition des fonctions tolérogènes par les Stat5b-CA.DCs qui se manifestent par un phénotype mature tolérogène, marquées par une forte expression de molécules immunorégulatrices (PD-L1 et PD-L2) et une grande production de cytokines anti-inflammatoire (TGF-β) et une baisse significative de la production de cytokines pro-inflammatoires (IL-12p70, TNF-α et d’IL-23). Par ailleurs, nous avons mis en évidence le rôle de STAT5B dans la régulation à la hausse d’IRF4 et l’implication du complexe STAT5B/EZH2 dans le contrôle de la régulation à la baisse d’IRF8. En effet, cette régulation différentielle de l’expression des gènes Irf4 et Irf8 est accompagnée du développement d’une sous population CD11c+ CD11b+ DCs. Nos études ont démontré que le potentiel tolérogène des Stat5b-CA.DCs à rétablir et à maintenir la tolérance périphérique du système immunitaire vis-à-vis des auto-antigènes est associe à leur grande capacité d’induire la conversion et l’expansion des Tregs ainsi que la différentiation de deux populations cellulaires régulatrices Th2 et Tc2. Nous avons aussi démontré in vivo qu’une injection intraveineuse unique de Stat5-CA.DCs (spléniques ou générés de la moelle osseuse) ou de Tregs des souris transgéniques NOD.CD11cStat5b-CA a induit une protection totale contre le diabète chez les souris NOD receveuses. Notre étude apporte donc une évidence claire que la correction du défaut de la voie de signalisation Jak-Stat5b au sein des DC chez la souris NOD induit une protection à long terme contre le diabète. Finalement, cette voie de signalisation peut constituer une cible thérapeutique éventuelle non seulement dans le contexte du diabète de type 1 mais également dans d’autres maladies auto-immunes. / The contribution of DCs in the initiation and progression of autoimmune diseases is well established. Several studies have reported that phenotypic and functional abnormalities of DCs, in Non Obese Diabetic (NOD), contribute to spontaneous type 1 diabetes (T1D) development. DCs are among the first cells that infiltrate the pancreatic islets, produce excessive amounts of pro-inflammatory cytokines, and contribute to the activation of T effector cells (Teff). This increased ability of DCs to activate Teff is regulated by several intracellular signaling pathways. STAT5 is among the critical transcription factors in the regulation of genes associated with the development, maturation and functions of DCs. The predisposition to T1D in NOD is determined by several regions of susceptibility to diabetes (idd1-20). Interestingly, the Stat5b gene is located in the idd4 susceptibility region in NOD mice suggesting its involvement in the development of diabetes. Recent studies have identified a dysfunction in the Jak-Stat5 signaling pathway in NOD mice, including the presence of a mutation (L327M) at the DNA-binding domain of Stat5b which alters its binding to DNA. Furthermore, previous studies from our laboratory have shown that the GM-CSF- or TSLP-conditioned DCs, which activate the Jak-Stat5 signaling pathway, is a potential pathway for immunotherapy in NOD mice. These data suggest a central role for Stat5b in the regulation of tolerogenic functions of the immune cells. Here, we generated a transgenic NOD mouse model (NOD.CD11cStat5b-CA) that constitutively express the active form of STAT5B from the C57BL/6 mouse specifically in DCs. Our results showed that these transgenic mice are completely protected against autoimmune diabetes. This long-term diabetes protection is associated with the acquisition of tolerogenic functions by Stat5b-CA.DCs, that exhibit a mature tolerogenic phenotype, overexpression of immunoregulatory molecules (PD-L1 and PD-L2) and produce anti-inflammatory cytokines (TGF-β) and a significantly decrease their production of pro-inflammatory cytokines (IL-12p70, TNF-α and IL-23). Moreover, we have highlighted the role of STAT5B in the upregulation of IRF4 and also the involvement of the STAT5B/ EZH2 complex in downregulation of IRF8. This differential regulation of the Irf4 and Irf8 genes expression is accompanied by promoting the development of CD11c+CD11b+ DC subset. Furthermore, we demonstrated that the tolerogenic Stat5b-CA.DCs were able to restore and maintain peripheral immune tolerance to autoantigens, which is associated with their high ability to induce conversion and expansion Tregs and to promote Th2 and Tc2 immune deviation. We also demonstrated that a single intravenous injection of Stat5-CA.DCs (splenic or bone marrow derived dendritic cells) or Tregs from transgenic mice NOD.CD11cStat5b-CA halted ongoing diabetes in recipient NOD mice. Thus, our study provides clear evidence that the correction of the Jak-Stat5b signaling pathway defect in DC of NOD mice induces long-term protection against diabetes suggesting that signaling pathway can be a potential therapeutic target not only in the context of type 1 diabetes but also in other autoimmune diseases.
102

Role of a Mitochondrial Micropeptide in Regulating Innate Immune Responses

Bhatta, Ankit 29 September 2020 (has links)
Short ORF-encoded peptides (SEPs) are increasingly being identified as functional elements in various cellular processes. The current computational methods and experimental molecular biochemistry allow us to discover putative SEPs or micropeptides from proteogenomic datasets and experimentally validate them. Here, we identified a micropeptide produced from a putative long noncoding RNA (lncRNA) 1810058I24Rik which is downregulated in both human and murine myeloid cells exposed to lipopolysaccharide (LPS), as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed this transcript is localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 amino acid micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene ‘Mitochondrial micropeptide-47 (Mm47)’. Functional studies using siRNA and Cripsr-cas9-mediated deletion in primary cells, showed that the transcriptional response downstream of TLR4 was not affected by Mm47 loss of function. In contrast, both the Crispr-cas9- and siRNA-targeted BMDM cells were compromised for Nlrp3 inflammasome responses. However, the primary macrophages derived from the Mm47 knockout mice do not require Mm47 for Nlrp3 activation, likely due to basal downregulation of a negative regulator microRNA of Nlrp3 called Mir-223. Notably, the Mm47-deficient mice are susceptible to influenza virus infection and succumb despite comparable antiviral and inflammatory response to wildtype mice. We hypothesize that the Mm47 deficiency may affect the antiviral resilience of mice due to secondary mitochondria dependent immunometabolic defect or failure of recovery from immune pathology, which warrants further investigation. This study therefore identifies a novel mitochondrial micropeptide Mm47 that is required for activation of the Nlrp3 inflammasome in cells and resistance to influenza virus infection. Broadly, this work highlights the presence of translatable ORFs is annotated noncoding RNA transcripts and underscores their importance in innate immunity and virus infection.
103

Modulation of T cell receptor signals during thymic selection

Dong, Mengqi 03 1900 (has links)
Les cellules T ɑβ conventionnelles expriment des récepteurs antigéniques qui peuvent reconnaître et répondre à une grande variété d’agents pathogènes. En parallèle, des mécanismes cruciaux sont en place pour empêcher les cellules T de réagir aux auto-antigènes afin de prévenir le développement d’auto-immunité. Dans le but d’assurer la génération d’un réservoir de cellules T fonctionnelles, diverses et tolérantes au soi, les récepteurs des cellules T (TCR) ɑβ appropriés sont sélectionnés dans le thymus en fonction de la quantité et de la qualité des interactions avec les peptides du soi présentés par le complexe majeur d’histocompatibilité (CMH) sur les cellules présentatrice d’antigènes (CPA). Chez les nouveau-nés, des mécanismes intrinsèques et extrinsèques aux cellules influencent les interactions entre le TCR et le complexe CMH-peptide du soi, résultant en un répertoire de cellules T qui possèdent des propriétés distinctes par rapport à leurs homologues adultes. Par ailleurs, les souris diabétiques non-obèses (NOD), qui ont des cellules T auto-réactives qui attaquent les cellules β du pancréas, responsables de la production d’insuline, sont porteuses de polymorphismes génétiques qui peuvent influencer la sélection thymique. Ainsi, nous avons émis l’hypothèse que des facteurs intrinsèques et extrinsèques aux cellules modulent la sélection thymique tout au long de la vie et peuvent ultimement contribuer à la fonction et au dysfonctionnement de cellules T effectrices. La force globale de la signalisation TCR perçue lors du développement des lymphocytes T peut être mesurée en évaluant le niveau d’expression de différentes molécules, telles que CD5. Nous avons découvert que, tant chez la souris que l’humain, le répertoire des cellules T néonatales est composé de cellules T exprimant des niveaux plus élevés de CD5 que ceux des adultes. Cette augmentation des niveaux d’expression de CD5 n’est pas due à des défauts de tolérance centrale. En fait, nous avons plutôt démontré que les thymocytes exprimant un TCR de faible affinité pour les antigènes du soi ne sont pas sélectionnés efficacement chez les nouveau-nés et donc ne font pas partie du répertoire des cellules T néonatales, alors que ces thymocytes se développent adéquatement chez les adultes. Cette modification dans les seuils de sélection thymique biaise le niveau basal d’auto-réactivité du répertoire de cellules T néonatales et pourrait expliquer en partie les différences de réponse aux infections observées entre les nouveau-nés et les adultes. En comparant les niveaux de CD5 sur les thymocytes et les cellules T périphériques de souris NOD, prédisposées au diabète, avec ceux de souris C57BL/6 (B6), résistantes au développement du diabète auto-immun, nous avons découvert que les populations de cellules T des souris NOD ne perçoivent pas nécessairement des signaux TCR plus forts lorsqu’ils interagissent avec des antigènes du soi. Au contraire, une plus grande proportion de cellules T CD4+ et régulatrices avec un plus faible niveau de CD5 se différencient chez les souris NOD. Ce phénotype est fortement dépendant du locus du CMH des souris NOD. En revanche, les niveaux de CD5 sur les cellules T CD8+ périphériques des souris NOD sont plus élevés que ceux des souris B6, en raison d’un biais de survie intrinsèque aux cellules. Ces différences en niveau d’expression de CD5 sur le répertoire de cellules T des souris NOD ont des conséquences fonctionnelles directes et pourraient contribuer au développement ou à la progression du diabète auto-immun. Enfin, nous avons évalué si la sélection thymique était modulée par une molécule de co-signalisation, le co-stimulateur inductible de cellules T (ICOS). ICOS appartient à la famille des molécules de co-signalisation de type CD28 et se lie au ligand de ICOS (ICOSL). Alors que d’autres molécules de co-signalisation ont été démontré comme étant impliquées dans la tolérance centrale, le rôle joué par ICOS n’est pas clair. Nous avons démontré que ICOSL est exprimé par une variété de CPA thymiques importantes dans l’induction de la tolérance centrale et que ICOS est exprimé à la hausse durant la sélection thymique, en fonction de la force du signal TCR perçue par les thymocytes. Nous fournissons également, pour la première fois, une preuve que la voie ICOS-ICOSL pourrait avoir un rôle dans la régulation fine de la sélection négative. En conclusion, les résultats présentés dans cette thèse démontrent que la sélection thymique être altérés dans le contexte de l’ontogénie et du diabète auto-immun, conduisant au développement de cellules T avec une auto-réactivité basale relativement plus élevée ou plus faible en comparaison aux animaux adultes en bonne santé. Ces modulations pourraient avoir des conséquences importantes sur la fonction immunitaire et doivent être considérées pour le développement de futurs vaccins ou approches thérapeutiques chez ces populations. / Conventional ɑβ T cells express antigen receptors that can recognize and respond to a wide range of foreign pathogens. In parallel, critical mechanisms are in place to prevent T cells from reacting to self-antigens causing autoimmunity. To ensure the generation of a functional, diverse yet self-tolerant T cell pool, appropriate ɑβ T cell receptors (TCR) are selected in the thymus based on the quality and quantity of their interactions with self-peptides presented by the major histocompatibility complex (MHC) on thymic antigen presenting cells (APC). In neonates, cell-intrinsic and -extrinsic mechanisms influence TCR and self-peptide-MHC interactions resulting in a T cell pool that exhibits distinct functions as compared to their adult counterparts. Similarly, non-obese diabetic (NOD) mice, which contain autoreactive T cells that attack insulin-producing pancreatic β cells, carry genetic polymorphisms that can influence thymic selection. Therefore, we hypothesized that cell-intrinsic and -extrinsic factors modulate thymic selection throughout life and may ultimately contribute to the function and dysfunction of effector T cells. The overall perceived strength of TCR signaling during T cell development can be measured by several molecules, including CD5. We found that, in both mice and humans, the neonatal T cell pool is composed of T cells with higher CD5 levels than their adult counterparts. The increased CD5 levels are not due to defects in central tolerance. Instead, we demonstrated that thymic selection is altered in neonates. Thymocytes expressing a TCR with low affinity to self-antigen that develop in adults are not efficiently selected into the neonatal T cell pool. This shift in thymic selection thresholds skews the basal self-reactivity of the neonatal T cell repertoire and may explain, in part, differences in the neonatal versus adult response to infections. By comparing CD5 levels on thymocytes and peripheral T cells from diabetes-prone NOD mice with those from autoimmune-resistant C57BL/6 (B6) mice, we found that T cell populations in NOD mice do not necessarily perceive stronger TCR signals when interacting with self-antigens. Rather, NOD mice allow the differentiation of more CD4+ T cells and thymic Tregs with lower CD5 levels. This phenotype is strongly dependent on the NOD MHC locus. In contrast, CD5 levels on peripheral NOD CD8+ T cells are higher than those in the B6 mice that is likely due to a cell-intrinsic survival bias. These differences in CD5 levels in the NOD T cell pool have direct functional consequences and may contribute to the development or progression of autoimmune diabetes. Lastly, we investigated whether thymic selection is modulated by the co-signaling molecule, inducible T cell costimulator (ICOS). ICOS belongs to the CD28 family of co-signaling molecules and binds to the ICOS ligand (ICOSL). While other co-signaling molecules have been implicated in central tolerance, the role played by ICOS is unclear. We demonstrated that ICOSL is expressed by an array of thymic APCs important for central tolerance induction, and that ICOS is upregulated during thymic selection relative to the strength of TCR signaling the thymocytes perceive. We also provide, for the first time, evidence that the ICOS-ICOSL pathway may fine-tune negative selection. In conclusion, the results presented in this thesis demonstrate that thymic selection appears to be altered within the context of ontogeny and autoimmune diabetes, leading to the development of T cells with relatively higher or lower basal self-reactivity as compared to healthy adult animals. These modulations may have significant consequences on immune function and require careful consideration for future vaccination and therapeutic approaches within these populations.
104

Exploration Strategies for Robotic Vacuum Cleaners / Strategier för utforskning med robotdammsugare

Navarro Heredia, Sofia January 2018 (has links)
In this thesis, an exploration mode for the PUREi9 robotic vacuum cleaner is implemented. This exploration would provide information for optimizing the cleaning path beforehand, and would allow the robot to relocalize itself or the charger more easily in case it gets lost. Two elements are needed in order to implement an exploration mode; first, an exploration algo-rithm which will decide the next position of the robot in order to obtain useful information about the environment (unknown areas, empty spaces, obstacles...), and second, an exploration map which stores that information and is updated each time a new relevant position is reached. These elements are related and generally both are required for performing successfully the exploration of a specific environment. A frontier-based strategy is adopted for the exploration algorithm, together with occupancy grid maps. This strategy has long been regarded as a key method for autonomous robots working in unknown or changing environments. The idea of frontier-based algorithms is to divide the environ-ment into cells of regular size and drive the robot to the frontiers between cells with no obstacles and cells for which no information has been gathered. It plans one step ahead by choosing a lo-cation which provides new environment information, instead of planning in advance all locations where the robot needs to acquire new sensor information. Based on frontier strategy, two different exploration algorithms are implemented in the project. The first one is called "random frontier strategy", which chooses arbitrarily the frontier to go among the frontiers set. The second is called "closest frontier strategy", which chooses the closest frontier as the NBV (Next Best View) the robot should drive to. A path planning algorithm, based on Dijkstra’s algorithm and a node graph, has also been implemented in order to guide the robot towards the frontiers. The two methods have been compared by means of simulations in different environments. In addition, both exploration strategies have been tested on a real device. It is found that the closest frontier strategy is more efficient in terms of path length between scanning points, while both methods give a similar exploration ratio, or percentage of fully explored cells within the final map. Some additional work is required in order to improve the performance of the exploration method in the future, such as detecting unreachable frontiers, implementing a more robust path planning algorithm, or filtering the laser measurements more extensively. / I den här rapporten har vi implementerat en utforskningsmod för robotdammsugaren Pure i9. Sådan utforskning skulle ge underlag för att optimera städmönstret i förhand och låta roboten relokalisera sig själv eller laddaren om den tappar bort sig. För att implementera utforskning behövs två saker. För det första krävs en algoritm för utforsk-ning, som bestämmer nästa position för roboten, med målet att samla användbar information om omgivningen (okända eller fria områden, hinder etc.) För det andra krävs en karta som lagrar informationen och uppdateras varje gång roboten når en relevant ny position. Dessa två hänger ihop och i allmänhet krävs båda för att framgångsrikt utforska ett specifikt område. Vi har valt en front-baserad strategi för utforskningsalgoritmen, tillsammans med en rutnäts-karta med sannolikheten för hinder. Denna strategi har länge betraktats som en nyckelmetod för autonoma robotar som arbetar i okända eller föränderliga miljöer. Idén med front-baserade strate-gier är att köra roboten till fronterna mellan celler utan hinder och celler där information saknas. Den planerar ett steg framåt genom att välja en plats som ger ny information om miljön, istället för att i förväg planera alla platser där roboten behöver samla in ny sensorinformation. Baserat på front-strategi, har vi implementerat två utforskningsalgoritmer i projektet. Den första är en slumpmässig strategi, som godtyckligt väljer en front att åka till, ur hela mängden av fronter. Den andra är en närmaste fronten-strategi som väljer den närmaste fronten som den nästa bästa utsiktspunkt som roboten ska åka till. Vi har också implementerat en algoritm för banplanering, baserad på Dijkstras algoritm och en nod-graf, för att styra roboten mot fronterna. Vi har jämfört de två metoderna genom simulering i olika miljöer. Dessutom har båda utforsk-ningsstrategierna testats på en riktig enhet. Närmaste fronten-strategin är effektivare med avse-ende på banlängd mellan skanningspunkter, medan båda metoderna ger liknande utforsknings-grad, eller samma procentandel av fullt utforskade celler inom den slutliga kartan.
105

Type 1 Diabetes Diagnostic Assay

Jackson, LaDonya L. January 2015 (has links)
No description available.
106

Mechanistic And Functional Insights Into Mycobacterium Bovis BCG Triggered TLR2 Signaling : Implications For Immune Evasion Strategies

Ghorpade, Devram Sampat 07 1900 (has links) (PDF)
Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Host immune responses during mycobacterial infection involve potent cell effector functions including that of CD4+, CD8+ and γδT cells, macrophages and dendritic cells (DCs). Further, the critical regulators of protective immunity to mycobacterial infection include IFN-γ, IL-12, IL-23, TNF-α, lymphotoxins, CD40, nitric oxide and reactive oxygen species. However, the success of mycobacterial infection often relies in its ability to evade immune surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immunoregulatory molecules. Therefore, the key to control mycobacterial growth and limit pathogenesis lies in the understanding the interactions between Mycobacterium and primary responders like macrophages and DCs. In this scenario, the role of pattern recognition receptors (PPRs) in orchestrating host immune responses assumes central importance. The cell surface receptors play crucial role in influencing overall immune responses. Of the PRRs, the Toll-like receptors (TLRs) form key immune surveillance mechanisms in recognition as well as control of mycobacterial infection. Among them, TLR2 is the primary interacting receptor on antigen presenting cells that recognize the invading mycobacteria. Mycobacterial cell wall constituents such as LAM, LM, PIM and 19-kDa protein have been shown to activate TLR2 signaling leading to proinflammatory responses. Recent reports have suggested that PE_PGRS antigens of M. tuberculosis interact with TLR2. For example, RV0754, Rv0978c, RV1917c have been implicated in modulation of human DCs. The 19-kDa lipoprotein, LpqH (Rv3763) and LprG (Rv1411c) utilize TLR2 signaling to inhibit macrophage responsiveness to IFN-γ triggered MHC class II expression and mycobacterial antigen presentation. Interestingly, recognition and amplification of pathogenic-specific signaling events play important roles in not only discriminating the invading microbes, but also in regulating explicit immune responses. In this context, integration of key signaling centers, which modulate host immunity to pathogenic mycobacterial infections, remains unexplored. In accordance to above observations, signal transduction pathways downstream to TLRs play a critical role in modulation of battery of host cells genes in terms of expression and production of immune modulatory cytokines and chemokines, recruitment of cellular machineries to site of infections etc. This suggests the decisive role for TLRs in modulation of host cell fate decisions. However, during the ensuing immunity to invading pathogens, beside TLR signaling pathways, various other signaling molecules are thought to execute specific functions in divergent cellular contexts. Recent studies from our laboratory have clearly demarcated a novel cross talk of TLR2-NOTCH1 and TLR2-Wnt signaling pathways during mycobacterial infections. The current study primary focuses on the broad range of cross talk of TLR2 and Sonic hedgehog (SHH) signaling pathways and its functional significance. The present investigation demonstrates that M. bovis BCG, a vaccine strain, triggers a robust activation of SHH signaling in macrophages compared to infection with diverse Gram-positive or Gram-negative microbes. This observation was further evidenced by the heightened SHH signaling signatures during in vivo scenario in cells /tissues from pulmonary tuberculosis (TB) individuals as well as tuberculous meningitis (TBM) patients. Furthermore, we show that the sustained TNF-α secretion by macrophages upon infection with M. bovis BCG is a critical necessity for SHH activation. Significantly, perturbation studies implicate a vital role for M. bovis BCG stimulated TLR2/PI3K/PKC/MAPK/NF-κB axis to induce TNF-α, that contributes to enhance SHH signaling. The TNF-α driven SHH signaling downregulates M. bovis BCG induced TLR2 signaling events leading to modulation of battery of genes that regulate various functions of macrophages genes like Vegf-a, Socs-3, Cox-2, Mmp-9 and M1/M2 genes. Importantly, utilizing whole-genome microRNA (miRNA) profiling, roles for specific miRNAs were identified as the molecular regulators that bring about the negative-feedback loop comprising TLR2-SHH signaling events. Thus, the current study illustrates how SHH signaling tightly regulates the kinetics and strengths of M. bovis BCG specific TLR2 responses, emphasizing a novel role for SHH signaling in host immune responses to mycobacterial infections. As described, variety of host factors contributes for ensuing effective host defenses and modulation of host cell fate decisions. Interestingly, avirulent pathogenic mycobacteria, including the vaccine strain M. bovis BCG, unlike virulent M. tuberculosis, cause extensive apoptosis of infected macrophages, which suggests a significant contribution of the apoptosis process to the initiation and subsequent amplification of innate as well as adaptive immune responses. Among various cues that could lead to apoptosis of host cells, the initiation of the apoptotic machinery by posttranscriptional mechanisms assumes significant importance. Among posttranscriptional control mechanisms, miRNAs are suggested to regulate several biological processes including immune responses. Various effectors of host immunity are known to be regulated by several miRNAs, and a prominent one among them, miRNA-155 (miR-155), often exhibits crucial roles during innate or adaptive immune responses. In this perspective, we identified a novel role of miR-155 during M. bovis BCG induced apoptosis of macrophages. The genetic and signaling perturbations data suggested that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in induced expression of the apoptotic genes as well as Caspase-3 cleavage and Cytochrome c translocation. Thus, augmented PKA signaling by M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, emphasizing a novel role for miR-155 in host immunity to mycobacterial infections. In perspective of these studies, important directives are often comprised of sequential and coordinated activation of TLR and NLR-driven signal transduction pathways, thus exhibiting foremost influence in determining the overall strength of the innate immune responses. As described, TLR2 exhibits dominant role in sensing various agonists including pathogen-associated molecular patterns (PAMPs) of microbes at the cell surface and generally considered as major effectuator of proinflammatory responses. Interestingly, NLRs like NOD1 or NOD2 often act in contrary, thus regulating anti-inflammatory responses as well as polarization of T cells towards skewed Th2 phenotype. This presents an interesting conundrum to functionality of DCs or macrophages in terms of effector functions during rapidly evolving immunological processes including effects originating from immunosuppressive effectors such as CTLA-4 or TGF-. DCs like macrophages are important sentinels of innate immunity, possesses array of PRRs that include TLRs and NOD-like receptors (NLRs). Signaling events associated with innate sensors like TLRs and NLRs often act as regulatory circuits that modulate the overall functions of DCs in terms of maturation process, cytokine or chemokine production, receptor expression, migration to secondary lymphoid organs for antigen presentation for effectuating Th polarization. TLR2, while acting as sensors for extracellular cues or endocytic network, drives signaling events in response to recognition of PAMPs including mycobacterial antigens like ESAT-6, PE_PGRS antigens, while NOD1 and NOD2 operate as cytosolic sensors initiating signaling pathways upon recognition of diaminopimelic acid (DAP) and muramyl dipeptide (MDP), components of bacterial peptidoglycan. Thus, TLRs or NOD receptors could trigger similar or contrasting immune responses by cooperative or non-cooperative sensing, consequently exhibiting immense complexity during combinatorial triggering of host DCs-PRR repertoire. In view of these observations, our current investigation comprehensively demonstrated that maturation process of human DCs were cooperatively regulated by signaling cascades initiated by engagements of TLR2, NOD1 and NOD2 receptors. Importantly, combined triggering of TLR2 and NOD receptors abolished the TGF-β or CTLA-4-mediated impairment of human DCs maturation, which required critical participation of NOTCH1-PI3K signaling cohorts. Thus, our data delineated the novel insights in modulation of macrophages and DCs effector functions by mycobacterial TLR2 or NOD agonists and broaden our understanding on the signal dynamics and integration of multiple signals from PRRs during mycobacterial infections. Altogether, our findings establish the understanding of conceptual frame work in fine tuning of TLR2 responses by SHH signaling as well as potential co-operativity among TLRs and NODs to modulate NOTCH1 dependent DCs maturation. Importantly, our study provides mechanistic and functional insights into various molecular regulators of macrophage cell fate decisions like miR-31. miR-150 and miR-155, which can fuel the search for attractive and effective drug targets and novel therapeutics to combat diseases of the hour like tuberculosis.
107

Money Laundering Detection using Tree Boosting and Graph Learning Algorithms / Detektion av Penningtvätt med hjälp av Trädalgoritmer och Grafinlärningsalgoritmer

Frumerie, Rickard January 2021 (has links)
In this masters thesis we focused on using machine learning methods for detecting money laundering in financial transaction networks, in order to demonstrate that it can be used as a complement or instead of the more commonly used rule based systems. The graph learning method graph convolutional networks (GCN) has been a hot topic in the field since they were shown to scale well with data size back in 2018. However the typical GCN models cannot use edge features, which is why this thesis combines the GCN model with a node and edge neural network (NENN) in order to solve this problem. This new method will be compared towards an already established machine learning method for financial transactions, namely the tree boosting method (XGBoost). Because of confidentiality concerns for financial transactions data, the machine learning algorithms will be tested on two carefully constructed synthetically generated data sets, which from agent based simulations resembles real financial data. The results showed the viability and superiority of the new implementation of the GCN model with it being a preferable method for connectivly structured data, meaning that a transaction or account is analyzed in the context of its financial environment. On the other hand the XGBoost method showed better results when examining transactions independently. Hence it was more accurately able to find fraudulent and non fraudulent patterns from the transactional features themselves. / I detta examensarbete fokuserar vi på användandet av maskininlärningsmetoder för att detektera penningtvätt i finansiella transaktionsnätverk, med målet att demonstrera att dess kan användas som ett komplement till eller i stället för de mer vanligt använda regelbaserade systemen. Grafinlärningsmetoden \textit{graph convolutional networks} (GCN) som har varit ett hett ämne inom området sedan metoden under 2018 visades fungera bra för stora datamängder. Däremot kan inte en vanlig GCN-modell använda kantinformation, vilket är varför denna avhandling kombinerar GCN-modellen med \textit{node and edge neural networks} (NENN) för att mer effektivt detektera penningtvätt. Denna nya metod kommer att jämföras med en redan etablerad maskininlärningsmetod för finansiella transaktioner, nämligen \textit{tree boosting} (XGBoost). På grund av sekretessanledningar för finansiella transaktionsdata var maskininlärningsalgoritmerna testade på två noggrant konstruerade syntetiskt genererade datamängder som från agentbaserade simuleringar liknar riktiga finansiella data. Resultaten visade på applikationsmöjligheter och överlägsenhet för den nya implementationen av GCN-modellen vilken är att föredra för relationsstrukturerade data, det vill säga när transaktioner och konton analyseras i kontexten av deras finansiella omgivning. Å andra sidan visar XGBoost bättre resultat på att examinera transaktioner individuellt eftersom denna metod mer precist kan identifiera bedrägliga och icke-bedrägliga mönster från de transnationella funktionerna.
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DSTYK Enhances Chemoresistance in Triple-Negative Breast Cancer Cells

Ogbu, Stella C., Rojas, Samuel, Weaver, John, Musich, Phillip R., Zhang, Jinyu, Yao, Zhi Q., Jiang, Yong 29 December 2021 (has links)
Breast cancer, as the most prevalent cancer in women, is responsible for more than 15% of new cancer cases and about 6.9% of all cancer-related death in the US. A major cause of therapeutic failure in breast cancer is the development of resistance to chemotherapy, especially for triple-negative breast cancer (TNBC). Therefore, how to overcome chemoresistance is the major challenge to improve the life expectancy of breast cancer patients. Our studies demonstrate that TNBC cells surviving the chronic treatment of chemotherapeutic drugs show significantly higher expression of the dual serine/threonine and tyrosine protein kinase (DSTYK) than non-treated parental cells. In our in vitro cellular models, DSTYK knockout via the CRISPR/Cas9-mediated technique results in apoptotic cell death of chemoresistant cells upon drug treatment. Moreover, DSTYK knockout promotes chemotherapeutic drug-induced tumor cell death in an orthotopic mouse model. These findings suggest that DSTYK exerts an important and previously unknown role in promoting chemoresistance. Our studies provide fundamental insight into the role of DSTYK in chemoresistance in TNBC cells and lay the foundation for the development of new strategies targeting DSTYK for improving TNBC therapy.
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The role of inducible T-cell co-stimulator in regulatory T cell homeostasis and function

Chang, Jinsam 12 1900 (has links)
Inducible T-cell co-stimulator (ICOS) is a member of the CD28 family of T cell costimulatory receptors that is induced upon activation in CD4+ and CD8+ T cells. It has been established that ICOS plays a critical role in humoral immunity by supporting the generation and function of T follicular helper cells. Thus, ICOS deficiency in humans and mice leads to immunodeficiency due to impaired germinal center reaction and antibody production. ICOS can also promote expansion, survival, and cytokine expression of inflammatory T cells. However, given that ICOS is also constitutively expressed in Foxp3+ regulatory T (Treg) cells, interruption of ICOS signaling may lead to different outcomes depending on the context of immune reactions. Due to the potential opposing roles of ICOS in overall T cell immune response, the T cell subsetspecific role of ICOS needs to be clarified. In order to address the intrinsic roles of ICOS in Treg homeostasis and function, we generated mice (termed ICOS FC) in which Icos gene is specifically deficient in Foxp3+ T regulatory cells. Using flow cytometry and single-cell transcriptome analysis, we show that ICOS FC mice do not have any severe alterations in the Tcon cell activation status and subset compositions of Treg cells under a steady state condition. Consistently, no spontaneous autoimmune symptoms developed in aged ICOS FC mice. In contrast, when the mice were challenged with chemically induced skin inflammation, ICOS FC mice mounted more severe inflammatory responses. In parallel, the number of Treg cells was reduced allowing increase of inflammatory CD4+ and CD8+ T cells in the draining lymph nodes and the skin. Although very small, our single-cell transcriptome analysis identified a cluster of Treg cells coexpressing T-bet and CXCR3 (termed Th1-Treg) in the draining lymph nodes in an ICOS-dependent manner. Therefore, Treg-intrinsic ICOS deficiency had minimal impact on the overall Treg homeostasis, but weakened Treg cells’ capacity to control Th1-driven skin inflammation likely due to the impaired differentiation of Th1-Treg cells. A dual role of ICOS in T cell-driven autoimmune disease has been modeled in NOD mice. In pure NOD mice in which polyclonal T cells control the disease, ICOS germline deficiency reduced disease progression by dampening activation of pathogenic autoreactive T cells. In contrast, when the TCR repertoire was highly restricted to autoantigens in BDC2.5 TCR transgenic III NOD line, ICOS-expressing Treg cells appear to play a dominant role by halting progression of insulitis to overt diabetes. However, previous studies could not exclude the possibility of altered TCR repertoire by ICOS deficiency in germline. In this study, we tested the impact of Icos gene deletion in adult NOD mice using an inducible ubiquitous Cre system once peripheral T cell repertoire had been established. We observed reduced incidence of diabetes in pure NOD mice but accelerated disease in BDC2.5-NOD mice, very similar to germline ICOS-deficiency. These results support the prevailing view that the main function of ICOS is to regulate effector and regulatory T cells in the periphery. In sum, we demonstrated that ICOS-deficient Treg cells retain the capacity to prevent spontaneous autoimmune disease but have a compromised ability to dampen Th1-driven skin inflammation. We further confirm the notion that ICOS mainly regulates mature T cells as opposed to thymic selection process. / Le co-stimulateur inductible des lymphocytes T (ICOS) est un membre de la famille CD28 des récepteurs co-stimulateurs qui est induit suite à l'activation des lymphocytes T CD4+ et CD8+. Il a été établi que ICOS joue un rôle essentiel dans l'immunité humorale en soutenant la génération et la fonction des cellules T auxiliaires folliculaires. Le déficit en ICOS chez l'homme et la souris conduit à une immunodéficience due à une altération de la réaction du centre germinatif et de la production d'anticorps. ICOS peut également favoriser l'expansion, la survie et l'expression des cytokines des cellules T inflammatoires. Cependant, étant donné que ICOS est également exprimé de manière constitutive dans les cellules T régulatrices Foxp3+ (Treg), l'interruption de la signalisation ICOS peut conduire à des résultats différents selon le contexte des réactions immunitaires. En raison des rôles potentiellement opposés de ICOS dans la réponse immunitaire globale des lymphocytes T, le rôle spécifique de ICOS chez les sous-ensembles de lymphocytes T doit être clarifié. Afin d'étudier les rôles intrinsèques de ICOS dans l'homéostasie et la fonction des Tregs, nous avons généré des souris (appelées ICOS FC) dans lesquelles le gène Icos est spécifiquement aboli chez les cellules T régulatrices Foxp3+. À l'aide de la cytométrie en flux et de l'analyse du transcriptome unicellulaire, nous démontrons que les souris ICOS FC ne présentent aucune altération grave de l'état d'activation des cellules Tcon et des compositions de sous-ensembles de cellules Treg dans des conditions d'équilibre. De plus, aucun symptôme auto-immun spontané ne s'est développé chez les souris ICOS FC âgées. En revanche, lorsque les souris ont été confrontées à une inflammation cutanée induite chimiquement, les souris ICOS FC ont présenté des réponses inflammatoires plus sévères. En parallèle, le nombre de cellules Treg a été réduits permettant une augmentation des cellules T CD4+ et CD8+ inflammatoires dans les ganglions lymphatiques drainants et la peau. Notre analyse du transcriptome unicellulaire a identifié un petit groupe de cellules Treg coexprimant T-bet et CXCR3 (appelé Th1-Treg) dans les ganglions lymphatiques drainants d'une manière ICOS-dépendante. Par conséquent, le déficit en ICOS intrinsèque aux Tregs a eu un impact minimal sur l'homéostasie globale des Tregs, mais a affaibli la capacité des cellules Treg à contrôler l'inflammation cutanée induite par les cellules Th1, probablement en raison de la différenciation altérée des cellules Th1-Treg. V Un double rôle de ICOS dans les maladies auto-immunes induites par les cellules T a été modélisé chez les souris NOD. Dans les souris NOD pures chez lesquelles des cellules T polyclonales contrôlent l’apparition du diabète auto-immun, un déficit de ICOS réduit la progression de la maladie en atténuant l'activation des cellules T autoréactives pathogènes. En revanche, dans un contexte où le répertoire du TCR est fortement restreint aux auto-antigènes (lignée NOD transgénique BDC2.5 TCR), les cellules Treg exprimant ICOS semblent jouer un rôle dominant en arrêtant la progression vers le diabète manifeste. Cependant, des études antérieures n'ont pas pu exclure la possibilité d'un répertoire de TCR altéré par un déficit en ICOS dans la lignée germinale. Dans cette étude, nous avons testé l'impact de la délétion inductible du gène Icos chez des souris NOD adultes à l’aide d’un système Cre ubiquitaire une fois que le répertoire des cellules T périphériques a été établi. Nous avons observé une incidence réduite du diabète chez les souris NOD pures, mais une accélération de la maladie chez les souris NOD BDC2.5 très similaire au phénotype causé par une ablation de Icos de la lignée germinale. Ces résultats soutiennent l'opinion dominante selon laquelle la fonction principale de ICOS est de réguler les cellules T effectrices et régulatrices dans la périphérie. En résumé, nous avons démontré que les cellules Treg déficientes en ICOS conservent la capacité de prévenir les maladies auto-immunes spontanées, mais démontrent une capacité réduite à atténuer l'inflammation cutanée provoquée par les cellules Th1. Nous confirmons de plus l’hypothèse que ICOS régule principalement les cellules T matures au lieu du processus de sélection thymique.

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