NRH quinone oxidoreductase 2 (NQO2) is regarded as a mammalian Phase I detoxifying enzyme responsible for reducing quinones to hydroquinones. NQO2 is highly expressed in different types of cancer such as breast and prostate cancer suggesting its participatory role in the progression of these diseases. A potential reason for this is that NQO2 has the ability to modulate the stability of cyclin D1 and activity of NF-ÃŽÂoB and it has been shown that inhibition of NQO2, either genetically or pharmacologically, can alter the pattern of proliferation of cancer cells. However, the biological roles of NQO2 in cancer progression are still ambiguous and need further investigation. A panel of seven ovarian cancer cell lines (OVCs) were screened for the presence and functionality of NQO2. SKOV-3 and TOV-112D cells expressing comparatively the highest and lowest levels of NQO2 were stably transduced to silence and overexpress NQO2 respectively. Pharmacological inhibition was achieved using resveratrol or a series of novel 4-aminoquinolines synthesised in-house. Cell proliferation was monitored by cell counting and clonogenic assays. Flow cytometric analysis was used to determine cell cycle distribution and levels of ROS following modulation of NQO2 function. The expression of cell cycle regulatory markers was determined by Western blot. The contributory roles of NQO2 in determining the cytotoxicity of Adriamycin (ADR) towards OVCs was investigated using MTT assay together with evaluation of P-gp expression and basal ROS levels. In the OVCs panel, NQO2 protein levels and enzymatic activity showed an excellent correlation; with activity varying 36-fold between the cell lines. The sensitivity of OVCs to CB1954 was significantly increased when combined with the NRH-like co-factor, EP0152R. This supports the notion that NQO2 mediates the toxicity of CB1954, which is further confirmed by the strong correlation between cellular NQO2 activity and the responsiveness of the OVC cell lines to CB1954. Hydrazone quinolines showed the highest inhibitiory potency against NQO2 in SKOV-3 when compared to the typical and in-house synthesised quinolines inhibitors. NQO2-overexpressing TOV-112D cells showed more aggressive growth pattern and higher capacity to form colonies than wild-type cells. This was consistently associated with an enhancement in the progression of cells through cell cycle phases and significant reduction in Rb expression. A reduction in ROS levels in NQO2-OE cells may also explain this enhancement in cell growth. Overexpressing NQO2 also resulted in destabilisation of CDK4 and cyclin D1 with significant reduction in their expression levels, and concomitant increase in p-cyclin D1 (Thr286). The involvement of NQO2 in controlling cyclin D1 turnover is also confirmed in SKOV-3 cells when genetic silencing of NQO2 was accompanied by significant reduction in p-cyclin D1 and subsequent stabilisation of cyclin D1 levels. In spite of this, no alterations in the growth pattern of SKOV-3 cells were observed highlighting the impact of cell type on the variations in cellular responses. The role of NQO2 in determining the toxicity of ADR treatment was not proved in OVC cells. This was despite that modulation of NQO2 levels caused significant changes in P-gp expression. The intracellular basal levels of ROS was found to affect the responsiveness of OVCs to ADR as demonstrated when treating SKOV-3 with resveratrol was accompanied by significant increase in ROS levels and concomitant enhancement in the cells’ response to ADR. In conclusion, NQO2 can profoundly alter the proliferation characteristics of OVCs and is a potential therapeutic target for the treatment of this disease. However, the biological functions of NQO2 and its contributory roles in particular pathways are varied among different types of cancer -in other words- are highly dependent on cancer type.

In the gastrointestinal tract, probiotics have been shown to promote host immunity and to regulate immune signaling pathways. This study used Caco-2 cell line to examine the effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu” a Kenyan traditional cultured milk, on the production inflammatory cytokines in enterocytes. Live Lactobacillus rhamnosus (MRS6AN), its cytoplasmic fraction (CF), filtered spent broth (FSB) or heat inactivated FSB (HIB) were used as treatments on differentiated Caco-2 cell monolayer in transwells. Cytokine content in the cell lysates, apical and basolateral supernatants were determined using ELISA. Caco-2 cell lysate treatments showed significantly increased anti-inflammatory TGF-β (ng/ml) levels on average about 100x more compared to the increase in pro-inflammatory IL-8 (pg/ml) levels. These levels were significantly reduced after inhibition of NF-κB. In conclusion, live Lactobacillus rhamnosus, its CF, FSB or HIB seemed to modulate the production of inflammatory cytokines in enterocytes partly via the NF-κB signaling pathway.

Nanoparticles, while included in a growing number of consumer products, may pose risks to human health due to heavy metal leaching and/or the production of reactive oxygen species following exposures. Subcellular mechanisms of action triggered as a result of exposure to various nanoparticles are still largely unexplored. In this work, an effort to elucidate such toxicological parameters was accomplished by evaluating oxidative stress generation, changes in gene and protein expression, and cell cycle status after low-dose exposures to a variety of metal and carbon-based nanomaterials in primary human dermal cells. Additionally, mitigation of nanoparticle toxicity via microencapsulation was investigated to assess the feasibility of utilizing nanomaterials in dermally implantable biosensor applications. Cellular immune and inflammatory processes were measured via qPCR and immunoblotting, which revealed gene and protein expression modulation along the NF-kappaB pathway after a variety of nanoparticle exposures. The role of immunoregulatory transcription factor NF-kappaB was examined in an oxidative stress context in cells exposed to a panel of nanoparticles, whereby glutathione conversion and modulation of oxidative stress proteins in normal and NF-kappaB knockdown human dermal fibroblasts were monitored. Results revealed decreased antioxidant response and corresponding increased levels of oxidative stress and cell death in exposed normal cells, compared to NF-kappaB incompetent cells. However, reactive oxygen species production was not an absolute precursor to DNA damage, which was measured by the comet assay, gamma-H2AX expression, and flow cytometry. Protein analysis revealed that map kinase p38, rather than p53, was involved in the halting of the cell cycle in S-phase after ZnO exposures, which caused DNA double strand breaks. Microencapsulation of fluorescent quantum dot nanoparticles, specifically, was utilized as a method to improve system functionality and surrounding cellular viability for the purpose of a dermal analyte detection assay. In vitro results indicated a functional localization of nanoparticles, as well as cessation of cellular uptake. Subsequently, cellular metabolism was unaffected over the range of time and concentrations tested in comparison to unencapsulated quantum dot treatments, indicating the usefulness of this technique in developing nanoparticle-driven biomedical applications.

Le virus T lymphotrope humain de type 1 (HTLV-1, Human T cell Leukemia Virus type 1) est l'agent responsable de la leucémie à cellules T de l'adulte, une prolifération maligne de lymphocytes T CD4+. L'activation constitutive de la voie NF-kB dans les lymphocytes T exprimant la protéine virale Tax s'est révélée primordiale pour la prolifération et la transformation induites par HTLV-1. Selon le modèle classique, Tax agit à deux niveaux de la voie NF-kB. Dans le cytoplasme, Tax active constitutivement le complexe IKK (IKB Kinase) en se liant à sa sous-unité régulatrice NEMO/IKKy. Dans le noyau, Tax interagit directement avec les dimères NF-kB dans des corps nucléaires Tax. L'ubiquitinylation et la SUMOylation de Tax ont été initialement décrites comme nécessaires pour l'activation de la phase cytoplasmique et de la phase nucléaire respectivement. Cependant, les mécanismes régulateurs des modifications post-traductionnelles de Tax restent difficiles à identifier car il n'a pas été possible d'étudier séparément l'ubiquitinylation et la SUMOylation de Tax.Au laboratoire, nous avons généré et caractérisé fonctionnellement un nouveau mutant de Tax qui nous a permis de découpler les rôles de l'ubiquitinylation et de la SUMOylation de Tax. Tax- P79AQ81A est ubiquitinylé de façon quantitativement similaire à Tax mais présente une forte réduction (80%) de SUMOylation. De plus, Tax-P79AQ81A ne forme pas de corps nucléaires. Néanmoins, ces deux défauts ne semblent pas préjudiciables pour la capacité du mutant à activer la voie NF-KB non seulement dans des lignées cellulaires mais également dans des lymphocytes T CD4+ primaires. En parallèle, nous avons montré que les corps nucléaires Tax sont rarement présents dans des lymphocytes T chroniquement infectés par HTLV-1, renforçant l'idée que ces structures ne sont pas requises pour l'activation de la voie NF-KB et probablement pas pour les autres fonctions de Tax. Enfin, nous avons démontré que les capacités d'activation de la voie NF-KB de différents mutants de Tax sont fortement corrélées à leur niveau d'ubiquitinylation mais pas de SUMOylation, confirmant que l'ubiquitinylation de Tax est la modification essentielle pour l'activation de la voie NF-KB.Le virus HTLV-2 ne possède pas les propriétés transformantes du virus HTLV-1 et les propriétés de la protéine Tax2 comparées à celles de Tax1 pourraient être à l'origine des différences de pathogénicité entre les deux virus. Notre étude a révélé que, de façon surprenante, l'activation de la voie NF-KB par la protéine Tax2 est non seulement indépendante de la SUMOylation et de la formation des corps nucléaires comme pour Tax1, mais également indépendante d'une quelconque ubiquitinylation, suggérant des mécanismes différents d'activation du complexe IKK parTax1 et Tax2.Nos études, aussi bien de la protéine Tax1 que de la protéine Tax2, nous ont donc permis de revisiter le modèle actuel d'activation de la voie NF-kB en démontrant l'impact mineur de la SUMOylation et en révélant une différence majeure en ce qui concerne le rôle de l'ubiquitinylation, distinguant les virus HTLV-1 et HTLV-2

Large signalosome assembly is a prerequisite for NF-κB signaling upon engagement of various immunoreceptors. Adaptor proteins containing protein-protein interaction domains oligomerise in response to such stimuli in order to propagate signaling. Each immunoreceptor uses distinct adaptors, as well as common ones, to achieve that. The main characteristic shared by these proteins is their ability to undergo poly-ubiquitinylation in a non-degradative manner, leading to optimal NF-κB activation. In this work, we aimed to identify novel deubiquitinylating enzymes that control ubiquitinylation status. That is how USP34 came up to be a negative regulator of NF-κB signaling in TCR-activated Jurkat cells, a T lymphocyte cell line. Our data suggest a model whereby USP34 prevents excessive NF-κB activation by acting rather late, directly or indirectly on the NF-κB:IκBα dimers, downstream of IKK, altering transcription factor DNA binding affinity. In parallel, studies of the endocellular membrane microenvironment that hosts mature signalosomes in response to TCR-, TNFR- and CD40 ligation led to the identification of an ER-residing protein, Metadherin (MTDH), which seems to globally integrate signaling before forwarding it to downstream pathway components able to activate IKK.

La protéine Damaged DNA-Binding 2 (DDB2) est connue pour son rôle dans la réparation de l'ADN lésé par les UV. Cependant, le laboratoire a montré que cette protéine est surexprimée naturellement dans les cellules tumorales mammaires non métastatiques et active leur prolifération, en favorisant leur entrée en phase de transition G1/S du cycle cellulaire. Il a été montré que cette nouvelle activité biologique de DDB2 dépend de sa capacité à intervenir dans la transcription de gènes cibles, comme celui codant l'enzyme anti-oxydante, la superoxyde dismutase à manganèse (SOD Mn). Sur la base que DDB2 est peu ou pas exprimée dans les cellules tumorales mammaires métastatiques, ce travail a consisté à étudier le rôle de cette protéine dans les capacités invasives de ces cellules. Dans un 1er temps, nous avons montré que les cellules tumorales mammaires hautement métastatiques (MDA-MB231 et SKBR3), lorsqu'elles surexpriment DDB2 après introduction de son gène, ont des capacités migratrices et invasives in vitro, ainsi que des propriétés in vivo à développer des métastases pulmonaires, fortement réduites, en association avec une diminution importante de l'expression de la métalloprotéase matricielle 9 (MMP-9). De même, lors d'une analyse rétrospective sur 92 échantillons cliniques provenant de patientes, une corrélation inverse entre l'expression de DDB2 et le haut grade (SBR>ou =3) des tumeurs mammaires est observée. Dans un 2ème temps, nous avons identifié le mécanisme moléculaire par lequel DDB2 agit négativement sur les capacités invasives des cellules tumorales mammaires. Nous avons montré que DDB2 intervient positivement sur l'expression du gène codant I kappa B alpha (IkBa), en se fixant sur une séquence d'ADN localisée dans la région proximale du promoteur, qui entraîne en conséquence une forte diminution de l'activité du facteur de transcription NF-kB. Ce dernier est connu pour son rôle dans les capacités invasives et migratrices des cellules tumorales mammaires métastatiques, en régulant de nombreux gènes cibles comme celui codant la MMP-9. Nous avons montré, que l?inhibition de l'expression d'IkBa, par ARN interférence restaure en partie les propriétés invasives des cellules tumorales mammaires métastatiques surexprimant DDB2, en association avec une réexpression de MMP-9. Dans un 3ème temps, nous avons également montré dans les cellules tumorales mammaires métastatiques, que l?expression constitutivement élevée de la SOD Mn, en l'absence de DDB2, dépend de l'activité conjointe des facteurs de transcription NF-kB et Sp1, révélant ainsi un autre mécanisme moléculaire impliqué dans les propriétés invasives de ces cellules. L'ensemble de ce travail contribue ainsi à mieux comprendre comment les cellules tumorales mammaires progressent vers un statut invasif et renforce également l'idée que DDB2 présente un intérêt clinique potentiel, comme marqueur prédictif de la progression métastatique des tumeurs mammaires. Enfin, la relation entre la DDB2, NF-kB et la SOD Mn représente une voie intéressante pour le développement de nouvelles thérapies anticancéreuses.

Le virus T lymphotropique humain HTLV-1 est l'agent étiologique de la leucémie-lymphome T de l'adulte (ATLL) et de nombreuses maladies inflammatoires. HTLV-1 est associée à de nombreuses modifications quantitatives de l'expression des gènes cellulaires. À ce jour, ces modifications ont été décrites essentiellement à l'échelle transcriptionnelle à travers notamment les effets de l'oncoprotéine virale Tax, et plus récemment HBZ. Outre leurs impacts sur les niveaux d'activité des promoteurs, certains facteurs apparaissent jouer également un rôle dans la régulation de l'épissage alternatif. Ce mécanisme essentiel à la diversité du transcriptome et du protéome cellulaire, apparait étroitement couplé à la transcription et ses dérégulations sont de plus en plus décrites dans les phénomènes cytotoxiques et pathogènes tels que les infections et les cancers. Dans ce contexte, mon travail s'est intéressé à caractériser les profils d'expression des exons des cellules T CD4+ infectées ou non, et transformée ou non par HTLV-1 in vivo. Dans une seconde étude, j'ai abordé les aspects mécanistiques des modifications d'épissage alternatif par HTLV-1. Mes données montrent que, outre ses effets sur la régulation quantitative de l'expression des gènes cellulaires, l'activation de la voie NF-kB par l'oncogène Tax est impliquée dans la reprogrammation de l'épissage alternatif de nombreux gènes. Ces données révèlent un nouveau degré de complexité dans les mécanismes de dérégulation de l'expression des gènes cellulaires par HTLV-1 et ouvre de nouvelles perspectives d'investigations dans la compréhension des processus leucémogènes associés à l'infection par le virus HTLV-1

Thesis (Ph.D. (Biochemistry)) -- University of Limpopo, 2021 / Introduction: Rift Valley fever virus (RVFV) is an arthropod-born RNA zoonotic virus causing Rift Valley fever (RVF) disease. RVFV is prevalent across sub-Saharan Africa and the Arabian Peninsula with no existing effective and approved antiviral remedies for humans or animals. RVFV has developed mechanisms to hide from immune recognition and induce anti-apoptosis processes to keep the infected host cells viable in an attempt to advance their viral progeny. RVFV is a single-stranded enveloped RNA genome virus composed of 3 segments; the L, M and S segments. The S segment is known to encode a non-structural protein (NSs) identified to be the main virulence factor promoting viral replication through immune suppression. RVFV elicits a set of diverse symptoms ranging from a febrile illness to more severe symptoms that usually culminate in life-threatening haemorrhagic fever with high fatality rates. Thus, this study was designed to investigate the efficacy of lithium as a potential drug for reduction of RVFV load and amelioration of imbalanced and dysregulated inflammatory responses observed in Huvec and Raw 264.7 macrophages infected with this virus. Methods and results: The MTT and Cyquant viability assays were used to demonstrate that lithium exerts no cytotoxic effects on non-infected Raw 264.7 macrophage cells but rather promotes cell growth and proliferation. Conversely, lithium was shown to significantly induce cell death in RVFV-infected Raw 264.7 macrophages. The Annexin-V/PI apoptosis assay was employed to demonstrate that RVFV induces apoptosis as a mode of cell death on Raw 264.7 cells. RVFV-induced apoptosis was accompanied by antagonistic Bax/Bcl-2 protein expression ratios. RVFV-infected cells treated with lithium resulted in higher levels of apoptosis signals compared to untreated RVFV-infected cells. Analysis of apoptosis stages using the real-time cell analyser (RTCA) also revealed that lithium induced early forms of apoptosis in RVFV-infected cells. Interestingly, induction of early apoptosis in these cells corresponds with lower viral load, probably as a result of early inhibition of viral progeny replication, as determined using viral titration assay. Immune response profiles elicited in Raw 264.7 macrophages infected with RVFV and treated with lithium were monitored. An ELISA assay was used to determine the effect of lithium on cytokines and chemokine production in this cell model. The results obtained showed that lithium significantly stimulated production of IFN-γ as RVFV-infected lithium-treated cells produced high levels of IFN-γ compared to lithium-free RVFV-infected control cells. Furthermore, in the same setting, the secondary pro-inflammatory cytokine, IL-6, and chemokine, RANTES, were stimulated by lithium 12 hrs post-infection (pi). Lithium was shown to significantly stimulate TNF-α production as early as 3 hrs pi. In addition to TNF-α expression, the expression of the regulatory cytokine, IL-10, was significantly stimulated by lithium with the highest expression peak at 12 hrs pi. As determined using the H2DCF-DA and DAF-2 DA florigenic assays, reduced production of the ROS and RNS was observed in RVFV-infected lithium-treated cells as opposed to untreated RVFV-infected controls. This was further supported by the Western blot assay results that showed low expression of the iNOS while upregulating expression of heme oxygenase and IκB in RVFV-infected lithium-treated cells. Results from immunocytochemistry and Western blot assays revealed that lithium inhibits NF-κB nuclear translocation in RVFV-infected cells compared to lithium-free RVFV-infected cells and 5 mg/ml LPS controls. This study hypothesises persistent and deregulated inflammation as the central phenomenon responsible for endothelial damage and haemorrhagic fever in RVFV pathogenesis. Supernatants were collected from RVFV-infected macrophage cells treated with lithium and their effects on the integrity of endothelial cells were evaluated. The xcelligence real-time cell analyser system (RTCA) and transwell assay that measure endothelial monolayer integrity were used to demonstrate that lithium protects endothelial cells from RVFV-induced cellular damage. Moreover, lithium was shown to upregulate expression of cytoplasmic molecules such as α and β-catenins involved in attaching the cadherin molecules to the actin cytoskeleton on the endothelial cell. Expression of α-catenins, talins, zyxins and vinculins that attach integrins to the extracellular matrix and to other cells were observed to be upregulated by supernatants from RVFV-infected Raw 264.7 macrophage cells treated with lithium. Endothelial cell monolayer exposed to supernatants from RVFV-infected lithium-treated Raw 264.7 cells displayed upregulated expression of transmembrane molecules such as E-cadherins and N-cadherins. However, expression of VE-cadherins was observed to be lower compared to those treated with supernatants from lithium-free RVFV-infected Raw 264.7 control cells. Conclusion: These findings propose that lithium limits viral replication and viral load in macrophages by inducing early apoptosis in RVFV-infected cells. Since lithium was shown to promote Raw 264.7 macrophage proliferation, it is thus suggested that the use of lithium as an RVFV antiviral drug is less likely to elicit leukocytopenia. Lithium seems to regulate excessive inflammation in RVFV-infected Raw 264.7 macrophages by modulating the NF-kB signalling pathway. The endothelial integrity observed in the permeability assays has been supported by the expression of the molecules involved in keeping the cell to cell adhesion intact. This study links endothelial integrity patterns exerted by lithium with lowered production of inflammatory mediators such as ROS and RNS as these molecules are involved in destabilisation of cell junctions. Results from this study point towards the use of lithium as a potential treatment for RVFV infections by limiting viral replication, restricting viral spread and restoring the inflammation-regulating machinery. Key words. Lithium, Rift Valley fever virus, NF-kB, endothelial integrity, inflammation and apoptosis / Poliomyelitis Research foundation and National Research Foundation

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19141号 / 生博第324号 / 新制||生||43(附属図書館) / 32092 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 米原 伸, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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