No description available.

No description available.

No description available.

No description available.

Rapid production of type I Interferon is pivotal to initiate cellular antiviral host defense and adaptive immunity. In order to facilitate innate immune processes, a cell harbors pattern recognition receptors (PRRs) which sense distinctive forms of pathogen associated molecular patterns (PAMPs). For example, Toll like receptors (TLRs) and RIG-I like receptors (RLRs) were discovered as PRRs for pathogen derived molecules and the production of type I Interferon (IFN). To induce type I IFN, several transcription factors such as nuclear factor-kappaB (NF-ĸB), interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), and activating protein-1 (AP-1) need to be stimulated through the specific signaling adaptors. Among them, our lab is interesting in the death domain (DD) containing proteins Receptor interacting kinase1 (RIP1) and Fas-associated death domain protein (FADD), which we showed were important for innate signaling processes. RIP1 and FADD were initially identified as Fas and TNFR interacting proteins which were involved in death receptor mediated apoptosis. Aside from apopotic function, recent publications indicate that RIP1 and FADD mediate cell survival, proliferation, and cytokine production through NF-ĸB activation. Here, we show that RIP1 and FADD are essential for efficient TLR-independent signaling. We report that RIP1 and FADD lacking MEF cells are sensitive to viral cytolysis and also exhibit impaired IFN production against dsRNA virus infection. RIP1 acts as a scaffolding protein for death receptor mediated apoptosis and NF-ĸB activation, necrosis, and innate immunity. As mentioned, we demonstrate that cells lacking RIP1 are sensitive to RNA virus infection. To understand the detailed mechanisms of RIP1 function in innate signaling, we first tested whether RIP1 is involved in RIG-I signaling. We found that RIP1 forms a complex with RIG-I in the presence of dsRNA. Additionally, we showed that RIP1 is required for optimal RIG-I and melanoma differentiation-associated protein 5 (MDA-5) activity. We also find that FADD, a RIP1 interaction protein, is implicated in innate immunity. To study the precise mechanisms of FADD in type I IFN signaling, we generated FADD variants and used luciferase reporter assays to indicate that the FADD death effector domain (DED) is crucial for IFN-β signaling. In order to identify interacting partners of FADD, yeast two hybrid assays were performed and indicated that FADD binds to protein inhibitor of activated STAT (PIAS1), part of the SUMO machinery. SUMOylation is a reversible post-translational modification of a protein by SUMO, a 100 amino acid protein. The consequence of SUMOylation alters specific proteins’ function by affecting activity, localization, stability or influencing molecular interactions by interfering with or linking to a target protein. To confirm FADD-PIAS interactions, we conducted in-vitro SUMOylation assays by using Ubc9 conjugated FADD and found possible FADD SUMOylation sites. We also discovered that FADD and SUMO are co-localized in the nucleus. This result reveals that FADD undergoes SUMOylations and its modification might regulate FADD’s function, including role in innate signaling. Furthermore, we report here that HTLV-1 Tax protein interacts with RIP1 and inhibits IFN-β inducing signaling by abrogating RIP1 and IRF7 interaction. This implies that RIP1 is involved in the regulation of IRF7 and is essential for IFN-β production. Collectively, our data demonstrate the significance of RIP1 and FADD in dsRNA recognition pathways in mammalian cells that are essential for the optimal induction of type I IFNs and other innate genes important for host defense.

Les anticorps monoclonaux (AcM) sont désormais considérés comme une alternative thérapeutique crédible pour traiter les infections virales graves. Comprendre leurs multiples mécanismes d’action est donc crucial pour améliorer leur effet thérapeutique. En utilisant un modèle d’infection virale chez la souris (leucémie induite par le rétrovirus FrCasE), l’équipe a montré qu’une immunothérapie courte par un AcM neutralisant induisait une immunité antivirale protectrice sur le long-terme (effets « vaccinaux ») qui est dépendante du fragment Fc de l’AcM. Ainsi, des immuns complexes (IC) formés à partir de l’AcM thérapeutique et de déterminants viraux, induisent l’activation de cellules immunitaires, notamment les cellules dendritiques (DCs), via leur interaction avec les FcRs exprimés à la surface des cellules. Cependant, ces interactions IC-FcR peuvent également concerner d’autres cellules du système immunitaire outre que les DCs, telles que les macrophages, monocytes ou bien encore les neutrophiles, qui expriment elles aussi les FcRs à leur surface et ce de façon différentielle. Dans ce contexte, il est important d’identifier quels FcRs et quelles cellules les exprimant sont essentiels à l’induction des effets vaccinaux par les AcM. C’est pourquoi mes travaux thèse se sont focalisés sur l’étude du rôle des neutrophiles et des FcγRs dans la modulation de la réponse immune par les AcM. Cette étude repose sur le caractère Fc-dépendant de l’induction d’une réponse immune protectrice par les AcM ainsi que sur les propriétés immunomodulatrices des neutrophiles, qui ont été décrites dans différents contextes pathologiques mais jamais étudiées dans le cadre d’une immunothérapie antivirale par AcM. Pour cela, j’ai utilisé différentes approches in vitro, ex vivo et in vivo.En utilisant le modèle d’infection par FrCasE, il a été montré que les neutrophiles ainsi que le FcγRIV ont un rôle crucial dans l’induction des effets vaccinaux par les AcM, notamment via l’induction d’une réponse humorale antivirale endogène protectrice à très long-terme. De plus lors d’expériences in vitro, il a également été souligné que les neutrophiles sont plus efficacement activés par les IC comparé au virus seul et que différentes cytokines pro-inflammatoires et/ou immunomodulatrices (telles que le TNF et les intérferons de type I et II) potentialisent l’activation des neutrophiles induite par les IC. Mes travaux ont aussi mis en évidence que l’infection virale et l’immunothérapie modulent l’expression des FcRs, et notamment induisent la surexpression du FcRIV sur deux populations distinctes de neutrophiles (différentiées par le niveau d’expression du marqueur de surface Ly6G: Ly6Ghi et Ly6Gint) et sur les monocytes inflammatoires. Enfin, mes travaux montrent que l’immunothérapie par AcM module les profils de sécrétion chimiokinique et cytokinique de ces 3 types cellulaires surexprimant le FcRIV, bien que la nature des profils de sécrétion varie en fonction du type cellulaire et évolue au cours du temps. Ces résultats suggèrent que l’effet immunomodulateur des AcM repose sur l’activation de différents acteurs de la réponse immunitaire précoce, en induisant la sécrétion de chimiokines et de cytokines nécessaires à l’orchestration de la réponse immune. Ils suggèrent aussi une coopération entre ces différents acteurs dans la mise en place d’une immunité protectrice.Pour finir l’ensemble de mes travaux ont mis en évidence un rôle immunomodulateur clé du FcyRIV, ainsi que des différentes cellules l’exprimant, dans l’induction d’une réponse immune protectrice induite par des AcM antiviraux. Ces révélations pourraient avoir des conséquences importantes dans l'amélioration des immunothérapies à base d'AcM. / Monoclonal antibodies (mAbs) are now considered as a true therapeutic alternative for treating severe viral infections. Figure out their multiple mechanisms of action is therefore crucial to improve their therapeutic effect. Using a mouse model of viral infection (the FrCasE retrovirus-induced leukemia), the team showed that a short immunotherapy with a neutralizing mAb induces long-term protective antiviral immunity ("vaccine" effects) which is Fc-dependent. Notably, immune complexes (IC) formed with therapeutic mAbs and viral determinants induce the activation of immune cells, especially dendritic cells (DCs) via their interaction with FcγRs expressed on the cell’s surface. However, IC-FcγR interactions can involve different cells of the immune system in addition to DCs, such as macrophages, monocytes or neutrophils, which differentially express FcγRs. In this context, it is important to identify which FcγRs and which FcγR-expressing cells are crucial in the induction of vaccine effects induced by mAbs. It’s the reason why my thesis work has focused on the study of the role of neutrophils and FcγRs in the modulation of immune response by mAbs. This study is based on the Fc-dependent nature of the induction of a protective immune response by mAbs and the immunomodulatory properties of neutrophils, described in different pathological situations but never studied in an mAbs antiviral immunotherapy context. To this end, I used different approaches in vitro, ex vivo and in vivo.By using the FrCasE infection model, it has been shown that neutrophils as well as FcγRIV have a crucial role in the induction of vaccine effects by mAbs, notably via the induction of a long-term protective antiviral humoral response. Moreover the in vitro experiments, highlighted that neutrophils are more effectively activated by IC compared to virus alone and that different pro-inflammatory and/or immunomodulating cytokines (i.e.TNFα and type I and type II interferons) potentiate the activation of neutrophils induced by IC. My work also revealed that viral infection and immunotherapy modulate the expression of different FcγRs, and notably they induce the overexpression of FcγRIV on two distinct populations of neutrophils (differentiated by their expression levels of the Ly6G surface marker: Ly6Ghi and Ly6Gint) and inflammatory monocytes. Finally, my work shows that immunotherapy with Mab modulates the chemokinic and cytokinic secretion profiles of these 3 FcγRIV-over-expressing cell, although the nature of the secretion profiles differs according to the cell type and evolves over time. These results suggest that the immunomodulatory effect of mAbs is based on the activation of different actors of the early immune response by inducing the secretion of chemokines and cytokines necessary for the orchestration of the immune response. They also suggest a potential cooperation between these different actors in the establishment of protective immunity.Altogether, these results show a key immunomodulator role of FcγRIV as well as of different cells expressing it in the induction of a protective immune response by antiviral mAb. They might have important consequences for the improvement of Mab-based immunotherapies.

ISG15 is an interferon-induced and anti-viral ubiquitin-like protein (Ubl). Ube1L, UbcH8, and Herc5 have been identified as the E1-E2-E3 enzymes for ISG15 conjugation, and, like ISG15, their expression is induced by type I interferons. Although Herc5 is the major E3 for ISG15, over 300 proteins have been identified as ISG15 target proteins in interferon-stimulated cells. In this work, I address two aspects of the human ISG15 conjugation system: 1) the specificity of the Ube1L-UbcH8 interaction and 2), the basis of substrate recognition by Herc5. Regarding the selection of UbcH8 by Ube1L, my experiments show that although UbcH8 had been reported to function as an E2 for both Ub and ISG15, UbcH8 is preferentially activated by Ube1L compared to Ube1 (E1[superscript Ub]). The basis of this preference is a result of specific interactions between the ubiquitin-fold domain (UFD) of Ube1L and the amino-terminal [alpha]1 helix and [beta]1 [beta]2 region within UbcH8. Examination of the interferon-induced and transfected expression levels of UbcH8, combined with the kinetic constants, suggest that UbcH8 is unlikely to function as a Ub E2 in most cell lines. In examining the selection of target proteins by Herc5, I show that the range of substrates extends far beyond the proteins identified in proteomics studies and includes many exogenously expressed foreign proteins. Furthermore, I show that ISG15 conjugation is restricted to newly synthesized pools of proteins and Herc5 is associated with polyribosomes. I propose a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a co-translational manner and suggest that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, I show that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses. These discoveries have greatly increased our understanding of the mechanism of ISG15 pathway and provide a framework for establishing an in vitro ISG15 conjugation system and further examination of the anti-viral function of ISG15. / text

Since the beginning of human civilization, medicinal plants have been used to treat a variety of infectious and non-infectious diseases. The therapeutic properties of phytochemicals have been recognized since ancient human history. The genus Helichrysum Mill. with its attractive flowers consist of an estimated 500‒600 species in the Asteraceae family. In South Africa and Namibia there are about 244‒250 species with tremendous morphological diversity. Several Helichrysum species are widely used by the indigenous population to treat various disorders such as wounds, infections, respiratory conditions, headaches, coughs, colds and fevers. Several of the Helichrysum species exhibit antiviral activity with the most relevant to this study being the discovery of anti-human immunodeficiency virus (anti-HIV) and anti-reverse transcriptase (anti-RT) activity of some species. Drug discovery and development, from the early stages of a promising compound to the final medication, is an intensive, expensive and incremental process. The ultimate goal is to identify a molecule with the desired effect in the human body and to establish its quality, safety and efficacy for treating patients. The ability to combine high-throughput analytical techniques like metabolomic and other experimental approaches with drug discovery will speed up the development of safer, more effective and better-targeted therapeutic agents. The rapidly emerging field of metabolomics and molecular docking analysis provides valuable information on drug activity, toxicity, customized drug treatments and can predict therapeutic outcomes. Extraction of the aerial parts of 32 Helichrysum species was done using polar [methanol (MeOH) 50%: distilled water (dH2O) 50%] and non-polar [hexane (Hex), dichloromethane (DCM) and acetone (Ace)] solvent systems. Anti-human immunodeficiency virus bioassays on the live HI virus revealed that polar extracts of H. mimetes and H. chrysargyrum at 2.5 μg/mL and 25 μg/mL, polar and non-polar extracts of H. infuscum at 25 μg/mL and polar and non-polar extracts of H. zeyheri, H. setosum, H. platypterum and H. kraussii at 2.5 and 25 μg/mL, had higher than 90% inhibitory activity. The polar extract of H. mimetes also exhibited reverse transcriptase (RT) inhibition as a possible indication of the mechanism of action. Proton nuclear magnetic resonance (1H NMR) spectra of the polar extracts exhibited the presence of aromatic compounds and carbohydrate moieties. Principal component analysis (PCA) of the polar extracts showed clustering related to the activity of the extracts with good predictability scores (Q2 > 0.5). However, orthogonal projections to latent structures discriminant analysis (OPLS-DA) predictability of the model was low based on the Q2 at approximately 0.25. Quinic acid (QA), isolated from H. mimetes showed promising anti-RT activity [50% inhibition concentration (IC50) = 53.82 μg/mL] which was comparable to the positive drug control, doxorubicin (IC50 = 40.31 μg/mL). The molecular docking study revealed the probable binding site and conformation of QA within cavity 4, with a docking score of -8.03. The docking score of doxorubicin within cavity 4 was -7.87. With this study, it was shown that metabolomic analysis as a tool to predict anti-HIV activity in Helichrysum species can be valuable to shorten the process. Moreover, the study of molecular docking revealed the mechanism action of quinic acid and doxorubicin against RT. / Thesis (PhD)--University of Pretoria, 2019. / Plant Production and Soil Science / PhD / Unrestricted

4’ -ester analogues of resveratrol were synthesized using reaction conditions developed to produce mono-ester products in the presence of two other unprotected phenols. Basic conditions were employed to deprotonate the most acidic 4’ phenol followed by addition of anhydrides or acid chlorides to give the ester product. The reaction favored 4’-ester formation in polar aprotic solvents with DMSO being the optimal solvent. (—)-Englerin A is a guaiane-type sesquiterpene containing two ester side chains. Mimics of (—)-englerin A were proposed that retained the ester side chains while replacing the non-polar core with less complicated structures. These proposed mimic cores would maintain the three-dimensional positioning of the esters which are responsible for the anti-cancer activity of (—)-englerin A. Three mimics were synthesized using the bicyclic terpenes borneol and fenchol. Installation of the second ester on the terpene core was accomplished throught the development and optimization of a unique methylene oxidation using chromium trioxide in glacial acetic acid. These mimics were screened against two kidney cancer cell lines. The compounds were shown to have IC50 (inhibitory concentration for 50 % of cells) values above 30 µM.

Healthcare-associated infections present an escalating worldwide issue, further intensified by the emergence of antimicrobial resistance and the spread of pathogens on surfaces. Current infection prevention methods have shown limited effectiveness, leading to several health issues, an overuse of antibiotics, and a continuous threat of surface recontamination. In response, extensive research has focused on bioinspired omniphobic smart coatings that effectively reduce the contact area available for pathogen attachment, achieved through an increase in surface roughness and apparent surface energy. This thesis introduces a new class of an omniphobic spray-coating, featuring hierarchical structured polydimethylsiloxane (PDMS) microparticles coated with gold nanoparticles, encompassing primary microscale (~0.23 𝜇m) and secondary nanoscale (~5 nm) buckyball and labyrinth wrinkles. This substrate-independent coating efficiently repels a wide range of liquids, including pathogens, even under harsh conditions like high temperatures, ultraviolet (UV) exposure, and abrasions. Repellency tests comparing coated and uncoated gloves revealed that uncoated gloves spread contamination to 50 secondary surfaces, while coated gloves transferred fewer bacteria and viruses to just three and two surfaces, respectively. The investigation extended to the coating's biocidal capabilities, incorporating gold nanoparticles functionalized with mercapto-silane to create a "Repel and Kill" coating. This process initiates chemisorption through thiol-gold bonding, allowing for the formation of diverse surface structures, including three-dimensional self-assembly, multilayers, and island structures. These modifications significantly enhance the roughness and hydrophobicity of the gold nanoparticles, amplifying their biocidal effectiveness. The wrinkled structure of PDMS contribute to repellency, while the functionalized gold nanoparticles play a crucial role in the antimicrobial property. This enhancement was evident in the antibacterial tests, which exhibited an immediate 99.90% reduction in bacterial adhesion for both MRSA and Pseudomonas aeruginosa (P. aeruginosa), followed by an additional 99.70% and 99.90% reduction in bacterial growth after 8 hours for MRSA and P. aeruginosa, respectively. Moreover, the coating's antiviral properties were confirmed, demonstrating a 98% reduction in the transfer of the bacterial virus Phi6. Recognizing the role of hospital fabrics as potential reservoirs for infection transmission, primarily due to their ability to sustain bacterial growth for extended periods, especially in the presence of bodily fluids, we took further steps to modify the wrinkled PDMS microparticles. This involved the incorporation of silver nanoparticles, capped with a positively charged ligand known as branched polyethyleneimine (bPEI). Additionally, we integrated a colorimetric sensor, giving rise to the "Repel, Kill, and Detect" smart coating. The transition of color from blue to green-yellow provided a tangible indicator of contamination detection based on the acidic mileu of the biofilms. To evaluate its realworld effectiveness, we conducted simulations of infection transmission in hospital environments, resulting in a remarkable reduction in pathogen adhesion from urine and feces by 99.88% and 99.79%, respectively, compared to uncoated fabrics. To further enhance the validation of our results, we employed a powerful deep learning network architecture, that determined whether the surfaces are contaminated or safe. In the face of evolving health challenges, this coating emerges as a resilient and adaptable solution, promising to enhance overall safety and alleviate the burden of infectious diseases. / Thesis / Doctor of Engineering (DEng) / The prolonged survival of pathogens on surfaces, significantly highlighted by the COVID-19 global pandemic, has intensified the urgency of addressing contamination on high-touch surfaces. Engineered surface coatings with repellent properties have emerged as a long-lasting and health-conscious solution for infection prevention and control. In this thesis, we introduce a new class of multifunctional engineered coatings featuring hierarchical structures adorned with biocidal nanoparticles and an integrated colorimetric sensor. We comprehensively investigate these coatings' multifunctional capabilities to repel, exterminate, and detect contaminants. Through specific characterization tests involving a wide range of pathogens, including viruses, bacteria, and fungi, within complex biological fluids like urine and feces, this research culminates in the development of surface coatings equipped with both antimicrobial and pathogen-sensing capabilities. In addition to advancing our understanding of surface hierarchy and chemical modifications for repellency and biocidal activity, this thesis yields insights into the dynamics of biofouling and pathogen transfer, with the overarching goal of reducing pathogen transmission via surfaces.