Rift Valley fever virus (RVFV) is a negative-sense RNA virus that is classified as an overlap select agent by the USDA and the HHS. It was first discovered in the Rift Valley of Kenya in the early 1930s. RVFV is an arbovirus that is transmitted by mosquitoes and infects ruminants and humans. RVFV in humans causes an acute self-limiting febrile illness but in a small percentage of cases, a severe version is noted by ocular disease, hepatitis, hemorrhagic fever, and death. In ruminants, the disease is similar with young livestock being the most susceptible. RVFV is also known to cause "abortion storms" where infected pregnant ruminants abort their fetuses with a near 100% fatality rate. Viruses are obligate intracellular parasites utilizing host-factors to replicate. This study identified three host-protein interactors of the viral Gn and L proteins that aid in viral replication. UBR4 was determined to be an interactor of Gn via immunoprecipitation followed by either LC/MS/MS or western blot analysis. Its inhibition via siRNA or CRISPR-Cas9 knockout showed a reduction of viral titers and viral RNA production. It was determined that UBR4 specifically affects viral RNA production and not entry or egress. Conversely, CK1α and PP1α were identified as binding partners of the L protein using similar methods. CK1α, a kinase, and PP1α, a phosphatase, were chosen for further verification due to data demonstrating the L protein is phosphorylated on at least one serine residue, in addition to PP1α already being shown to impact RVFV replication. Inhibition of CK1 and PP1 via small molecule inhibitors, D4476 and 1E7-03, respectively, showed a decrease in viral titers and RNA production. Strand-specific RT-qPCR demonstrates that CK1 and PP1 impact genomic replication. Upon treatment with D4476 a decrease in L protein phosphorylation was observed. Additionally, it has already been shown that treatment with 1E7-03 increases L protein phosphorylation. These data indicate that CK1 and PP1 modulate L protein phosphorylation, contributing to changes in RVFV replication. This study identifies three host-proteins that affect viral replication, which could be used as a foundation for host-based therapeutic and vaccine development. / Doctor of Philosophy / Rift Valley fever virus (RVFV) is a major biological threat due to its ability to infect both livestock and humans and be passed by mosquito bite. RVFV was first discovered in Africa in the early 1930s. To date, there is no approved therapeutic or vaccine. RVFV usually causes very mild disease but in a small percentage of cases, it progresses to include liver disease, vision loss, swelling of the brain, bleeding, and death. A virus itself is not alive; it needs a living host in order to replicate. To do this, it utilizes things naturally occurring inside the host. The purpose of this study is to identify host-factors that the virus uses in order to efficiently make more viruses. The first viral protein of interest is the glycoprotein, Gn, which is important for viral entry and assembly of the viral particles. It was determined that the host-protein UBR4 is an interactor of Gn and that the inhibition of UBR4 decreases the amount of infectious virus being produced. Similarly, the host-proteins, CK1α and PP1α, were found to be interactors of the viral L protein. The L protein is responsible for synthesizing the building blocks of the virus. It was determined that when CK1 and PP1 are inhibited, the L protein is less efficient at making these building blocks. Understanding the host-factors the virus utilizes is important to the basic understanding of how RVFV infects the host and the development of therapeutics to combat an outbreak.

No / Objectives It is hypothesised that endothelial cell reactive antibodies (ECRA) play a role in the progression of PAD through activation of endothelial cells and the release of inflammatory cytokines. We aimed to test this hypothesis by assessing levels of ECRA, E-selectin and IL-6 in patients with PAD of varying severity in a case controlled study. Design, materials, methods Patients were assessed clinically and with ankle¿brachial pressure indices. Patients with critical ischaemia (CI, n=30), stable claudicants (SC, n=30), and age-matched controls (AMC, n=20) were studied. Antibody, E-selectin and IL-6 levels were measured using ELISA. Results ECRA levels were significantly raised in the CI group over AMC. IL-6 levels were significantly elevated in both SC and CI over the control group and in CI over SC. There were no significant differences in E-selectin levels between the AMC, SC and CI. Conclusion Our findings support the hypothesis that autoantibodies play a role in promoting PAD by elevating IL-6. The absence of an elevation in E-selectin in this study may be due to its short half-life, and casts doubt on its value as a marker of inflammation in atherosclerosis.

BACKGROUND:Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide.METHODS:To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS).RESULTS:3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters.CONCLUSIONS:The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-kappabeta and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures. A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides. Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated. Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.

Herpesviruses can enter host cells by pH-dependent endocytic pathways in a cell-specific manner. The role of pH in herpesvirus endocytosis is unclear. Herpes simplex virus (HSV) is a paradigm for virus membrane fusion via a complex of glycoproteins. HSV glycoproteins B, D and the heterodimer H-L are necessary and sufficient for membrane fusion. This work analyzes the structure and function of HSV glycoproteins B, D, and H-L at neutral pH, and at the physiological low-pH encountered during endocytic entry. It is demonstrated that mildly acidic low pH triggers specific conformational changes in HSV gB at a pH of 5.7 to 6.0. The antigenic structure of gB functional region I that is critical for fusion is specifically altered by mildly acidic pH both in vitro and during entry into host cells. Point mutations within gB functional region 1 that block membrane fusion still allow conformational changes in region 1. This suggests that specific hydrophobic residues are essential for fusion domain insertion into the host cell membrane but not conformational change. The detected conformational changes were reversible, similar to other class III fusion glycoproteins. Exposure to mildly acidic pH directly triggered the fusion function of HSV glycoproteins and caused gB, but not other glycoproteins, to become more hydrophobic. The oligomeric conformation of gB is altered at a similar pH range. In addition, several approaches were used to monitor gB throughout glycoprotein synthesis and maturation. It is shown that gB may cotranslationally fold and oligomerize as it is synthesized on the ribosome. As gB matures it then alters conformation and/or binding partner to form antigenically distinct populations of gB within the cell and virion. I conclude that intracellular low pH induces changes in gB conformation that, together with additional triggers such as receptor-binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.

La problématique de la neutralisation par les anticorps constitue un enjeu majeur dans la perspective de la conception d’un vaccin efficace contre le VIH et les connaissances récemment acquises conforte NT l’absolue nécessité de maintenir une recherche cognitive fondamentale sur le sujet. L’un des objectifs de ce travail de thèse a été de documenter les propriétés biologiques en terme de sensibilité à la neutralisation de variants présents chez certains patients asymptomatiques à long terme (ALT) et présentant des caractéristiques particulières. Nous avons pu identifié certains déterminants moléculaires associés à la sensibilité ou à la résistance à l’anticorps monoclonal 2G12 tels le site potentiel de glycosylation (PNGS) en position N302 et la longueur de la boucle V1V2 du gène env. Nous avons ensuite caractérisé la relation entre l’évolution du gène env et la sensibilité à la neutralisation dans un contexte d’évolution tardive chez un patient ALT. Ces travaux ont permis de mettre en évidence une poursuite de l’évolution du gène env plus de 10 ans après l’infection et ceci malgré la présence d’anticorps largement neutralisants et d’une réponse autologue croissante au cours du temps. Le contexte de la transmission mère enfant (TME) constitue un modèle de choix afin d’étudier le rôle des anticorps neutralisants. Afin d’identifier d’éventuels corrélats de protection, mon travail a consisté à étudier la réponse neutralisante dans une population de 114 couples mères-enfants. Nous avons pu confirmer que le spectre de neutralisation des sérums maternels n’était pas associé à une moindre TME du VIH-1, mais que les anticorps neutralisant certains isolats pourraient constituer des indicateurs d’intérêt associé à un moindre risque de transmission. L’ensemble de ces travaux souligne à nouveau la complexité et la pertinence à poursuivre les investigations relatives à l’identification d’éventuels corrélats de protection. / Basic research on neutralizing antibodies. still remains relevant in term of HIV vacccine development. One of the aim of this thesis was to document the neutralization sensitivity of particular HIV-1variants from long term non progressor (LTNP) patients. We first identified molecular signatures associated with sensitivity to 2G12, such as a potential N-linked glycosylation site (PNGS) at N302 and a longer V1V2 loop of gp120. We also studied the relationship between long-term evolution of the virus and neutralization sensitivity in a LTNP patient. We showed that HIV-1 may continue to evolve in presence of both broadly neutralizing antibodies and increasing autologous neutralizing activity more than 10 years post-infection. Mother-to-child transmission provides a natural model for studying the role of neutralizing antibodies. In previous studies, we showed that the presence or high titers of neutralizing antibodies against a CRF01_AE strain, MBA, was associated with a lower rate of HIV-1 intrapartum transmission in Thailand (Barin et al., 2006; Samleerat et al., 2009). In order to confirm this observation and to identify potential correlates of protection in the MTCT context, we examined the breadth and levels of neutralizing antibodies in 57 transmitting and 57 non-transmitting untreated HIV-1 infected mothers. Our study confirmed that the breadth of maternal neutralizing antibodies was not associated with protection of infants from infection, but that neutralizing antibodies to particular strains might be associated with a lower rate of MTCT of HIV-1.

Le VIH-1 est l'agent infectieux qui cause le SIDA. De nombreuses thérapies existent pour combattre le SIDA mais aucune ne permet son éradication et des résistances apparaissent. Le développement de nouvelles thérapies est donc nécessaire. Les anticorps simple-domaine (sdAb) de lamas présentent les propriétés idéales pour le développement de molécules neutralisantes. Des lamas ont donc été immunisés avec Vpr et les formes native, ou induite par un miniCD4, du trimère de gp140 (partie extracellulaire de l'enveloppe (Env)). Des banques de sdAbs ont ensuite été construites et des sélections par phage display et par double hybride ont été réalisées. Trois sdAbs se liant au site de liaison du co- récepteur de l'Env et un sdAb se liant au site de liaison du CD4 ont ainsi été sélectionnés. Ces sites sont conservés mais difficile d'accès pour des immunoglobulines conventionnelles. Ces sdAbs ont ensuite été caractérisés par ELISA, SPR et cytométrie de flux pour leur capacité de liaison à différentes Env, et en « single round assay » pour leur capacité de neutralisation d'un large spectre (LS) de pseudovirus. Des protéines multidomaines (plusieurs sdAbs reliés par un linker) ont ensuite été construites et testées pour leur neutralisation. Plusieurs de ces molécules, neutralisant un LS de virus, pourraient être utilisées dans des microbicides. La stabilité caractéristique des sdAbs, même en absence de formation de pont disulfure, par exemple dans un environnement réducteur tel que le cytoplasme, est primordiale dans le développement d'anticorps intracellulaires (intrabodies). / HIV-1 is the infectious agent of AIDS. Numerous therapies exist to fight AIDS, but they are not able to eradicate it, and resistances appear. So, new therapy development is necessary. Single-domain antibodies (sdAb) of llamas have ideal properties to develop neutralizing molecules. So, llamas have been immunized with Vpr and with free or miniCD4 induced trimeric gp140 (extracellular part of the envelope (Env)). SdAb libraries have been built and selections were done by phage display and yeast two hybrid. Three sdAbs targeting the co-receptor binding site of the Env and one sdAb targeting the CD4 binding site have been selected. These sites are conserved but inaccessible by conventional immunoglobulins. These sdAbs have been characterized by ELISA, SPR and FACS for their ability to bind different Env and by single-round assay for their neutralization ability. Multimeric proteins (linked sdAbs) have been built and tested for their neutralization ability. Several of these molecules are able to neutralize a broad spectrum of pseudoviruses. They can be used in microbicides. The characteristic stability of these sdAbs, even without disulfide bound formation, ie into reducing environment, as the cytoplasm, is primordial for intracellular antibody (intrabody) development. One sdAb anti-Vpr has been selected using the Sos Recruitment System (SRS), an yeast two-hybrid system allowing detection of cytoplasmic protein-protein interactions. This sdAb is able to alter the localization of its antigen into eukaryotic cells. It is a proof of concept ot the use of SRS in the selection of intracellularly functional sdAbs.

5 Anglický abstrakt Experimental autoimmune encephalomyelitis (EAE) is widely accepted as a murine model of human multiple sclerosis autoimmune disease. Murine EAE is usually actively induced by immunization with a suitable myelin antigen. Following immunization, CD4+ T helper lymphocytes Th1 and Th17 accumulate in the nervous tissue and via the production of cytokines, they mediate an inflammatory reaction and the subsequent destruction of myelin. The main goal of this study was the induction of EAE with clinically observable symptoms and the observation of changes in the counts and phenotypes of cells, mainly NK and T cells. NK cells express a wide range of inhibitory and activation receptors from the C-lectin-like receptor superfamily. The specific ligand of the activating NKR-P1C isoform is still unknown and thus this receptor's involvement in EAE was also observed. Another goal was the use of medication with regard to the disease progress improvement. For the purposes of this study, two inbred murine strains with distinct NKR-P1 surface expression were used - the SJL/J strain (expressing inhibitory NKR-P1B) and C57BL/6 (expression activating NKR-P1C). SJL mice elicited a relapse-remitting of EAE, while C57BL/6 had chronic EAE. Both mouse strains exerted changes in the counts of NK...

NG2 is a transmembrane glycoprotein mainly expressed in developing tissue, and often re-expressed in tumor cells. NG2 glycoprotein is an important regulator of cell migration and adhesion. Increased expression of NG2 enhances the metastatic potential of cancer cells. However, the molecular mechanisms of these processes are still not fully understood. An increasing number of evidences, in recent years, have shown that NG2 can be responsible for Rho/ROCK activation, which is essential for effective amoeboid invasiveness. In this thesis, we analysed the role of NG2 glycoprotein, especially the role of its PDZ- binding motif on amoeboid phenotype induction, and activation of Rho/ROCK signaling. Our results demonstrate the importance of the NG2 PDZ-binding motif on mesenchymal- amoeboid transition of cells in a 3D environment. Surprisingly, they show that the expression of both the NG2 cytoplasmatic domain and the truncated version, lacking the PDZ-binding motif, do not change the amount of Rho-GTP or the activation of the Rho/ROCK signaling pathway in 2D.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Radek Machan Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Determination of permeability and active transport of selected butyrylcholinesterase inhibitors in vitro European Medicine Agency (EMA) and Food and Drug Administration agency (FDA) emphasise drug membrane permeability and drug-drug interactions on ABC transporters expressed in physiological barriers should be investigated for compounds in preclinical studies or for those already clinically used but evidence free. In this work we aimed to assess the capability of several experimental butyrylcholinesterase inhibitors that had been designed to treat dementia to permeate blood-brain barrier and to elucidate role of ATP-binding (ABC) cassette transporters in this transport. For this purpose, we employed in vitro bidirectional transport study across monolayers formed by polarized and highly differentiated Caco-2 cells. The permeability values gained from measurements were similar to values of several commonly used drugs for treatment of CNS disorders (e.g. antidepressants, antiepileptics). In addition, the compounds showed values of efflux ratio (basolateral- to-apical/apical-to-basolateral) approximately one...