The RNA binding protein heterogeneous nuclear ribonucleoprotein L-Like (hnRNP LL) is known to regulate the alternative splicing of various physiologically important precursor messenger RNAs (pre-mRNAs). It undergoes a wide range of post-translational modifications (PTMs), including phosphorylation, ubiquitination and acetylation. However, the target amino acids and effects of these PTMs on the functions of hnRNP LL have not been characterized so far. In this study, we show for the first time that the endogenous hnRNP LL is phosphorylated upon depolarization. Using phosphopeptide mapping followed by the generation of a custom-made phospho-site specific antibody, we further show that phosphorylation at Ser308 of hnRNP LL is induced by depolarization though it is probably not the major phospho-amino acid target of depolarization/CaMKIV. The residue is critical for the nuclear localization and its phosphorylation essential for the CaMKIV-caused perinucleolar localization of the hnRNP LL protein in HEK293T cells. The residue is likely also critical in the regulation of nuclear functions like pre-mRNA splicing. / February 2016

Antimicrobial peptides are key actors in organisms€™ immune systems. They play an important role in phagocytosis, breaking bacteria membranes. They destroy the microbes, keeping them from repairing themselves, and therefore do not promote antimicrobial resistance. LL37 is a peptide produced by the human body. It is a short amino acid chain that is particularly active on the skin and mucous membranes. It has antimicrobial and fungal activity as well as wound healing properties, which makes it a very interesting active substance in wound treatment. However, its fragile and sensitive structure is a challenge to its use. Nowadays, encapsulation in a biocompatible polymer system is a promising technique in drug delivery, and presents a solution to LL37 administration and delivery. LL37 is a hydrophilic active substance, it will be trapped in PLGA (poly (lactic-co-glycolic acid)) by double emulsion and the microspheres will be shaped and stabilized by solvent evaporation. The capsules will be characterized by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy. Their main features, drug loading, encapsulation efficiency and release profile, are determined using the Bradford assay. Since the peptide is expensive and delicate, it is important to optimize its encapsulation. For that reason, we will adapt the process to have the best drug loading as possible using water in oil in oil emulsions. For an external use, the capsules would be used over a few days, so having a fast release is very relevant. The larger the specific surface area, the faster the diffusion. For that reason, we will also study the impact of porosity on the release profile. As a result, different types of capsules will be synthesized, with higher porosity and by two processes: aqueous double emulsion and oil double emulsion. Their characteristic features and impact on bacterial pathogens will be determined and compared in order to determine their optimal synthesis process and formulation in given conditions of use.

The work outlined herein presents an investigation of synthetic model complexes of relevance to the active sites of Ni(II)-containing enzymes, particularly urease, glyoxalase I, and acireductone dioxygenase. The research focuses on studying the structural and reactivity features of nickel complexes with biologically relevant substrates. The anion of acetohydroxamic acid is a well-known inhibitor of urease enzymes, including those isolated from Klebsiella aerogenes and Bacillus pasteurii. A precursor to the acetohydroxamate coordination in ureases is proposed to be an interaction between Ni(II) and acetohydroxamic acid. By using a novel supporting chelate ligand capable of secondary hydrogen bonding interactions a novel pseudo-octahedral, Ni(II) acetohydroxamic acid complex has been isolated and characterized. Detailed analysis of the structural features and acetohydroxamic displacement reactivity of this complex has provided fundamental chemical insight toward understanding of the inhibition mechanism in urease enzymes. Glyoxalase I (Glx I) catalyzes one step of the cellular detoxification pathway for α-ketoaldehydes (e.g. methylglyoxal) in humans and bacteria. The GlxI enzyme from E. coli is a Ni(II)-containing enzyme that catalyzes the isomerization of a hemithioacetal to produce a thioester. Of relevance to this enzyme, the first example of a Ni(II) complex that promotes a hemithioacetal isomerization is reported herein. In order to monitor this type of reaction a new approach involving a a deuterium-labeled hemithioacetal (PhC(O)CH(OH)SCD3) and 2H NMR was employed. Acireductone dioxygenases (ARDs) catalyze aliphatic oxidative C-C bond cleavage of an acireductone (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene) intermediate in the methionine salvage pathway. A unique aspect of these enzymes is that the regioselectivity of the dioxygenase reaction depends on the metal ion bound in the active site. Outlined herein are descriptions of the synthesis, characterization, and O2 reactivity of a novel trinuclear Ni(II) enediolate complex of relevance to the proposed enzyme/substrate adduct in Ni(II)-ARD. Efforts have also been made toward the preparation of C(1)-H acireductone compounds using a combined synthetic/enzymatic approach. A phenyl appended-C(1)-H acireductone was isolated and introduced to a Ni(II) precursor complex. This reaction produced spectroscopic changes consistent with the formation of a new Ni(II) acireductone complex. Preliminary studies of the O2 reactivity of this complex are reported.

Microbial infections are a global problem. Due to the over and misuse of antibiotics, drug-resistant pathogens are becoming more common. It is imperative to explore broad spectrum antimicrobial approaches. In this work, we modified collagen/hyaluronic acid polyelectrolyte multilayers (PEMs) with the natural antimicrobial peptide, LL-37 to study hepatic infections. LL-37 was physisorbed and covalently linked to the surface of the PEMs. Escherichia coli DH10B were cultured in the presence of LL-37modified PEMs in bacterial adhesion and contact killing models. Physisorbed LL-37 PEMs prevented bacterial adhesion and could also kill pathogens in the surrounding environment due to the release of LL-37 from the film. Immobilized LL-37 PEMs resulted in less bacterial adhesion on the surface due to the presence of the peptide. Films were then placed in contact with primary rat hepatocytes as well as in hepatocyte/bacteria co-cultures. LL-37 input concentrations up to of 16μM did not exhibit cytotoxic effects on hepatocytes. The LL-37 modified PEMs exhibited a hepatoprotective effect on albumin and urea secretion functions in co-cultures. The hepatoprotective effects were dependent on the ratio of hepatocytes and bacteria as well as the concentration of LL-37. These findings are encouraging and demonstrate that LL-37 modified PEMs can be used to investigate hepatic infections caused by bacteria. / Master of Science

Recurrent infection with human respiratory syncytial virus (RSV) is one of the most common causes for lower respiratory tract illness (LRI) in infants, the elderly, and immunocompromised individuals. Due to lack of vaccines and therapeutic interventions, medical care of acute RSV bronchiolitis is mostly limited to supportive measures. Thus, novel treatment options to control RSV infection are desperately required. The cationic host defence peptide human cathelicidin LL-37 possesses both microbicidal and immunomodulatory properties. This essential effector of the innate immune system holds potent antiviral activity against a variety of viruses, including influenza virus, and has been proposed as a promising candidate for antiviral drug development. Previous studies revealed that lower cathelicidin levels put RSV infected infants at risk for more severe RSV disease, while infection of lung epithelial cells induced cathelicidin up-regulation. These findings suggest that LL-37 might possess antiviral activity against RSV. However, its potential antiviral function on RSV remains to be elucidated. This thesis therefore aimed to evaluate the antiviral activity of cathelicidins against RSV, by assessing its relevance in vitro and in vivo and elucidating the underlying antiviral mechanism. Firstly, the antiviral effects of human cathelicidin LL-37 against RSV were addressed in vitro. Presence of LL-37 during infection potently reduced viral titres and protected cells against virus-associated cytopathic effects. Experiments revealed that only the core region of LL-37 holds antiviral activity against RSV. Antiviral effects were also observed for the murine LL-37 orthologue mCRAMP. Administration of LL-37 at different stages in the infection cycle provided evidence that LL-37 can be used preventatively, protecting against RSV infection by directly acting on both cells and viral particles. When given therapeutically, once an infection was established, LL-37 also limited viral spread. Next, the molecular mechanism mediating the peptide’s antiviral activity was investigated. It was demonstrated that LL-37 does not affect the interferon-mediated cellular antiviral immune response to RSV. Experiments established that LL-37 does not contribute to viral clearance by inducing epithelial cell death. Further mechanistic studies revealed that the peptide directly binds to RSV particles, destabilises the integrity of the viral envelope, and prevents adsorption of RSV to epithelial cells during the entry stage of infection. Finally, the in vivo relevance of LL-37 treatment and endogenous cathelicidin expression was examined, employing both murine and human model systems. It was established that LL-37 has protective antiviral effects against RSV in vivo. In contrast to the cell culture model, only co-administration of LL-37 and RSV, but not treatment prior or post infection, protects mice from clinical signs of infection. Levels of the murine LL-37 orthologue mCRAMP were increased in RSV infected lungs, pointing towards its importance in antiviral defence. In keeping with this, mCRAMP-deficient mice were more susceptible to RSV induced disease. Equally, individuals with low nasal LL-37 baseline levels that were experimentally challenged with RSV, were more susceptible to infection. This highlights the importance of endogenous cathelicidin expression to fight and control RSV infection. Overall, these results identify LL-37 as an important antiviral agent against RSV in vitro and in vivo, and emphasise the role of endogenous cathelicidins in the defence against this pathogen. Moreover, unravelling the underlying antiviral mechanism of LL-37 against RSV adds to our understanding of how CHDP act on enveloped viruses, thus supporting the development of new antiviral treatment options.

The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.

The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.

No description available.

The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.

The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.