Következő Generációs Analitikai Glikomika: Automatizálható Biomarker Kutatás

A glikoziláció biomarker potenciálja került bemutatásra ebben a tézisben, a haptoglobin és IgG glikoproteinekre fókuszálva. A haptoglobin glikozilációjának változásai már számos korábbi publikációban leírásra kerültek, mi viszont a glikánok elágazási és fukozilációs fokának változásaira fordítottunk kiemelt figyelmet. A talált eredmények megegyeznek a korábbi tanulmányokban leírtakkal minthogy emelkedett fukozilációs és elágazási fokot sikerült kimutatni a haptoglobin glikánjain a gyulladásos és daganatos tüdőbetegségekben. A válaszadók azonosítása rendkivül fontos olyan hosszan tartó költséges kezelések esetén mint a biológiai terápiák. RA-ben és CD-ben a jelenlegi pontozós rendszerek segítségével hónapokba telik a válaszadók azonositása. Az IgG glikoziláció köztudottan változik ezekben a betegségekben, mely magában hordozza a lehetőséget, hogy markerként hasznosítsuk. A CD páciensekben 2 héttel az anti-TNFα kezelés után sikerült kimutatni szignifikáns változást mely alapján megkülönböztethetők a válaszadók és nem-válaszadók. Ez az eredmény magában hordozza annak lehetőségét, hogy a jövőben az IgG glikozilációt esetleges biomarkerként használják ebben a betegségben. A jelenlegi glikánpreparálási technológiákat több napos inkubálás, manuális pipettázás és a vákum-centrifuga nélkülözhetetlensége jellemzi. Erre jelenthet megoldást az általunk újonnan kifejlesztett mágneses gyöngy alapú mintapreparálási eljárás mely mindössze 4 óra alatt képes akár 96 mintát elkészíteni vákumcentrifuga szükségessége nélkül és teljes mértékben automatizálható. Ez nagy mértékben megkönnyítheti a kutatók dolgát a jövőben, mindamellett megnövelheti a kísérletek precizitását és reprodukálhatóságát.The biomarker potential of glycosylation is presented in this work focusing on haptoglobin and IgG. Haptoglobin glycosylation was reportedly altered in inflammatory and malignant conditions. Fucosylation- and branching-degrees were examined and the importance of linkage-specific fucosylation-degrees was pointed out. The preliminary results were in agreement with the literature as increased branching and fucosylation was found in various pathological conditions. Prediction of patient response for any therapy is critical in inflammatory diseases such as CD and RA, as currently used scoring systems require months for responder identification. The importance of reliable biomarkers in these diseases is essential as the efficacy of biological therapies can vary between patients. Utilizing the high resolving power of CE-LIF, 26 IgG glycoforms were examined in RA and CD before and after anti-TNFα treatment. In RA, three low abundant galactosylated structures were found to be significantly different before the treatment and all responders showed higher galactosylation levels. No significant alteration was detected in RA in response to the treatment. In CD FA2G2S1 level was significantly increased in response to anti-TNFα therapy, thus being a possible candidate marker for responder identification. This finding was also supported by transcriptomics analysis of the corresponding glycosyl-transferase and glycosidase enzymes, as higher sialyl-transferase and lower sialidase activity was found. For reliable biomarkers huge number of samples has to be analyzed which necessitates automated high-throughput methods. A magnetic bead based protocol was developed for N-glycosylation analysis of glycoproteins not requiring hard-to-automate centrifugation and vacuum-centrifugation steps. Glycan release, APTS-labeling and clean-up were optimized resulting in a 4 hours magnetic bead based process with excellent yield and high reproducibility. The next step of this work is to apply this optimized magnetic bead based protocol with all steps from PNGase F digestion, through optimized fluorophore labeling and clean-up to high throughput sample processing in 96 well plates format with simple liquid handling robots allowing full automation.N

Nyelvtechnológiai módszerek a Budapesti Szociolingvisztikai Interjú lexikai és szintaktikai vizsgálatában

A dolgozat célja a Budapesti Szociolingvisztikai Interjú társalgási moduljainak lexikai és szintaktikai elemzése nyelvtechnológiai módszerekkel. Az elemzés a gépi eljárással annotált szövegeket elsősorban statisztikai módszerekkel vizsgálja. A BUSZI társalgási nyelvhasználatát a Magyar Nemzeti Szövegtárból vett minta segítségével az írott nyelvhasználat jellemzőivel veti össze. Ahol erre mód nyílik, a BUSZI2 által vizsgált társadalmi csoportok közötti lexikális és mondatszerkesztésbeli nyelvhasználati különbségeket is vizsgálunk

Mennyiségből minőséget : nyelvtechnológiai kihívások és tanulságok az MNSz új változatának elkészítésében

A Magyar Nemzeti Szövegtár egymilliárd szavas új változatának fejlesztése során egyrészt a szövegek mennyiségéből, másrészt a nyelvi elemzés minőségével kapcsolatos elvárásokból adódóan számos olyan feldolgozási kérdés merült fel, melyekre nem lehetett a jelenleg hozzáférhető nyelvi elemző eszközök „polcról levett” alkalmazásával kielégítő választ adni. A tanulmány azokat a megoldásokat és javaslatokat mutatja be, melyek hozzájárulnak ahhoz, hogy az olyan jelentős méretű korpuszokban is, ahol a manuális hibajavítás nem lehetséges, az annotáció minősége megfeleljen a felhasználói elvárásoknak

Cryopreservation of gander semen in cryovials – Comparative study

The aim of the study was to find a practical and inexpensive method for freezing goose semen for use in routine inseminations under farm conditions. Two basic freezing protocols [(1) dynamic, programmable freezing and (2) static, nitrogen vapour method] were evaluated with varying concentrations of dimethylformamide (DMF) plus additional osmoprotectants such as betaine, trehalose, and sucrose, using cryovials as containers. Altogether eight different treatments were compared. sperm viability before freezing and after thawing was examined by in vitro tests and, in the case of the simplest effective method, also by in vivo fertility test. There were no significant differences in sperm survival either in the dynamic (48–50%) or in the static protocol (43–46%), except for the treatment where the lowest DMF concentration was used without any osmoprotectant in the dynamic protocol (42.6%). The addition of osmoprotectants did not improve thawed sperm viability in any case. Fertility with frozen/thawed sperm using the simplest method was 58.5%, while that obtained with fresh, diluted semen was 66.9%. The study proved that the simple freezing of gander semen in nitrogen vapour with 9% DMF in cryovials could produce acceptable fertility. The newly elaborated method can be successfully used for routine inseminations by small- and large-scale goose breeders

Hard nut to crack: Solving the disulfide linkage pattern of the Neosartorya (Aspergillus) fischeri antifungal protein 2

As a consequence of the fast resistance spreading, a limited number of drugs are available to treat fungal infections. Therefore, there is an urgent need to develop new antifungal treatment strategies. The features of a disulfide bond-stabilized antifungal protein, NFAP2 secreted by the mold Neosartorya (Aspergillus) fischeri render it to be a promising template for future protein-based antifungal drug design, which requires knowledge about the native disulfide linkage pattern as it is one of the prerequisites for biological activity. However, in the lack of tryptic and chymotryptic proteolytic sites in the ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is not easy with traditional mass spectrometry-based methods. According to in silico predictions working with a preliminary nuclear magnetic resonance (NMR) solution structure, two disulfide isomers of NFAP2 (abbacc and abbcac) were possible. Both were chemically synthesized; and comparative reversed-phase high-performance liquid chromatography, electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility and efficacy tests indicated that the abbcac is the native pattern. This knowledge allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum through the modulation of the evolutionarily conserved gamma-core region, which is responsible for the activity of several antimicrobial peptides. Disruption of the steric structure of NFAP2 upon gamma-core modification led to the conclusions that this motif may affect the formation of the biologically active three-dimensional structure, and that the gamma-core modulation is not an efficient tool to improve the antifungal efficacy or to change the antifungal spectrum of NFAP2

The Membrane Activity of the Amphibian Temporin B Peptide Analog TB_KKG6K Sheds Light on the Mechanism That Kills Candida albicans

Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Temporin B (TB) is a 13-amino-acid-long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We recently showed that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low micromolar concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions, showing a preference for anionic lipids, and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using fluorescein isothiocyanate-labeled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy-independent manner. Peptide-treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures had disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and supports its great potential as a future anti-Candida therapeutic. IMPORTANCE Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Small, cationic, membrane-active peptides are promising compounds to fight against resistance development, as many of them effectuate rapid fungal cell death. This fast killing is believed to hamper the development of resistance, as the fungi do not have sufficient time to adapt to the antifungal compound. We previously reported that the synthetic variant of the amphibian TB peptide, TB_KKG6K, rapidly kills C. albicans. In the current study, the mechanism of action of the TB analog was investigated. We show that this TB analog is membrane-active and impairs cell membrane function, highlighting its potential to be developed as an attractive alternative anti-C. albicans therapeutic that may hinder the development of resistance