Inhibition Effect of Lactic Acid Bacteria against Food Born Pathogen, Listeria monocytogenes

In recent years due to changes in lifestyle and eating behavior of the human populations, disease caused by contaminated food has increased significantly. Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enteric are three of the most important food borne bacterial pathogens and can lead to food borne diseases. Also today wide spread of resistance to antibiotics among bacteria occurs due to increased consumption of antibiotics. Therefore, there is a dire need for development of new types of safe antimicrobial compounds. In this field, the most extensive research and commercial practices are based on probiotic bacteria. Probiotics, specifically lactic acid bacteria, are broadly used in the food industry for fermentation. Furthermore, probiotics produce valuable antimicrobial products that results to health effects. Now, the use of probiotic for treatment of disease is thought to be an effective way to improve the gut health and an alternative for treatment by antibiotics. Probiotics contribute to food safety by inhibition of the growth of other bacteria. Lactic acid bacteria can be used as protective cultures to compete with several pathogens and undesired organisms. Since food safety has become a significant international concern, here we investigated application of lactic acid bacteria for controlling the growth of Listeria monocytogenes

Internet as a tool of the present time

Diplomová práce je výsledkem sedmiletého studia na Technické Univerzitě v Liberci. Během této doby jsem přestoupil z fakulty Vizuální komunikace, po dvou letech bakalářského studia a neshodách s panem docentem Stanislavem Zippem. V zápětí jsem našel útočiště na fakultě Environmentálního designu, která byla ještě vedená panem profesorem Bořkem Šípkem. Zde jsem poprvé pocítil porozumění v plném rozsahu a dostal volnost, kterou jsem se snažil využít. Po prvotním seznámení s tehdejším vedením ateliéru jsem začal většinu projektů realizovat pod vedením pana profesora Jaroslava Brabce, který byl nejen fotograf ale také kameraman. Přesto, že jsme měli oba diametrálně rozdílné metody a nástroje tvorby, vždy jsem si odnášel dokonalou představu, jak by práce měla ve výsledku vypadat.První dva semestry magisterského studia mi vedení poskytl pan architekt Saman Saffarian. Zde jsme se sešli velmi dobře v moderních technologiích a zaměřili jsme se spíše na formu a následnou úroveň prezentace projektu.Po celou dobu studia na fakultě Environmentálního designu jsem měl možnost věci především ohledně prostoru konzultovat s panem docentem Janem Stolínem, u kterého vznikla i hlavní linie konečné diplomové práce.Období studia na vysoké škole mi poskytlo prostor, možnosti a nástroje pro dotvoření svých hodnot, názorů a cílů, kterých bych chtěl dosáhnout. Také mi byl dopřán prostor a vedení při přemýšlení o problému ze všech stran. Díky pobytu v Liberci jsem poznal mnoho spolužáků a přátel. Někteří stejného smýšlení jako já, kde jsem mohl kultivovat, a jiní rozdílného, od kterých jsem se mohl učit. Děkuji všem výše zmíněným za oporu, vedení a vzdělávání, a z mých přátel především Ladislavu Hofmanovi, který mne vždy poslouchal a pomáhal hlavně po technické stránce.The diploma thesis is the result of a seven-year study at the Technical University in Liberec. During this time I moved from the Faculty of Visual Communication, after two years of bachelor studies and disagreements with associate professor Stanislav Zippe. I found refuge in the Faculty of Environmental Design, which was still led by Professor Bořek Šípek. Here, for the first time, I felt an understanding in full and got the freedom I tried to use.After the initial introduction to the studio, I started to implement most of the projects under the guidance of Professor Jaroslav Brabec, who was not only a photographer but also a cameraman. Although we had both diametrically opposed methods and tools of creation, I always had a perfect idea of what the job should look like.The first two semesters of my master's degree were provided by architect Saman Saffarian. Here we met very well in modern technologies and focused more on the form and subsequent level of project presentation.Throughout my studies at the Faculty of Environmental Design, I had the opportunity to consult mainly with the associate professor Jan Stolín about the space.The period of university studies gave me space, possibilities and tools for completing my values, opinions and goals that I would like to achieve. I was also given space and guidance to think about the problem from all sides.Thanks to my stay in Liberec I met many classmates and friends. Some of the same minds as I, where I could cultivate, and others different from which I could learn.I would like to thank all of the above mentioned for their support, guidance and education, and especially for my friends Ladislav Hofman, who always listened to me and helped me mainly in technical part

Molecular functions of endogenous and heterologous genetic RNA elements in the tick-borne encephalitis virus genome

Flaviviren sind kleine lipidumhüllte Viren mit einem positiv-strängigen RNA Genom, denen eine Reihe von humanpathogenen Krankheitserregern, wie das Dengue-Virus, das Gelbfieber-Virus und das FSME (Frühsommer-Meningoencephalitis)-Virus angehören. Die Epidemiologie dieser Viren wird weitestgehend durch die ökologischen Anforderungen ihrer Insektenvektoren (Stechmücken oder Zecken) bestimmt und die Krankheitsbilder, die sie hervorrufen, reichen von milden, fieberhaften Symptomen über Hirnhautentzündung bis hin zu hämorrhagischem Fieber. Das Genom aller Flaviviren besteht aus einem einzigen RNA-Molekül, das die kodierende Sequenz für das virale Polyprotein enthält und das selbst als infektiöse mRNA fungiert. Der einzige offene Leserahmen auf diesem RNA-Molekül ist an den beiden Enden von nichtkodierenden Regionen flankiert, die eine entscheidende Rolle in der Translation, der Replikation und möglicherweise auch in der Verpackung der Virus-RNA spielen. Verglichen mit den kodierenden Sequenzbereichen des Genoms, sind diese nicht-kodierenden Regionen zwischenden beiden Hauptgruppen, den Insekten- und den Zecken-übertragenen Flaviviren, allerdings nur sehr schwach konserviert. Das Hauptziel der vorliegenden Arbeit war die Charakterisierung von endogenen wie auch von heterologen Sequenzelementen im FSME-Virus Genom. Die innerhalb der Flaviviren einzigartige, nicht-kodierende Region am 3’-Ende der viralen RNA (3’-NCR) wird in eine hoch konservierte „Kernregion“ und eine hinsichtlich ihrer Sequenz flexible „variable Region“ unterteilt. Die näher am 3’-Ende liegende „Kernregion“ enthält hoch konservierte RNA-Sekundärstrukturen, die unter anderem essentiell für die Virusreplikation sind. Die weiter innen angeordnete „variable“ Region differiert stark hinsichtlich ihrer Länge und ist auch zwischen einzelnen Stämmen innerhalb der FSME-Virus Familie nicht konserviert. Die Funktion der variablen Region, die in manchen FSME-Virus Stämmen durch eine Poly-A Sequenz gekennzeichnet ist, ist im Wesentlichen unbekannt. Im ersten Teil dieser Arbeit gingen wir der Frage nach, ob die variable Region in der 3’- nicht-kodierenden Sequenz des FSME-Virus einen Einfluss auf die Replikation und/oder Translation der viralen RNA ausübt. Dazu führten wir Mutationen in diesen Sequenzbereich ein und analysierten in einem sensitiven Luziferase-Reporter-Replikon System potentielle Effekte auf Translation und Replikation. Unsere Ergebnisse zeigten, dass eine Verkürzung oder Entfernung der Poly-A Sequenz, aber auch eine Deletion der gesamten variablen Region, keinen signifikanten Effekt auf einen dieser beiden Prozesse ausübt. Darüber hinaus wurde klar, dass die variable Region ohne Beeinträchtigung der Translation oder Replikation durch heterologe Sequenzelemente ersetzt werden kann. Letztere Erkenntnis lieferte den Grundstein für unser zweites Projekt, in dem wir untersuchten, ob das FSME-Virus in der Lage ist, funktionelle microRNAs zu kodieren. MicroRNAs sind eine Klasse von kleinen, nicht-kodierenden RNAs, die essentielle regulatorische Funktionen in der eukaryotischen Genexpression innehaben. Sie vermitteln dabei die sequenz-spezifische, post-translationale Inhibierung oder den Abbau von mRNAs. Obwohl kürzlich gezeigt werden konnte, dass DNA-Viren eigene microRNAs kodieren und diese in der Wirtszelle auch zu ihren Gunsten einsetzen können, gibt es keine Berichte über microRNAs von Viren mit einem RNA-Genom und einem zytoplasmatischen Lebenszyklus. Dem momentanen Wissensstand zufolge beginnt die Biogenese von microRNAs im Zellkern und zeichnet sich dort unter anderem durch einen Schnitt im RNA-Molekül aus, der den microRNA Vorläufer für die weitere Prozessierung freisetzt. Es wird daher allgemein angenommen, dass dieser Biogenese-Weg für Viren mit einem RNA-Genom und einem zytoplasmatischen Lebenszyklus weder zugänglich noch nutzbar ist. Unser Ziel war es, diese Annahme hinsichtlich ihrer Gültigkeit in einem Modellsystem zu überprüfen. Wir klonierten dazu einen heterologen Herpesvirus microRNA-Vorläufer in die nicht-kodierende 3’-Region des FSME-Virus. In der nachfolgenden Charakterisierung dieser chimären Mutante gelang uns der erstmalige Nachweis, dass eine funktionelle microRNA auch von einem zytoplasmatisch replizierenden Virus mit einem RNA-Genom generiert werden kann, ohne dass das notwendigerweise mit einer signifikantenBeeinträchtigung der RNA Replikation einhergehen muss. Im dritten Teil der vorliegenden Arbeit befassten wir uns mit konservierten RNA-Sekundärstrukturen am 5’-Ende des viralen RNA Moleküls. Kürzlich konnte der 5’-Teil der FSMEVirus Zyklisierungssequenz einer Haarnadelstruktur in diesem Genombereich zugeordnet werden. Um die Rolle dieses und anderer Sequenzelemente hinsichtlich ihrer Funktion zu untersuchen, führten wir in unserem Luziferase-Reportersystem eine Mutationsanaylse durch, die auch eine gezielte Veränderung der thermodynamischen Stabilität beinhaltete und so neue Einblicke in die Wirkungsweisen dieser Elemente in der viralen Replikation und Translation liefern konnte. Zusammenfassend lässt sich sagen, dass die Ergebnisse dieser Arbeit den derzeitigen Wissensstand über die Funktion endogener RNA-Elemente im Genom des FSME-Virus erweitern, dass sie wesentlich zu einem besseren Verständnis der komplexen Interaktion von RNA-Viren mit der RNA-Interferenz-Maschinerie der Wirtszelle beitragen, und dass sie darüber hinaus eine wertvolle Grundlage für das Design von RNA-Virus Vektoren liefern.Flaviviruses are small enveloped viruses with a positive-stranded RNA genome that include important human pathogens such as Dengue virus, yellow fever virus and tick-borne encephalitis virus. The epidemiology of these viruses is largely determined by the ecological needs of the corresponding insect vectors, i.e. mosquitoes or ticks. The disease patterns they evoke range from mild febrile illness, to encephalitis and hemorrhagic fever. The genome of all flaviviruses consists of a single RNA molecule that per se acts as an infectious messenger RNA and contains the coding sequence for the viral polyprotein. The single open reading frame is flanked by noncoding regions (NCRs) that reside on the terminal ends of the viral RNA strand and occupy important functions in RNA translation, replication and possibly also packaging. Compared to the protein-coding region, the noncoding regions are not well conserved between mosquito- and tick-borne flaviviruses. The main objective of this thesis was the characterization of endogenous as well as heterologous sequence elements in the genome of tick-borne encephalitis virus (TBEV). The unique tick-borne encephalitis 3’-NCR is divided into a highly conserved core region, which comprises essential secondary structures that are involved in RNA replication, and a variable region of inconsistent length that completely lacks sequence conservation. The function of the variable region that is characterized by a poly-A stretch in some but not all TBEV strains is essentially unknown. In our first approach we addressed the question whether the variable part of the 3’-NCR region has any effect on the efficiency of RNA replication or RNA translation. For this purpose we analyzed the impact of various manipulations of the 3’-NCR in a sensitive luciferase-based reporter replicon system. Our results revealed that truncation or complete removal of the poly-A stretch or even the deletion of the entire variable region does not cause a significant effect on any of these processes. Furthermore we observed that the replacement of the variable region with heterologous sequence elements was well tolerated during RNA replication and did not impair viral input RNA translation. These findings provided the basis for our second study in which we examined the capability of tick-borne encephalitis virus to encode functional microRNAs. MicroRNAs are a class of small noncoding RNAs that have essential regulatory functions in eukaryotic gene expression by mediating the sequence-specific translational inhibition or degradation of mRNAs. Although DNA viruses have recently been shown to encode and exploit their own microRNAs in the complex interactions with their mammalian host cells, no such molecules have so far been identified from viruses with an RNA genome and a cytoplasmic replication cycle. Based on the current understanding that microRNA biogenesis is initiated in the nucleus and characterized by an RNA cleavage event, it is generally reasoned that this pathway is not available and unusable for viruses that are confined to the cytoplasm and comprise an RNA genome. We addressed this issue experimentally and introduced a heterologous herpesvirus microRNA-precursor element into the TBEV 3’-NCR. In the subsequent characterization of this chimeric virus we were able to demonstrate for the first time that a functional microRNA can indeed be produced from such a virus without an impairment of viral RNA replication. Additional studies of this thesis concentrated on the conserved RNA secondary structure elements in the 5’-NCR of TBEV. Recently the 5’-part of the TBEV cyclization sequence has been mapped to one of these motifs. To further determine the role of this and other hairpin elements we performed a mutational analysis in our luciferase-reporter system and screened for defects in viral RNA replication and translation. This approach also included the manipulation of the thermodynamic stability of these elements and revealed several new insights into the functional importance of the TBEV 5’-terminal stem-loop structures. Taken together the results of this thesis extend the current knowledge on endogenous genetic RNA elements of TBEV, contribute to the inceptive understanding of the complexinterplay of RNA viruses with the RNA silencing machinery, and also provide a rational basis for RNA virus vector design

Study on effect feedings with probiotics in increasing resistance to Aeromonas hydrophila and Changes in gut bacterial communities Sander lucioperca

Introduction: This study evaluated the effect of dietary administration Lactobacillus brevis MF01 on survival rate of total bacteria, lactic acid bacteria intestinal tract fish and changes in gut bacterial communities Sander lucioperca. Materials and methods: Lactobacillus brevis was isolated and identified from intestine of Sander luciopercaby biochemical and molecular tests.Fish were fed with dietary administration containing A1 (L.brevis MF01 10 10 CFU / g), A2 (L. brevis MF01 10 8CFU / g) and the control group (without Lactobacillus) for 45 days. At the end of the feeding period, fish were challenged with 4.5× 10 8CFU /ml Aeromonas hydrophila. The intestinal micro biota includes lactic acid bacteria and total bacteria at different times (15,30, 45 days) and 15 days after stopping feedings with probiotics were performed by using MRS agar, Plate count agar media. Results: The lactic acid bacteria levels were significantly increased compared to the control group following probiotics administration in diet (P,< 0.05). The highest number of intestinal micro biota was observed in Lactobacillus brevis 1010Cfu /g treatment (A1)(P < 0.05). Any lactic acid bacteria of the intestine, was not detected in the control group after 15 days. After the end of feeding, the number of total bacteria and Lactic acid bacteria in all groups was decreased. The highest and lowest survival rate, respectively were treatments A1 (86%) and the control group (60%). Discussion and conclusion: The results showed that addition of Lactobacillusbrevis as an additive in feeding of sander lucioperca, significantly increased the lactic acid bacteria, the intestinal bacteria and the survival rate was compared to the control group (injected)

Effect of floor type on the performance, physiological and behavioural responses of finishing beef steers

peer-reviewedBackground:The study objective was to investigate the effect of bare concrete slats (Control), two types of mats [(Easyfix mats (mat 1) and Irish Custom Extruder mats (mat 2)] fitted on top of concrete slats, and wood-chip to simulate deep bedding (wood-chip placed on top of a plastic membrane overlying the concrete slats) on performance, physiological and behavioral responses of finishing beef steers. One-hundred and forty-four finishing steers (503 kg; standard deviation 51.8 kg) were randomly assigned according to their breed (124 Continental cross and 20 Holstein–Friesian) and body weight to one of four treatments for 148 days. All steers were subjected to the same weighing, blood sampling (jugular venipuncture), dirt and hoof scoring pre study (day 0) and on days 23, 45, 65, 86, 107, 128 and 148 of the study. Cameras were fitted over each pen for 72 h recording over five periods and subsequent 10 min sampling scans were analysed. Results: Live weight gain and carcass characteristics were similar among treatments. The number of lesions on the hooves of the animals was greater (P < 0.05) on mats 1 and 2 and wood-chip treatments compared with the animals on the slats. Dirt scores were similar for the mat and slat treatments while the wood-chip treatment had greater dirt scores. Animals housed on either slats or wood-chip had similar lying times. The percent of animals lying was greater for animals housed on mat 1 and mat 2 compared with those housed on concrete slats and wood chips. Physiological variables showed no significant difference among treatments. Conclusions: In this exploratory study, the performance or welfare of steers was not adversely affected by slats, differing mat types or wood-chip as underfoot material

Synthetic rubber surface as an alternative to concrete to improve welfare and performance of finishing beef cattle reared on fully slatted flooring

open8noopenBrscic, M.; Ricci, R.; Prevedello, P.; Lonardi, C.; De Nardi, R.; Contiero, B.; Gottardo, F.; Cozzi, G.Brscic, Marta; Ricci, Rebecca; Prevedello, P.; Lonardi, Chiara; DE NARDI, Roberta; Contiero, Barbara; Gottardo, Flaviana; Cozzi, Giuli

Functional microRNA generated from a cytoplasmic RNA virus

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play a pivotal role in the regulation of posttranscriptional gene expression in a wide range of eukaryotic organisms. Although DNA viruses have been shown to encode miRNAs and exploit the cellular RNA silencing machinery as a convenient way to regulate viral and host gene expression, it is generally believed that this pathway is not available to RNA viruses that replicate in the cytoplasm of the cell because miRNA biogenesis is initiated in the nucleus. In fact, among the >200 viral miRNAs that have been experimentally verified so far, none is derived from an RNA virus. Here, we show that a cytoplasmic RNA virus can indeed encode and produce a functional miRNA. We introduced a heterologous miRNA-precursor stem-loop sequence element into the RNA genome of the flavivirus tick-borne encephalitis virus, and this led to the production of a functional miRNA during viral infection without impairing viral RNA replication. These findings demonstrate that miRNA biogenesis can be used by cytoplasmic RNA viruses to produce regulatory molecules for the modulation of the transcriptome