The effect of a single genetic mutation can vary greatly between different types of cells. The mutated gene may not be expressed in one tissue but may cause a devastating loss of function in another. To learn about disease mechanisms and generate novel therapies, genetic disorders must be studied in the types of cells where the mutations are most deleterious. Recently, scientists have begun manipulating cellular identity to create the cell types most affected by various genetic diseases. This dissertation describes the experience of generating reprogramming models for three genetic disorders: Ring 14 syndrome, Pearson syndrome, and Fanconi anemia.

No description available.

OBJECTIVES: Evaluate the effect of different veneering techniques, Y-TZP core thicknesses, and firing cycles with different heat rates and holding times on the mechanical properties of veneering porcelain. MATERIALS AND METHODS: Biaxial flexural strength and Thermal shock resistance tests were conducted to evaluate the mechanical properties of veneer porcelain. Core material, Y-TZP and two porcelain veneers, IPS e.max Ceram and VITA VM9, were used in this study. Vita YZ zirconia blocks were sectioned and sintered to provide slabs of 1.65, 3.25, and 6.50mm in thickness. Two techniques were used to fabricate VITA VM9 and e.max Ceram porcelain veneer porcelain discs, Hand Layered, mixing powder with manufacturer’s molding liquid, condensed in a mold, and Pressed, pressing powder uniaxially in a mold. A layer of carbon paint was applied to the zirconia to allow removal of the porcelain discs. For thermal shock test the veneering porcelain fired on the Y-TZP core with a wash layer. After the veneer discs were fabricated, they were sintered: (1) According to the manufacturer’s instructions; (2) Two different cycles with slower heat rate and longer holding than the recommended value. Biaxial flexural strength was determined using a universal mechanical tester. Thermal shock tests were performed using a Pober thermal shock device. Statistical analysis was conducted for all tests using ANOVA and Tukey post hoc test at p= 0.05. RESULTS: There was significant effect from changes of YZ Core thickness, firing cycle, and veneer technique on the mechanical properties of porcelain veneer. The veneering technique had the largest impact on the mechanical properties of veneer porcelain followed by firing cycle, followed by YZ core thickness. In using different firing cycles with different heat rates and holding times, slowing the heating rate had more influence on the mechanical properties of veneer porcelain. In measuring the surface temperature of the Y-TZP/VITA VM9 assembly, the specimen surface temperature was coincidental with the furnace programmed firing cycle using a slower heating rate firing cycle. CONCLUSIONS Veneering technique, slower heat rate firing cycle, and Y-TZP core thickness have significant impact on veneering porcelain mechanical properties. / 2018-09-28T00:00:00Z

This thesis deals with the bark beetle population gradation which resulted in dieback of montane spruce forest in the central part of the Bohemian Forest, Czech Republic, during 1991 - 2000. A spatio-temporal model of changing land cover has been made using remote sensing and GIS methods. The statistical analyses have been made using generalized linear models (GLM). The possible effect of various conditions and environmental factors at landscape as well as the stand level has been discussed.

This thesis evaluates the effectiveness of defensive techniques against the spruce bark beetle (Ips typographus L.) with a primary focus on pheromone traps and selected factors affecting their efficacy. The factors tested are the setup system of pheromone traps and type of pheromone dispenser baits, the effect on the efficacy of pheromone traps of increasing evaporated pheromones, a comparison of the biological efficacy of different types of pheromone dispensers and an assessment of their impact on the swarming curve. In addition, an evaluation of non-traditional methods of forest protection against the spruce bark beetle (water spraying of whole landings, efficacy of insecticidal sraying of whole landings, and the use of insecticidal nets) was conducted and the possibility of the spruce bark beetle s sex determination was also examined. When comparing the systems of pheromone traps installation, (1) the standard way was along the stand edge with 20 m space and (2) traps concentrating in the centre of clear cut adjacent to each other with different baiting, the most effective way was found to be the standard trap arrangement and the system with traps installed in line with the dispenser in each pheromone trap. Efficacy decreased with a lower number of pheromone dispensers. The influence of increased amounts of evaporated pheromone on efficacy was tested. Pheromone traps with one, two or three dispensers were compared and it was found that a trap s efficacy was not increased with a higher amount of released pheromone. The Pheagr IT and IT Ecolure Tubus dispensers showed unchanged effectivity throughout the flight activity of spruce bark beetle, but other dispensers need to be replaced according to the instructions since their efficacy decreases, thus affecting the swarming curve. Trying to determine the sex of the spruce bark beetle by selected morphological parameters shows that the gender can only be reliably determined by an autopsy of genitals.

Thesis topic concerns the issue of information security in corporate environments. Literature search includes information obtained by studying articles and literature in the field of information security. Resources were selected with a focus on the security risks, security technologies and legislative regulation. Attention is focused on technology that supports monitoring of communication flows in the data network. Overview of traffic operating a data network provides important information for the prevention or investigation of security incidents. Monitoring also serves as a source of information for the planning of the network infrastructure. It can detect faults or insufficient transmission capacity. The practical part is dedicated to implementation of the monitoring system in the real corporate networks. Part of the experience is the analysis of the network structure and choice of appropriate tools for actual implementation. When selecting tools, you can use the scoring method of multicriterial analysis options. The integration of the monitoring system is also the configuration of active network elements. Subsequent analysis of network traffic provides information about the most active users, most used applications or on the sources and targets of data transmitted. It provides a source of valuable information that can be used in case of failure on the network or security incident. The conclusion is a summary of the results and workflow.

The thesis describes the development and survival of immature Ips typographus specimens at low temperatures under laboratory and field conditions. Further, the focus was identifying and characterizing the digestive enzymes present in the gut of adult I. typographus, their location in the gut and enzymatic fluctuation over a full calendar year, with a specific focus on digestion of cellulose.

Arthropod predators are able to influence the population dynamics of forest insects such as bark beetles. Two common predators of bark beetles are Temnochila virescens (F.) (Coleoptera: Trogositidae) and Thanasimus dubius (F.) (Coleoptera: Cleridae). Studies on T. virescens ecology have been few and not quantitative. Also, in recent years there has been increased focus on multiple-predator effects on the population dynamics of a single prey species, which better describes a natural system. Therefore, a series of three studies was conducted to provide a general overview of T. virescens ecology and its potential use for biological control. The first study examined the functional response of T. virescens toward its Ips prey. The second evaluated the impacts of intraguild competition between T. virescens and T. dubius adults on predation of a mutual prey species, I. grandicollis. The third determined if there were differences in pheromone preference among individual T. virescens adults. To evaluate the functional response, T. virescens and Ips grandicollis (Eichhoff) adults were combined in varying densities. Ips grandicollis survival was assessed by examining the number of prey eaten in relation to predator and prey densities. I found that a type II functional response model incorporating interference provided a good fit for the data. The interference was strong enough that a ratio-dependent model fit the data just as well as the full model. The effect of competition between T. dubius and T. virescens adults on I. grandicollis survival was determined by placing varying numbers of each predator with a set prey number. I found a significant interaction between densities of the two predators, which indicates possible emergent predator effects. The combined effect of the predators was risk reducing for prey, likely because of intraguild predation. The predator T. dubius alone was more efficient at reducing the number of prey adults successfully entering the log. This study indicates the importance of low density releases of these predators for augmentative biological control, to reduce the effects of intraspecific competition. To determine if there were differences in pheromone preference among individual T. virescens adults, a mark-release-recapture experiment was combined in the field to provide a choice test between ipsenol and ipsdienol pheromones. Temnochila virescens adults that were initially captured on ipsenol were more frequently recaptured with this pheromone, while those first captured on ipsdienol were recaptured on both pheromones. This suggests populations of T. virescens are composed of specialists attracted only to ipsenol and generalists attracted to both ipsdienol and ipsenol pheromones.

Les cellules somatiques peuvent être reprogrammées en cellules pluripotent en sur-exprimant 4 facteurs de transcriptions: OCT4, SOX2, KLF4 et c-MYC. Ce processus nécessite en théorie un remodelage des organelles et un changement drastique du métabolisme. De plus, la reprogrammation cellulaire possède une composante stochastique qui est peu comprise et conduit à une faible efficacité. Nous avons fait l'hypothèse que cette variabilité est en partie due aux variations de la régulation de l'homéostasie protéique. Nous nous attendons à ce que la première phase de reprogrammation active les voies de stress qui régulent l'homéostasie protéique, ce qui impacterait l'efficacité de reprogrammation. Notre attention s'est dirigée vers le rôle de la réponse aux protéines dépliées du réticulum endoplasmique. Nous avons découvert que cette voie est active pendant la reprogrammation cellulaire et que son activation peut augmenter l'efficacité de ce processus. Par ailleurs le niveau d'activation de cette voie peut prédire l'efficacité de reprogrammation. Ces résultats suggèrent que la faible efficacité de reprogrammation cellulaire est en partie due à l'incapacité des cellules à activer cette voie de stress afin de pouvoir correctement répondre à la nouvelle charge de protéines synthétisées qui changera l'état de cette cellule. / Somatic cells can be reprogrammed into a pluripotent stem cells state and is achieved by the forced expression of 4 transcription factors: OCT4, SOX2, KLF4 and c-MYC. This process theoretically requires a global remodeling of the organelles and a drastic change in metabolism. Furthermore, reprogramming has an inherent property of stochastic variation that is limiting and largely unknown. We hypothesize that this variation is due, in part, by variable regulation of the protein homeostasis network. We therefore postulated that the early steps of reprogramming would result in the activation of a variety of stress pathways that regulate the protein homeostasis network, which might in turn impact the efficiency of reprogramming. We focused in particular on the endoplasmic reticulum unfolded protein response (UPRER). We find that the UPRER is activated during reprogramming and that its activation can increase the efficiency of this process. We find that stochastic activation of the UPRER can predict reprogramming efficiency. These results suggest that the low efficiency of cellular reprogramming is partly the result of the cell’s inability to initiate a proper stress response to cope with the newly expressed load of proteins that will eventually change the fate of this cell.

Flaviviruses are important emerging and re-emerging arthropod-borne pathogens that cause significant morbidity and mortality in humans. It consists of globally distributed human pathogens such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZIKV). Depending on type, flaviviruses can cause a variety of symptoms ranging from haemorrhage to neurological disorders. Virus infection is detected by host pattern recognition receptors (PRRs), and through downstream signalling it leads to the production of interferons (IFNs). These IFNs then act in an autocrine or paracrine manner on the cells to induce various IFN-stimulated genes (ISGs), which have antiviral roles. However, the amount of IFN produced depends on the nature of the PRRs used by host cells to detect a particular virus. Although there are many PRRs present in the host cells, their relative contribution in different cell types and against a specific virus may vary. In the first study, we determined the importance of IPS-1 signalling in immunity and pathogenicity of tick-borne flaviviruses. This is an adaptor protein for cytoplasmic RIG-I-like receptors. Using IPS-1-deficient mice, we showed its importance against TBEV and Langat virus (LGTV) infection (the LGTV model virus belongs to the TBEV serogroup). Absence of IPS-1 leads to uncontrolled virus replication in the central nervous system (CNS), but it has only a minor role in shaping the humoral immune response at the periphery. LGTV-infected IPS-1-deficient mice showed apoptosis, activation of microglia and astrocytes, an elevated proinflammatory response, and recruitment of immune cells to the CNS. Interestingly, we also found that IFN-b upregulation after viral infection was dependent on IPS-1 in the olfactory bulb of the brain.  Thus, our results suggest that local immune microenvironment of distinct brain regions is critical for determination of virus permissiveness. Interferons can upregulate several ISGs. Viperin is one such ISG that has a broad-spectrum antiviral action against many viruses. However, the importance of cell type and the significance of viperin in controlling many flavivirus infections in vivo is not known. Using viperin-deficient mice, we found that viperin was necessary for restriction of LGTV replication in the olfactory bulb and cerebrum, but not in the cerebellum. This finding was also confirmed with primary neurons derived from these brain regions. Furthermore, we could also show the particular importance of viperin in cortical neurons against TBEV, WNV, and ZIKV infection. The results suggested that a single ISG can shape the susceptibility and immune response to a flavivirus in different regions of the brain. Although viperin is such an important ISG against flaviviruses, the exact molecular mechanism of action is not known. To understand the mechanism, we performed co-immunoprecipitation screening to identify TBEV proteins that could interact with viperin. While viperin interacted with the prM, E, NS2A, NS2B, and NS3 proteins of TBEV, its interaction with NS3 led to its degradation through the proteosomal pathway. Furthermore, viperin could reduce the stability of other viperin-binding TBEV proteins in an NS3-dependent manner. We screened for viperin activity regarding interaction with NS3 proteins of other flaviviruses. Viperin interacted with NS3 of JEV, ZIKV, and YFV, but selectively degraded NS3 proteins of TBEV and ZIKV, and this activity correlated with its antiviral activity against these viruses. The last study was based on in vivo characterization of the newly isolated MucAr HB 171/11 strain of TBEV which caused unusual gastrointestinal and constitutional symptoms. This strain was compared with another strain, Torö-2003, of the same European subtype of TBEV but isolated from the different focus. Here we found unique differences in their neuroinvasiveness and neurovirulence, and in the immune response to these two strains. In summary, my work shed some light on the interplay between tick-borne flavivirus and the innate immune system. I have shown two examples of CNS region-specific differences in innate immune response regarding both in IFN induction pathways and antiviral effectors. Furthermore, we have investigated the in vivo pathogenesis of a strain of TBEV that caused unusual gastrointestinal and constitutional symptoms. / Flavivirus finns spridda över hela världen och orsakar miljontals infektioner varje år. Några av de medicinsk mest viktiga flavivirusen är fästingburen encefalit virus (TBEV), West Nile virus (WNV), Japansk encefalit virus (JEV), gula febern (YFV) och Zika virus (ZIKV). Dessa virus kan orsaka olika komplikationer till exempel blödarfeber och hjärninflammation. Vid en infektion så upptäcker värdcellen virusinfektionen med hjälp av speciella receptorer, så kallade PRRs. Dessa finns i alla celler och känner igen viruskomponenter som normalt inte finns i en oinfekterad cell. När PRRs detekterar en virusinfektion svarar cellen med att tillverka ett signal protein interferon (IFN). IFN skickas ut ur cellen och hämmar virusinfektioner genom att sätta igång ett försvarsprogram i andra celler bestående av hundratals försvarsproteiner som kan motverka virusinfektionen. Vilka PRRs som behövs för att detektera ett virus är olika vid olika virusinfektioner. I första studien fann vi att IPS-1 är av yttersta vikt för skydda mot fästingburna flavivirus. IPS-1 är ett så kallat adapter protein som behövs för att två PRRs, RIG-I och MDA-5, ska kunna förmedla signaler som leder till IFN tillverkning. Med hjälp av möss som saknar IPS-1 fann vi att IPS-1 behövs för att tillverka IFN protein och skydda mot fästingburna flavivirus. IPS-1 var särskilt viktigt för interferon produktion inom luktloben i hjärnan. Därför kunde vi dra slutsatsen att immunresponsen regleras olika inom olika delar av hjärnan. Ett försvarsprotein som visat sig vara särskilt viktig vid virusinfektion är viperin. Viperin har visat sig kunna hämma en rad olika virus men den specifika rollen av viperin in vivo vid flavivirus infektion var inte fullt känd. Vi fann att viperin behövs för att hämma LGTV i lukloben och storhjärnan men inte i lillhjärnan. Vi kunde bekräfta detta med hjälp av primära nervceller isolerade från dessa hjärnregioner. Vi fann även att viperin var av yttersta vikt för att kontrollera TBEV, WNV och ZIKV infektion i nervceller från hjärnbarken (del av storhjärnan). Därför kunde vi dra slutsatsen att ett enskilt försvarsprotein kan avgöra mottagligheten mot flavivirus inom olika hjärnregioner. Trots att viperin är så viktig för att skydda mot flavivirus så vet vi inte hur viperin åstadkommer detta. Därför ville vi undersöka hur viperin kan förmedla sin antivirala effekt. Vi fann att viperin kan binda till flera TBEV proteiner, men att viperin specifikt kan bryta ner ett virusprotein som heter NS3. NS3 är väldigt viktigt för att flavivirus ska kunna etablera en infektion och kunna föröka sig. Eftersom vi visste att viperin kan hämma andra flavivirus ville vi veta om viperin även förstör NS3 från JEV, ZIKV och YFV. Vi upptäckte att viperin kunde binda till NS3 hos alla dessa flavivirus men att viperin specifikt förstörde TBEV och ZIKV NS3, intressant nog så kunde viperin endast hämma dessa virus infektioner men inte JEV och YFV. I den sista studien ville vi karaktärisera en ny TBEV stam som bara orsakar magoch tarmbesvär men inga neurologiska symptom. TBEV har aldrig tidigare visat sig kunna orsaka detta och därför ville vi undersöka saken vidare. Vi fann att denna TBEV stam skiljde sig mot en närbesläktad stam genom att orsaka en starkare immunrespons men mildare sjukdomsförlopp. Sammanfattningsvis har jag undersökt samspelet mellan fästingburna flavivirus och det medfödda immunförsvaret. Jag har även visat att immunresponsen regleras olika inom olika hjärnregioner, både beträffande IFN inducering och antivirala proteiner. Vidare har jag hittat mekanismen för hur viperin proteinet hämmar TBEV och ZIKV, vilket var genom att förstöra NS3. Dessutom har jag karaktäriserat sjukdomsförloppet hos möss efter infektion med en ovanlig TBEV stam som orsakar mag och tarm besvär.