Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens

The innate immune system is the first line of host defense against invading pathogens. In multicellular organisms, specialized innate immune cells recognize conserved pathogen-associated molecular patters (PAMPs) with germ-line encoded pattern recognition receptors (PRR). Thereby, the organism discriminates between self and non-self and engages mechanisms to eliminate the invader. Beside PAMPs, PRRs recognize mislocalized self-molecules, so called danger-associated molecular patterns (DAMPs), which are indicators of tissue or cellular damage. Upon PAMP or DAMP recognition, PRRs induce innate immune signaling pathways leading to the activation of pro-inflammatory genes and interferon production, which are important mediators of inflammation. Therefore the recognition of invading pathogen and thereby activation of innate immune signaling pathways determines the success of the immune system to eliminate the potential threat. Innate immune signaling pathways largely depend on phosphorylation cascades. Today, global phosphorylation changes are analyzed by mass spectrometry, however the number of detected phosphopeptides remains unchanged despite technical improvements. Therefore, we investigated the issue of phosphopeptide detection in mass spectrometry. The analyses of phosphopeptide-enriched samples have revealed lower signal intensities in MS1 spectra compared to total cell lysate samples, which results in poor phosphopeptide detection with mass spectrometry. Based on these observations, we hypothesized that the phosphate groups of phosphopeptides account for this poor detection. Indeed, we significantly increase the signal intensities in MS1 spectra after enzymatic removal of phosphate groups from phosphopeptides, and consequently we detect three-times more peptides in phosphatase-treated samples. Validation experiments elucidate that most of the newly detected peptides have been initially phosphorylated. Moreover, the newly detected peptides enlarge the activated signaling network upon Salmonella infection. Importantly, we identify known innate immune signaling pathways, which were missing in the analyses of phospho-enriched samples. Taken together, the phosphate groups of phosphopeptides globally suppress peptide ionization efficacy and therefore account for the low phosphopeptide detection rate by mass spectrometry. By removing the phosphate groups, we identify three times more peptides after phosphatase treatment. The newly detected peptides enlarge the network of activated innate immune signaling pathways upon Salmonella infection and include signaling pathways that are important but have not been detected in phospho-enriched samples. Therefore our findings improve the analyses of innate immune signaling pathways by mass spectrometry and consequently the understanding of innate immunity. One of the main mechanisms to eliminate invading microbes is by phagocytosis and degradation within phago-lysosomes. However, professional pathogens have developed various defense mechanisms to resist intracellular killing and can even use innate immune cells as replicative niches. For example, the bacterial pathogen Francisella tularensis causes a severe and life-threatening disease called tularemia in humans, because Francisella can survive and replicate in macrophages and dendritic cells. Critical for Francisella pathogenicity is the ability of the phagocytosed bacteria to escape from the phagosome to the host cytosol. Even though we know that genes encoded on the Francisella pathogenicity island (FPI) are essential for escaping from the phagosome, the mechanism is unknown. Homology analyses have suggested that the FPI encodes a type 6 secretion system (T6SS). However experimental evidence is missing, which show that the FPI encode a functional T6SS. Therefore, we investigated whether the FPI encodes a functional T6SS and what impact a functional T6SS has on Francisella virulence in vitro and in vivo. We show that the FPI of Francisella novicida (F. novicida) encodes a functional T6SS that assembles exclusively at bacterial poles. T6SS function depends on the unfoldase ClpB, which specifically recognizes contracted T6SS sheaths leading to their disassembly. Furthermore we have characterized FPI genes that show no homology with known T6SSs. We have identified IglF, IglG, IglI and IglJ as structural components of the T6SS and PdpC, PdpD, PdpE and AnmK as potential T6SS effector proteins. Whereas PdpE and AnmK are dispensable for phagosomal escape, AIM2 inflammasome activation and virulence in mice, pdpC- and pdpD-deficient bacteria are impaired in all aforementioned analyses. This suggests that PdpC and PdpD are bacterial effector proteins involved in phagosomal escape and thereby in the establishment of a F. novicida infection. Taken together, F. novicida uses its T6SS to deliver the effector proteins PdpC and PdpD into host cells. PdpC and PdpD are involved in phagosomal rupture and consequently in bacterial escape to the cytosol. These findings are a major breakthrough in the understanding of Francisella pathogenicity and could lead to new vaccination strategies to eradicate the life-threatening human disease Tularemia

Optimization of energy efficiency in data and WEB hosting centers

Mención Internacional en el título de doctorThis thesis tackles the optimization of energy efficiency in data centers in terms of network and server utilization. For what concerns networking utilization the work focuses on Energy Efficient Ethernet (EEE) - IEEE 802.3az standard - which is the energy-aware alternative to legacy Ethernet, and an important component of current and future green data centers. More specifically the first contribution of this thesis consists in deriving and analytical model of gigabit EEE links with coalescing using M/G/1 queues with sleep and wake-up periods. Packet coalescing has been proposed to save energy by extending the sojourn in the Low Power Idle state of EEE. The model presented in this thesis approximates with a good accuracy both the energy saving and the average packet delay by using a few significant traffic descriptors. While coalescing improves by far the energy efficiency of EEE, it is still far from achieving energy consumption proportional to traffic. Moreover, coalescing can introduce high delays. To this extend, by using sensitivity analysis the thesis evaluates the impact of coalescing timers and buffer sizes, and sheds light on the delay incurred by adopting coalescing schemes. Accordingly, the design and study of a first family of dynamic algorithms, namely measurement-based coalescing control (MBCC), is proposed. MBCC schemes tune the coalescing parameters on-the-fly, according to the instantaneous load and the coalescing delay experienced by the packets. The thesis also discusses a second family of dynamic algorithms, namely NT-policy coalescing control (NTCC), that adjusts the coalescing parameters based on the sole occurrence of timeouts and buffer fill-ups. Furthermore, the performance of static as well as dynamic coalescing schemes is investigated using real traffic traces. The results reported in this work show that, by relying on run-time delay measurements, simple and practical MBCC adaptive coalescing schemes outperform traditional static and dynamic coalescing while the adoption of NTCC coalescing schemes has practically no advantages with respect to static coalescing when delay guarantees have to be provided. Notably, MBCC schemes double the energy saving benefit of legacy EEE coalescing and allow to control the coalescing delay. For what concerns server utilization, the thesis presents an exhaustive empirical characterization of the power requirements of multiple components of data center servers. The characterization is the second key contribution of this thesis, and is achieved by devising different experiments to stress server components, taking into account the multiple available CPU frequencies and the presence of multicore servers. The described experiments, allow to measure energy consumption of server components and identify their optimal operational points. The study proves that the curve defining the minimal CPU power utilization, as a function of the load expressed in Active Cycles Per Second, is neither concave nor purely convex. Instead, it definitively shows a superlinear dependence on the load. The results illustrate how to improve the efficiency of network cards and disks. Finally, the accuracy of the model derived from the server components consumption characterization is validated by comparing the real energy consumed by two Hadoop applications - PageRank and WordCount - with the estimation from the model, obtaining errors below 4:1%, on average.This work has been partially supported by IMDEA Networks Institute and the Greek State Scholarships FoundationPrograma Oficial de Doctorado en Ingeniería TelemáticaPresidente: Marco Giuseppe Ajmone Marsan.- Secretario: Jose Luis Ayala Rodrigo.- Vocal: Gianluca Antonio Rizz

Imaging the onset of the segmentation clock during mouse gastrulation

A fundamental requirement for embryonic development is tight spatiotemporal coordination between a steadily increasing number of cells. A notable example of highly coordinated cellular activity occurs during body axis segmentation of the vertebrate embryo. Organized oscillatory signaling activity produces regular waves of gene expression that traverse the tissue. These oscillations are thought to define the timing of segmentation and the positioning of embryonic segments, the somites. The striking rhythmicity of the process is ascribed to a molecular oscillator in undifferentiated cells, the “segmentation clock”, involving the Notch, Wnt and FGF signaling pathways in mouse. While the segmentation clock and its intercellular coordination mechanism have been studied in some detail, the start of the process, the establishment of an initial synchrony between several hundreds of presomitic cells, remains largely unknown. I chose a live imaging approach to examine the onset of synchronous signaling oscillations in the developing embryo. To this end, I established a mounting and culture method to enable long-term multi-sample post-implantation mouse embryo imaging on a light-sheet microscope. This new setup enables routine embryo culture and imaging from early gastrulation on embryonic day 6.5 for up to two days, closely recapitulating in utero development. Using genetically encoded signaling reporters to visualize the cellular oscillation status, I was able to capture the very first observable coherent oscillations of the cyclic gene Lfng as well as the preceding dynamics. I found concurrent upregulation of Lfng expression across nascent mesoderm and the primitive streak at mid gastrulation, marking the first synchronous activity pattern of this segmentation clock gene. This expression pulse was also seen for the FGF signaling target Dusp4 but not the Wnt target Axin2. Despite Lfng being a Notch signaling target in the context of somitogenesis, I found the Lfng pulse to occur in the presence of Notch signaling inhibitor DAPT as well as in the absence of Notch transcription factor RBP-J κ and the Notch signaling core oscillator HES7. These findings argue for an initial synchronization signal independent of the Notch pathway. Quantification of early wave dynamics identified a gradual buildup of phase waves and revealed that a period gradient, which is present from the very first observable oscillation onwards, functionally underlies the establishment of waves in the gastrulating embryo. Taken together, this study provides detailed and quantitative insight into the dynamics of the early segmentation clock in the mouse embryo, pioneering future studies on the molecular mechanism of the synchronization process. The post-implantation embryo culture and imaging method described here opens up new possibilities for the study of the gastrulation-to-organogenesis stages, which in the past have been difficult to follow in real time. Further technical developments of multi-sample mammalian embryo microscopy, which I also discuss in this work, will continue to make an important contribution to the investigation of the establishment of the segmentation clock, and beyond can help to better understand many aspects of mammalian embryonic development

Management strategies to control sexual maturation in sea-reared Atlantic salmon (Salmo salar L.): Biomass management, light-manipulation and sterility

Pre-harvest sexual maturation in farmed Atlantic salmon, Salmo salar, remains a key biological bottleneck compromising biomass and financial output, production predictability, environmental respect, stock welfare and the overall sustainability of the on-growing industry. The management practices currently in place are not optimized and events of high maturation rate are still sporadically observed. From an ecological perspective, the escape of reproductively competent, domesticated Atlantic salmon constitutes a threat to the integrity of wild stocks. The forecasted expansion of the Scottish salmon industry compels the need for a comprehensive and more reliable control of sexual maturation. The general aim of this research project was to optimize the current management strategy (windows of light-manipulation and quality grading) and test alternative practices (lighting-technologies, selective harvest and triploidization) in the control of pre-harvest sexual maturation within the Atlantic salmon on-growing industry. In that end, a number of trials were performed using stock reared in sea-cages on a full commercial-scale or in tanks on an experimental scale. The results of this project are organized around three experimental chapters dealing consecutively with body-size dimorphism, grading and harvest quality; light manipulations and triploidy. In each chapter, two original manuscripts either published or in review are included. In addition to these experimental results, a literature review chapter composed of two review papers on the photoperiodic synchronization and developmental regulation of maturation in salmonids and on morphological skin colour changes in teleosts (published) are presented. In the first experimental chapters, we aimed at investigating the possibility of detecting and selectively harvesting a high proportion of sexually recruited fish before flesh quality deterioration. Results clearly showed that body-size dimorphisms between maturity cohorts at the end of the anabolic window of reproduction (June/July) are strong and standard predictors of maturation among related populations with the same freshwater history. Dimorphism can therefore be modelled to easily and accurately estimate maturation rate in a number of discrete rearing-units. If required, a high proportion of sexually recruited fish can be selectively harvested as superior quality product while leaving the immature fish for further on-growing. This provides an alternative to visual grilse grading that is not feasible in large-scale aquaculture systems, prevents downgrading and increases production predictability as compared to emergency harvests. Furthermore, our results showed immature males grow faster than immature females which should be further investigated to fully determine gender specific performances and nutritional requirements. Weight-grading performed earlier in the cycle affects the sex-ratio within individual pens and in turn apparent performance. This work also revealed that Atlantic salmon can exhibit significant variations in skin colouration resembling the onset of nuptial display but that are not related to sexual recruitment and do not correlate with reduced flesh quality. This originates from a lack of purine (silver) pigments which was also identified, to a larger extent, as characteristic of the nuptial display. This suggests a degree of desmoltification in these histologically immature fish. The instrumental colouration of the altered phenotype was shown to be improved towards a more silver-like appearance by direct ice-contact. This knowledge could facilitate post-harvest quality grading towards the most appropriate market channel and increase product acceptance and attractiveness. The second experimental chapter investigated the possibility of improving photoperiodic manipulation used to suppress early maturation, currently applied for 6-months during the second winter at sea using wide-spectrum, high-intensity lighting systems. Our results showed that the window of continuous artificial-light (LL) exposure can be reduced to 4-months following its onset in early January without compromising its efficiency in suppressing pre-harvest maturation. In addition, alternative lighting technologies were also highly potent at suppressing sexual maturation. The mean-irradiance (intensity) generated within a commercial sea-cage was inversely proportional to the suppression of nocturnal plasma melatonin (light perception hormone) and negatively correlates with the maturation rate within the commercial sea-pen. Threshold levels of light-intensity required to achieve optimal (total) suppression of sexual maturation are suggested. Alternative, narrow band-width lighting-technologies (cold cathode and light-emitting diodes) present an array of technical, practical, economic and welfare benefits comparing to the system currently in use. Clear improvements of the photoperiod-manipulation strategy were demonstrated and these would reduce economic and environmental costs but also potential impacts on animal welfare. The third experimental chapter showed the strong potential of sterile-triploid Atlantic salmon stocks both in freshwater and seawater. Triploid out-of-season smolts were produced for the first time using a classical accelerated "square-wave" photoperiod. Triploidization affected the smoltification pattern but had no detrimental effects on freshwater and early seawater performances under both a S0+ and S1 regime. This illustrates the need to adapt the timing of seawater transfer for successfully producing triploid Atlantic salmon post-smolts. Following one year of seawater rearing, the prevalence of external deformities was higher in triploids but remained within acceptable levels. Importantly, the incidence of vertebral deformities and ocular cataract was higher in triploids possibly due to their specific requirements. It is suggested that tailoring the diet to the nutritional requirements of triploids holds strong potential for remediation. This must be addressed if the use of sterile-triploid stock is to become a commercial reality. The present research project provides means to optimize the maturation management strategy within the Atlantic salmon on-growing industry through light-manipulation, maturation detection and selective harvest, and quality grading. Proposed improvements have the potential to increase biomass and financial output, production predictability, environmental respect and animal welfare and will allow standardization of the overall control of pre-harvest sexual maturation. Their implementation provides a comprehensive strategy likely to favour a sustainable expansion of the Atlantic salmon industry. From a longer term perspective, the rearing of sterile-triploid stocks is promising and should be actively investigated to isolate domesticated strains from their wild conspecifics. This would also eliminate the need for on-growers to deploy a maturation management strategy that that might still affect stock welfare and remains, despite the strong improvements demonstrated, not 100% reliable, costly, technical and protracted

High-Throughput Process Development in the Field of Protein Purification - Method Development, Application, and Characterization

High-throughput methods were developed for the quantification of mAb aggregate/monomer ratios, and the analysis of content, purity, and activity of the egg white protein avidin. Furthermore, by using high-throughput screenings, a new avidin purification process was developed using precipitation, aqueous two-phase extraction and mixed-mode chromatography. Finally, method-specific effects present in high-throughput column chromatography were evaluated using both experimental and simulation data

Thermal Comfort in Sun Spaces: To what extend can energy collectors and seasonal energy storages provide thermal comfort in sun space?

Preparation for fossil fuel substitution in the building sector persists as an essential subject in architectural engineering. Since the building sector still remains as one of the three major global end energy consumer – climate change is closely related to construction and design. We have developed the archetype sun space to what it is today : a simple but effective predominant naturally ventilated sun trap and as well as living space enlargement. With the invention of industrial glass orangery’s more and more changed from frost protecting envelopes to living spaces from which we meantime expect thermal comfort in high quality. But what level of thermal comfort provide sun spaces? And to what extend may sun spaces manage autarkic operation profiting from passive solar gains and, beyond that, surplus energy generation for energy neutral conditioning of aligned spaces? We deliver detailed information for this detected gap of knowledge. We know about limited thermal comfort in sun spaces winter times. This reasons the inspection of manifold collector technologies, which enable to be embedded in facades and specifically in sun space envelopes. Nonetheless, effective façade integrated collectors are ineffective in seasons with poor irradiation. Hence, the mismatch of offer and demand we have experienced with renewable energies ignites thinking about appropriate seasonal energy storages, which enlarges the research scope of this work. This PhD thesis project investigates on both, a yearly empirical test set up analysis and a virtual simulation of different oriented and located sun spaces abroad Germany. Both empirical and theoretical evaluation result in a holistic research focusing on a preferred occupation time in terms of cumulative frequencies of operational temperature and decided local discomfort, of potential autarkic sun space operation and prospective surplus exergy for alternative heating of aligned buildings. The results are mapped geographically for Germany. Fossil fuel substitution, as far as this thesis elaborated, is closely related to quality of thermal comfort, sun space orientation and energetic standard of the aligned building. Unexpectedly, spaces, which define envelopes incorporating collectors in combination with storage technologies both profit and suffer to some extend in respect to thermal comfort. Essentially, we can conclude, that the more area-wise efficient and the more integral the collector technology is incorporated into façade design, the more distinct significance of thermal comfort quality and fossil fuel substitution is. Eventually, this dissertation determines the potential of a new generation of sun spaces in the context of energy transition

Thermal comfort in sun spaces:

Preparation for fossil fuel substitution in the building sector persists as an essential subject in architectural engineering. Since the building sector still remains as one of the three major global end energy consumer – climate change is closely related to construction and design. We have developed the archetype sun space to what it is today : a simple but effective predominant naturally ventilated sun trap and as well as living space enlargement. With the invention of industrial glass orangery’s more and more changed from frost protecting envelopes to living spaces from which we meantime expect thermal comfort in high quality. But what level of thermal comfort provide sun spaces? And to what extend may sun spaces manage autarkic operation profiting from passive solar gains and, beyond that, surplus energy generation for energy neutral conditioning of aligned spaces? We deliver detailed information for this detected gap of knowledge. We know about limited thermal comfort in sun spaces winter times. This reasons the inspection of manifold collector technologies, which enable to be embedded in facades and specifically in sun space envelopes. Nonetheless, effective façade integrated collectors are ineffective in seasons with poor irradiation. Hence, the mismatch of offer and demand we have experienced with renewable energies ignites thinking about appropriate seasonal energy storages, which enlarges the research scope of this work. This PhD thesis project investigates on both, a yearly empirical test set up analysis and a virtual simulation of different oriented and located sun spaces abroad Germany. Both empirical and theoretical evaluation result in a holistic research focusing on a preferred occupation time in terms of cumulative frequencies of operational temperature and decided local discomfort, of potential autarkic sun space operation and prospective surplus exergy for alternative heating of aligned buildings. The results are mapped geographically for Germany. Fossil fuel substitution, as far as this thesis elaborated, is closely related to quality of thermal comfort, sun space orientation and energetic standard of the aligned building. Unexpectedly, spaces, which define envelopes incorporating collectors in combination with storage technologies both profit and suffer to some extend in respect to thermal comfort. Essentially, we can conclude, that the more area-wise efficient and the more integral the collector technology is incorporated into façade design, the more distinct significance of thermal comfort quality and fossil fuel substitution is. Eventually, this dissertation determines the potential of a new generation of sun spaces in the context of energy transition

MicroRNA and Cancer

MicroRNAs (miRs) are small noncoding RNAs that function as post-transcriptional regulators of gene expression and have important roles in almost all biological pathways. Deregulated miR expression has been detected in numerous cancers, where miRs act as both oncogene and tumor suppressors. Due to their important roles in tumorigenesis, miRs have been investigated as prognostic and diagnostic biomarkers and as useful targets for therapeutic intervention. From a therapeutic point of view, two modalities can serve to rectify gene networks in cancer cells. For oncomiRs, a rational means is downregulation through antagomirs. Moreover, observations of the pathological reductions in tumor-suppressive miRs have inspired the concept of “miR replacement therapy” to enhance the amount of these miRs, thereby restoring them to normal levels. However, the clinical applicability of miR-based therapies is severely limited by the lack of effective delivery systems. Therefore, to understand the role of this new class of regulators, we need to identify the mRNA targets regulated by individual miRs as well as to develop specific, efficient, and safe delivery systems for therapeutic miRs