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Production of pectolyase from Rhizomucor pusillus by solid-state fermentation
No full textRhizomucor pusillus DSM 1331 is recognized to produce several industrial enzymes by solid-state fermentation (SSF). In pectinases research, little work was performed on the pectolyase enzyme production or molecular level using R. pusillus. Pectinases have been exploited in various industrial applications e.g. juice clarification and wine production. This project deals with the exploration of R. pusillus as pectolyase producer, with the emphasis on pectin lyase (PNL) production under solid-state conditions. PNL catalyzes the degradation of highly esterified pectin via a β-elimination mechanism. The study conducted a rational bioprocess development using different strategies. R. pusillus exhibited the ability to produce pectolyase using submerged and solid-state fermentation systems. SSF is showed an efficient fermentation mode using agroindustrial material reaching maximum PNL activity of 100 U/g. The process was optimized at the laboratory scale and scaled-up 200X utilizing at rotating drum bioreactor (with a productivity of 20,000 U/kg/d). Strain development strategy using the genome shuffling approach was selected to enhance enzyme production. The genome shuffling system was successfully developed. AR9-fusant was obtained with significant enhancement in pectolyase production showing a PNL activity of 580 U/g, five times higher than the parental strain. Optimal biochemical properties and pectinolytic activities of enzyme complexes revealed different patterns between R. pusillus and AR9-fusant. A preliminary attempt for protein identification was performed using MALDI-TOF. For the first time, the Rppnl gene, which encodes the pectin lyase of R. pusillus was isolated.Nucleotide and deduced amino acid sequence of Rppnl gene were compared with the reported PNL sequences and significant homology was confirmed. The results display the potential of R. pusillus as a promising pectolyase producer on inexpensive substrates, opening the way for several industrial applicatio
Personality Adjustment and Growth as Antecedents and Correlates of Wisdom
No full textPersonality development has been implicated in theory as one important antecedent to the development of wisdom, but there is very little longitudinal evidence directly relevant to the proposition. This dissertation sets out to investigate the longitudinal relationship between wisdom and two trajectories of positive personality development, personality adjustment and personality growth. The first of three empirical papers establishes partial measurement invariance for unique new Personality Adjustment and Personality Growth scales across adulthood in a longitudinal sample from approximately 34 to 73 years of age. The second paper uses a latent growth curve modeling approach in the same sample to establish trajectories for Personality Adjustment and Growth, and then latent class growth analysis to test for the presence of groups whose personality development paths towards later life wisdom are relatively similar. In the final paper, the same conceptual framework was applied to two adolescent samples to establish pre-adult validity. The key findings were as follows: a) the distinction between personality adjustment and growth is viable and useful throughout adulthood and in adolescence, with a consistent cross-sectional association between personality growth and wisdom; b) there are adolescence-specific features of personality growth and adjustment; and c) there is a longitudinal relationship between early adulthood personality growth and later life wisdom, and a class of people who had high levels of Personality Growth throughout adulthood and increasing levels of Personality Adjustment also had relatively high levels of wisdom at later life. Together, these findings indicate that personality development indeed plays a substantial role in the development of wisdom: As expected, personality growth even in early adulthood is an important antecedent of wisdom development, and personality adjustment has an important role as a scaffold to sustain personality growth
Elongator dependent anticodon modifications in Dictyostelium discoideum and the effect on glutamine codon translation
No full textTransfer RNAs are among the most posttranscriptionally modified RNA molecules in nature. Anticodon modifications at wobble position U34 are particularly important, since they help to modulate the codon - anticodon interaction and wobble recognition in the process of translation.
The eukaryotic Elongator complex consists of 6 different proteins (Elp1-Elp6) and catalyzes the first step in the Elongator dependent U34 modification pathway. Elongator dysfuncion has severe effects on different cellular processes and Elongator mutants in yeast and Arabidopsis display specific stress phenotypes.
This thesis presents the first investigation on Elongator dependent anticodon U34 modifications in the evolutionary supergroup of Amoebozoa. The first part of the thesis focused on the identity of the complex. Pulldown studies with ectopically overexpressed Elongator subunits revealed that the Dictyostelium discoideum Elongator complex also consists of 6 proteins. Furthermore, it was shown that the Elp3 protein is unstable.
The second part of the thesis focused on the Elongator dependent modifications. A series of mutant strains was generated that interrupted the Elongator dependent modification pathway at all major steps. Different analytic techniques were applied to investigate the modifications. It could be shown that the Elongator dependent modifications ncm5U, mcm5U and mcm5s2U are conserved in D. discoideum. The effect of loss of the mcm5s2U modifications was tested on the decoding capability of tQ(UUG) towards glutamine codon translation with artificial gene constructs. PolyQ leaders with different
CAA/CAG codon ratios fused to GFP were expressed in the background of different mutant strains. The expression differences were measured via flow cytometry and Western blot. Furthermore, the first unconditional tQ(CUG) null mutant in an eukaryotic model organism was generated. These findings provide evidence that wobbling of tQ(UUG) over CAG codon can occur in D. discoideum
A Facilitated Method to Characterize Rapidly Substrate Binding to Membrane Channel
No full textThe bacterial outer membrane porins are regarded as one of the main pathways for small hydrophilic antibiotic molecules to penetrate through the bacterial outer membrane. To understand the interaction mechanisms of antimicrobial molecules with the porins, the latter are extracted from their natural environment, reconstituted into free standing artificial lipid bilayer and characterized by electrophysiology. A broad range of general diffusion porins, i.e. OmpF and OmpC from E. coli, and their orthologs Omp35, Omp36 from E. aerogenes; OmpK35, OmpK36 from K.pneumoniae; OmpE35, OmpE36 from E. cloacae were characterized to study the biophysical properties of porins in different bacterial species. Electrophysiology together with MIC (Minimum Inhibitory Concentration) determination, high-resolution protein crystal structure and all atom MD (Molecular Dynamic) simulation were used to study the interaction mechanisms of ten ß-lactam molecules. A further research question was about the effect of naturally abundant divalent ions like calcium and magnesium on fluoroquinolone molecules. Although these molecules easily chelate in presence of divalent ions, the chelation appears to be unstable at the porin lumen and the single fluoroquinolone follow the electrostatic properties at the porin lumen.
Within this thesis we have in particular applied a relatively rapid screening approach by means of membranes-on-chip. This provides the potential to study the molecule-porin interaction at single molecule level in a higher throughput manner. β-lactamase inhibitors, though have little intrinsic antibacterial activity, inhibit the activity of massive plasmid-mediated β-lactamases. These agents are normally dosed in combination with β-lactams to tackle with MDR (Multi-Drug Resistant) bacteria. We found that the combinations of β-lactam and β-lactamase inhibitor combination gave a lower interaction rate than pure ß-lactam substrates via electrophysiology characterization
Cytomegalovirus gp40/m152 uses TMED10 as ER anchor to retain MHC class I
No full textIn a virus infection, major histocompatibility complex (MHC) class I molecules present viral antigens from infected cells to CD8+ cytotoxic T cells in a process called antigen presentation. Recognition of the viral epitope in the context of the class I molecule activates T cells to kill infected cells and thus eradicate viral infection. Viruses have evolved to perturb antigen presentation on MHC class I, and thus thwart the host immune response against the infection. For example, they encode proteins, called immunoevasins, that adversely affect one or several steps of the antigen presentation pathway. One such immunoevasin is m152/gp40 from the murine cytomegalovirus. In the presence of gp40, MHC class I molecules fail to leave the early secretory pathway(the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), and the cis-Golgi), and this prevents T cell recognition. Our work here shows for the first time that gp40 and class I molecules interact. The complex of gp40/class I circulates in the early secretory pathway. For this ER/ERGIC/cis-Golgi localization of the complex, the linker connecting the lumenal to the transmembrane domain of gp40 turned out to be necessary. This linker most likely binds to TMED10, a member of the p24 family of ER/Golgi transmembrane proteins, which anchors gp40 in the early secretory pathway. Deletion of TMED10 restores cell surface class I levels in the presence of gp40, most likely by destabilizing gp40 in a yet unknown fashion. We also show that the trafficking signals in the cytosolic tail of TMED10 (FF and KKxxx) are required for the ER localization of gp40. We have thus discovered the first viral client protein of the p24 family, and we have shown the first evidence that the p24 proteins can be exploited for viral evasion strategies
A Computational Study of Antibiotics and Substrates Transport through Bacterial Outer Membrane Channels
No full textThe outer membrane of the Gram-negative microbes imposes a permeability barrier for solutes located in the external environment. Integral membrane channels positioned along the outer membrane fulfill the task of nutrient translocation to the periplasm. Additionally, these channels are also known to be involved in the antibiotics uptake. However, the permeation routes taken by most classes of antibiotics remain unknown since their discovery. The studies reported in this thesis have been carried out to elaborate the molecular level understanding of the translocation of large solutes, i.e., substrates and antibiotics, through specific and non-specific channels using "state of the art" methods in all-atom molecular dynamics (MD) simulations.
The first part of the thesis includes an investigation on the translocation of the fluoroquinolone class of antibiotics, especially of ciprofloxacin and enrofloxacin molecules, through the porin OmpC of Escherichia coli. In the second part, the permeation of bulky cylcodextrin molecules through the specific channel CymA from Klebsiella oxytoca is investigated. Moreover, we have estimated the EOF using various salts and illustrated its influence on the α-CD interaction with the ∆CymA channel at an atomistic scale. In the final study, we have investigated the transport properties of the putative dicarboxylate specific channel DcaP from the pathogen Acinetobacter baumannii.
Overall, the findings presented in this thesis improve the atomistic understanding of the permeation of substrates and antibiotics through outer membrane channels of Gram-negative bacteria. Moreover, building such structure-function relationship of various outer membrane channels will help to develop future antimicrobials with enhanced permeability
Operation of Vessel Traffic Services covering international passages
No full textBased on the research question how to improve traffic in longer tidal waterways in terms of efficiency and risk mitigation, it is the objective of this PhD Thesis to approach an analysis towards the optimization of the Vessel Traffic Management (VTM) in longer tidal waterways. The theoretical relevance is the result of a lack of research to model, e.g. the sequence of vessels in waterways, for optimizing the entire traffic flow. The practical relevance is shown by using the River Elbe of the Port of Hamburg as an exemplary subject of interest for longer tidal waterways, because it is argued that the River Elbe is the most complicated river in Europe. Its dynamics and uncertainties of the waterway with recent developments in vessel sizes makes the traffic organisation more complex so that the VTM should be adapted to these increased challenges to support efficiency and safety. It is discussed by using the framework of Systems Engineering that such an adaptation needs a holistic perspective and must comprise the positioning of the problem-solving capacity of the current VTM organization regarding the external requirements in terms of the complexities of the system. Therefore, the development of a generic model is needed that can serve as the basic for a computer-based traffic prediction and management for the support of decision-making within a system of increasing complexity. To develop such a generic model, expert interviews are selected as the research methodology for the empirical data collection. The empirical data collections confirm that an overarching comprehensive system which is consistent and reliable is needed. With the help of the framework of the Business Process Modelling (BPM), the entire waterway-traffic-environment-system of the Rive Elbe is depicted and illustrated. The analysis of these process diagrams helps moving towards creating a precise system and optimizing the complexity of it. As a result, a conceptual model is outlined
Focused Ultrasound Therapy for Abdominal Organs During Respiratory Motion: Numerical Modeling and Simulation and In-Silico First-Stage Evaluation of a Novel Treatment System
No full textFocused ultrasound (FUS) is a noninvasive method for tissue ablation that has the potential for complete and local tumor destruction with minimal side effects. Already being used for the treatment of static organs, compensating target motion is not yet clinically available due to the complexity of the treatment. We here propose a numerical model of the therapy effects during respiratory motion to study FUS for moving liver targets. A focus lies on incorporating the motion and the computational efficiency of the simulations. A temperature model is proposed predicting the temperature distributions efficiently on the graphics processing unit by mapping the problem from the moving physical world to a static motion reference state. We also investigate the accuracy of ultrasound modeling in the highly heterogeneous propagation domain including ribs. A novel angular spectrum approach for heterogeneous media is proposed as the widely used hybrid angular spectrum method is found to be ineffective. For real-time applications, we propose an approximate ultrasound propagation model. An integrated FUS model is developed combining these model with an abdominal motion model, tissue damage, and a parameter model. The patient anatomy is automatically derived from CT images. Two clinical use-cases of the integrated model are given: A simulation-driven planning tool allows a clinician to interactively explore treatment options. And a study is performed using the model to optimize the placement of the FUS device. The model is furthermore used to study a novel motion-compensation FUS treatment system by replacing hardware and patient by model predictions. We estimate the efficiency of the treatment system in combination with a clinically available FUS device and MR imaging device (6.67 Hz image rate, 20 Hz FUS control rate) to be above 80%. This estimated efficiency of the new treatment system is expected to be already suited for clinical applications
Comprehensive Analysis of Cocoa Lipidomics – unraveling the unknown chemistry of cocoa butter
No full textTheobroma cacao is a tree that produces pods containing high valuable seeds, referred to as cocoa beans. About 50 million people around the world depend on cocoa as a source of income. From tree to bar, cocoa beans undergo an intricate processing chain leading to various chemical changes within the seed. Lipids constitute half of the dry weight of a cocoa bean and the most significant part of the entire cocoa market value taking the name of cocoa butter. Cocoa butter is composed of about 95% triacylglycerols (TAGs) and 5% of other minor components. The physical and chemical characteristics of this fat have been at the forefront of many research studies in the past decade. However, there are several knowledge gaps regarding the chemical composition and modifications that cocoa butter undergoes during the production process. This research aimed at investigating the chemistry of lipids in cocoa samples from different production steps and geographical regions, thus enriching the present knowledge of cocoa chemistry. Furthermore, the evolution of cocoa lipids during the factory production process was studied.
In the first place, analytical methods for the extraction, separation, and detection of cocoa lipids were developed and optimized. The overall lipid profile of unfermented, dried and roasted cocoa beans was investigated, and a solid phase extraction method, separating cocoa lipid extract in five fractions was established to aid the evaluation of the all cocoa lipid constituents.
A large part of the research was carried out aiming at product valorization and exploration of new possible industrial applications performing multiple analysis on different samples provided by the company. The outcomes give insights into the potential of cocoa butter and open new horizons for future studies and industrial applications
Designing Manufacturing Systems for Distributed Control
No full textThe distribution of control capabilities and functions among autonomous system components has attracted extensive research in the fields of logistics and production planning & control (PPC). Their emergent nature, however, renders much of the traditional, reductionist knowledge about the design of manufacturing systems and their control void, opening a gap in the understanding that is already threatening the industrial adoption of distributed PPC approaches. The current thesis addresses this particular research gap. It is driven in especially by the frequently expressed hypothesis that a combination of classical, centralized production control and new, distributed forms can yield optimal performance. This hypothesis is explored through a combination of interdisciplinary literature review and minimal model investigations. Cellular Automata on networks of different structure are applied to investigate the role of control network hierarchy on the performance of agents in simple, distributed problem solving settings, finding not only a performance peak at “medium” levels of hierarchy, but also developing a mechanistic understanding for it. The second quantitative model borrows from findings in algorithmic game theory to explore how the emergent behavior of selfish agents can be reconciled with the established ideal in manufacturing system design to set target utilization levels for machines. The findings of this thesis support a design approach for distributed Production Planning & Control (PPC) systems based on evidence and analysis, instead of experience and experimentation. It enhances our understanding of the success factors of distributed control in production environments and beyond. It can advance the development of “emergence engineering” by providing a deeper understanding of the target-driven design of Complex Adaptive System (CAS)