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Cloning and expression of the aspartic proteinase from Mucor mucedo structural characterization and technological properties in comparison with the native enzyme
No full textThis project explored the suitability of the aspartic proteinase from Mucor mucedo DSM 809 as novel microbial rennet. The native proteinase was produced via submerged cultivation in shaking flasks. Under optimized conditions, the peak of production (130 U/ml) was observed after 48 h of cultivation at 24°C and 220 rpm. The chromatographically purified enzyme depicted a molecular weight (MW) of 32.7 kDa, an isoelectric point (pI) value of 4.29, and no evidence of N-linked glycosylation was found. When acting on milk to provoke curd formation, the M. mucedo DSM 809 proteinase showed maximum potency at pH 5.0 and at 40°C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55°C for 10 min. The cDNA encoding the aspartic proteinase of M. mucedo has been identified (GenBank accession # JN660818). The proteinase gene was cloned into pGAPZαA vector and expressed in Pichia pastoris X-33. Maximum enzyme production was observed when initial medium pH was 3.5 at 20°C and utilizing 4% glucose as a carbon and energy source. The enzyme was secreted in glycosylated form; two potential N-glycosylation sites were elucidated by site directed mutagenesis. All of the generated mutants have exhibited increase in milk clotting activity. The maximum activity obtained was 624 U/ml. Effect of copy number of the expression cassette on the activity has been discussed. Thermal stability studies of the purified proteins revealed that there was almost no effect of glycosylation on the thermal stability of the enzymes. The ratio of milk clotting activity to proteolytic activity for the unglycosylated enzymes was almost 3-fold higher than the glycosylated counterparts. The M. mucedo proteinase / P. pastoris expression system has potential for the industrial production of a novel milk-clotting enzyme for cheese manufacturing
Assessing the Genetic Accessibility of Rhodopirellula baltica SH 1 T
No full textThe marine bacterium Rhodopirellula baltica SH 1 T is a member of the phylum Planctomycetes which are distinguished from other bacteria by their unique properties such as reproduction by budding, intracellular compartments and cell walls lacking murein. In this study, I have worked on two method development projects to gain insights into this unique bacterium. The main aim of my thesis was to develop methods to deliver DNA into Rhodopirellula baltica. A chemical transformation method where chromosomal DNA from chloramphenicol-resistant mutants is used to transform the wild-type competent cells was developed. Other transformation methods, as well as replication of broad host range plasmids and fluorescence-protein expression were explored. A protoplast-formation protocol using enzymatic treatment with lysozyme and osmotic pressure was developed, which can be used in future cell biology studies to further investigate the cell wall structure of Rhodopirellula baltica, as well as for DNA transfer by protoplast transformation. This work is the first demonstration of DNA transfer into Rhodopirellula baltica and one of the few examples of protoplast formation of a Gram-negative bacterium. My second project was to develop an amplification protocol for prokaryotic transcriptomes to be later used for next-generation sequencing. The method was applied to both Rhodopirellula baltica and bacterial communities from the North Sea surface water. The protocol has successfully amplified both the pure-culture transcriptome and the metatranscriptome. This protocol can be used to amplify prokaryotic cDNA for next-generation sequencing and microarray studies where sample size is limited
Polynuclear Transition Metal-Oxo Complexes Stabilized by Heteropolytungstates
No full textPolyoxometalates (POMs) are a remarkable class of inorganic compounds with enormous structural and compositional diversity and potential applications in various fields such as catalysis, analytical chemistry, magnetism, nanotechnology, and medicine. My doctoral work is mainly concerned with incorporation of multinuclear transition metal complexes in polyoxometalate frameworks. In the course of this work we were able to develop a novel, simple and one-pot synthetic strategy for the preparation of high-nuclearity, transition metal-containing POMs. We have now succeeded in preparing several novel paramagnetic and diamagnetic metal substituted polyoxoanions in simple one-pot reactions and structurally characterized in detail using analytical methods like FT-IR, single crystal XRD, powder XRD, multinuclear NMR (1H, 13C,89Y 183W), elemental analysis and thermal analysis (TGA-DTA). We were able to synthesized MnII-containing polyanions [Mn19(OH)12(SiW10O37)6]34- (1), [{Mn(H2O)3}2{Mn2(a-GeW10O38)}3]20- (2), and [{Mn3(B-β-PW9O33(OH))(B-β-PW8O29(OH)2)}2]22- (3). Polyanion 1 represents the highest nuclearity manganese-containing POM to date. The synthesized 16-CoII-containing [{Co4(OH)3PO4}4(A-α-PW9O34)4]28- (8) also represents the largest cobalt aggregate in polyoxotungstate chemistry, and the first example of a POM-based cobalt-core possessing single molecule magnetic behavior. The tetrameric 14-NiII-containing [(Ni14(H2O)10(OH)6(PO3(OH))4(α-P2W15O56)4]34- (5) represents the largest nickel aggregate in polyoxotungstate chemistry. The asymmetric 5-NiII-containing [Ni5(H2O)4(OH)4(b-GeW9O34)(b-GeW8O31)]14- (6) and the dimeric, sandwich-type polyanion [(NaOH2)2Ni2(P2W15O56)2]18- (7) were also isolated. The synthesized 15-CuII-containing [(A-a-SiW9O34)4Cu15O4(OH)8Cl]27- (8) represents the polyanion with the second largest copper-oxo-hydroxo core. The YIII-containing polyanions [{Y(H2O)(B-a-H2SbW9O33)}3(CH3COO)3(WO4)]17- (9a) and [{Y(H2O)3}8(As2W19O68)3(As2W19O66)(W2O6)2(WO6)]43- (10) have also been synthesized and structurally characterized. The solid-state frameworks, composed of cocrystallized trinuclear ruthenium cations and polyanions were also synthesized. These materials exhibit nanosized voids filled with crystal waters. These water molecules can be removed reversibly upon heating under a vacuum, and powder XRD measurements demonstrated that the crystallinity of the compound was preserved. Sorption studies on ethanol and methanol were also performed
A network characterization of metabolix flux predictions, medium-dependant essentiality and metabolic inconsistency
No full textThe thesis presented here is summarizing and interconnecting a series of articles on the network organization of metabolic and gene regulatory processes. These publications are the product of my PhD work. They cover the following scientific questions: (i) Does the spatial distribution of genes on a chromosome deviate from randomness, and if so, does it contribute to the transcriptional regulation of genes? (ii) Is the transcriptional regulation of metabolic processes predominantly accomplished by the classically conceived form of control that is exerted by the digital actions of transcription factors, or does the homeostatic control of chromosome and chromatin structure play a significant role, which is an analog type of regulation? (iii) Is it generally possible to quantify the impact of perturbations to the regulatory machinery and environment of an organism by the integration of gene expression profiles and genome-scale metabolic reconstructions? (iv) Can we assign topological markers to different forms of perturbations in the systems under investigation, e.g., the medium-dependent essentiality of reactions or metabolically altered system states in cancer cells? An introduction to these topics is provided in the first part of this thesis. In addition, concluding remarks and a future outlook will be given at the end of the thesis
Microbial Community Ecology of Temperate Coastal Sands
No full textEmerging high-throughput molecular techniques coupled with community ecology theories offer promising opportunities to better understand microbial ecology. In this thesis, patterns of diversity, community structure and ecology were investigated on temperate coastal sands over a 2-year period. Contextual environmental parameters were measured and bacterial diversity was described by applying automated rRNA intergenic spacer analysis (ARISA) and 454 massively parallel tag sequencing (MTPS) on sandy samples. In the first chapter, comparing ARISA with 454 MPTS led to high differences in community turnover (i.e. 50% with ARISA and 70-80% with 454 MPTS), but to similar patterns in community structure and its response to the environment. This study validates the robustness of applying ARISA together with 454 MPTS for a high resolution description of microbial ecology. The second chapter proposes a pipeline (MultiCoLA, www.ecology-research.com), which systematically truncates proportions of rare or dominant bacterial types from complex community data sets and tests its effect on the resulting ecological interpretation. With about 40% of the rare bacterial types removed from the original data set, a similar ecological signal was still obtained. This confirmed the importance of defining subsets of the microbial community for a consistent ecological interpretation. The third chapter gives a more thorough interpretation of each fraction of the bacterial community in temperate coastal sands. Dominant bacterial types presented similar ecological patterns as that of the overall community while rare types patterns were different. Rare types fluctuations were driving the really high turnover of the microbial community through depth and time and such fluctuations were linked to biogeochemical parameters. The accomplishments of this PhD thesis shed light on main processes shaping microbial communities, by constructing robust bases in microbial community ecology
Minimal Energy Control of an ESN Pattern Generator
No full textIn this report we present a method of adding a feedback control mechanism to an echo state network (ESN) pattern generator in order to modulate its output patterns with the purpose of tracking slowly varying control targets, e.g. shift, amplitude, or frequency of an oscillatory pattern. A proofof- principle case study is presented where a basic ESN is trained to produce a stable sinewave oscillation with fixed shift, amplitude and frequency. With the controller in place, the system demonstrates that the shift, amplitude and frequency of the produced sine waveform can be modulated simultaneously by suitably generated slow varying control signals inserted into the network. Furthermore, an equilibration procedure is introduced to relearn ESN weights such that the equilibrated ESN pattern generator can approximately reproduce the reservoir dynamics across the controllable range, with the feedback control loop switched off. As a result, when reconnecting the feedback control loop to the equilibrated ESN, the energy of the control signals are many orders of magnitude smaller compared to the native system
Verbalization and Communication Effects on Mental Representations and Judgments
No full textWays and means of verbalization and communication are diverse and so are their effects on mental representations and judgments. Accordingly, the present thesis aimed at investigating effects of verbalization and communication on mental representations and judgments from various angles using a multimethod approach. Three different papers are presented: In the first paper, two experiments demonstrate the saying-is-believing effect using visual target material: Participants remembered a target persons everyday behavior (Experiment 1) and a forensically relevant event (Experiment 2) presented in videos in accordance with their audience-tuned message. Participants communication had a greater impact on their mental representations in the evaluation of a previously presented event when they experienced a socially shared reality with their audience. The research reported in the second paper investigated the impact of linguistic abstraction used in communicated descriptions on mental representations in the linguistic category model framework. In this experiment, participants applied the level of linguistic abstraction from their descriptions of behavioral events to abstraction levels in their mental representations of the events, only when they communicated with an in-group (vs. out-group) audience. For the first time, an "abstracting-is-believing" effect, depending on the audiences group membership, was revealed. In the third paper, two experiments examined retrieval ease effects on subsequent memory judgments in the applied field of eyewitness memory. It was demonstrated that centrality of recalled information can have a strong impact on individuals memory judgments: When recalled information was peripheral, participants mnemonic certainty declined. In summary, the present five experiments provide evidence that mental representations and mnemonic judgments of individuals can be profoundly shaped by their own verbalization and communication of events
Central visual pathways and neurotransmitters in the freshwater molluscs Lymnaea stagnalis and Planorbarius corneus
No full textThe Mollusca are a large and extremely varied animal phylum and especially species of the class Gastropoda are popular subjects for neurobiological research. Eyes of gastropods have already received a great deal of attention, but their central visual pathways are still largely unknown for most of the species. Investigation in this direction should be extremely useful for our understanding of the structural basis of vision and behavioural reactions in gastropods. The aims of this thesis were, therefore, to investigate the central visual pathways and possible neurotransmitters in two species of freshwater pulmonate gastropods: Lymnaea stagnalis and Planorbarius corneus. We discovered that: 1) the central visual pathways in L. stagnalis and P. corneus are extensive, neurons and fibres present in all ganglia except the buccal ones; 2) the distribution of the identified cells in both species is similar; 3) processes of photoreceptors form a dense bundle of many fine axons, i.e., the optical neuropil; 4) the two eyes are connected through the cerebral ganglia; 5) some identified central neurons are probably polymodal; 6) the central pathways of pallial and intestinal nerves are widespread and include projections to the optic nerves as well; 7) structural connections between visual pathways and neurons of the pedal ganglia, as well as the statocysts are present; 8) the optic nerve in L. stagnalis enters the cerebral ganglion under a common sheath with tentacular and small labial nerves and that these three nerves exchange some fibres; 9) serotonin and FMRF-amide are present in the eyes and in the optic nerves of both species and likely perform efferently controlled modulation of the adaptational state of the retinas; 10) the chemical nature of the connection of two eyes in both species is neither serotonin nor FMRF-amide. The obtained results are analyzed and discussed in the light of data available on some known behavioral reactions in these species
Optics in thin-film silicon solar cells with periodic surface texture
No full textFor silicon based thin-film solar cells, photon management strategies such as efficient light incoupling and light trapping within the absorbing material are imperative for realizing efficient solar cells. In this thesis, the optical enhancements in microcrystalline thin-film silicon solar cells with periodic surface texture were investigated. Using Finite Difference Time Domain (FDTD) and Rigorous-Coupled Wave Analysis (RCWA) algorithms, the optical wave propagation in the solar cell structure was calculated by rigorously solving the Maxwells equations in two- and three-dimensions. By studying the influence of the period and height of the surface texture, the design of the structures were optimized to achieve higher short circuit currents and quantum efficiencies. Enhancement of the short circuit current in the blue part of the spectrum (wavelengths 300-500 nm) is achieved for smaller periods of the texture (P<200 nm), whereas the short circuit current in the red and infrared part of the spectrum (wavelengths 700-1100 nm) is increased for periods of the texture (P=900nm) comparable to the optical wavelength. Furthermore, the influence of the solar cell thickness on the upper limit of the short circuit current was investigated. The numerically simulated short circuit currents were compared to fundamental light trapping limits based on geometric optics. Additionally, the parasitic absorption losses in the solar cell were analyzed. By identifying these key losses, strategies to minimize the losses were also discussed. The results from this thesis show that efficient light trapping techniques, which can guide the propagating light within the absorber layer, becomes more crucial as the solar cells get thinner
Identifying Novel Substrates by Specificity Profile Analysis of Protein Lysine Methyltransferases
No full textIn the cell nucleus the DNA binds to histone proteins and forms a compact structure called chromatin. Both the components of chromatin are subjected to several post-translational modifications which regulate the gene expression. Enzymes (histone acetyltransferases and histone lysine methyltransferases) known to methylate histone protein have also been shown to act on non-histone proteins and methylation and acetylation of non-histone proteins carries many important biological signals, but not many non-histone methylation substrates of protein lysine methyltransferases are known. In this study we have characterised the substrate specificity of histone lysine methyltransferases and based on the specificity data, we identified several novel histone and non-histone substrates. The NSD1 enzyme is a histone lysine methyltransferase enzyme. Mutations of this protein cause the Sotos syndrome. We studied the substrate specificity of NSD1 using the H3 (30-50) sequence as a template. With the obtained consensus sequence motif we identified several novel histone and non-histone NSD1 substrates. We showed that NSD1 could not methylate H4K20, instead it methylates K44 in H4 protein, which is in agreement with our specificity profile. For the first time we showed NSD1 methylates H1 proteins in a variant specific manner; NSD1 methylates K168 in H1.2, H1.3 and H1.5 proteins but not in H1.4. Apart from the novel histone substrates, we also identified several non-histone proteins containing the NSD1 consensus sequence motif and confirmed methylation of 45 novel non-histone peptides and of the (ATRX and Probable U3 Small Nucleolar RNA-associated Protein) proteins. Based on the candidate screening approach, we also identified an automethylation site in NSD1 and confirmed the loss of methylation signal with the corresponding predicted lysine; NSD1-K1769R mutant protein. We also show that the NSD1 Sotos SET domain mutants impair its methyltransferase activity and thus establish a possible deregulation of signalling networks in Sotos patients. SUV39H1 is a H3K9 methyltransferase enzyme which plays a vital role in the formation of heterochromatin. We derived the specificity profile of this enzyme and showed that it mainly recognises an 'RK' motif corresponding to R8 and K9 in the H3 tail. In addition, lysine 4 of the H3 tail is very important for substrate recognition. With the derived specificity profile of SUV39H1 we identified several novel non-histone peptide substrates and confirmed methylation of RAG2, SET8, Jumonji and Sex comb on midleg protein 2 proteins at the protein level, albeit methylation on Jumonji and Sex comb on midleg protein 2 were weak. Similar to the K4 recognition on H3 tail, we have also observed lysine at -5 position with respect to the target lysine is important for SUV39H1 to methylate the newly identified targets RAG2 and SET8. We have shown that methylation of RAG2 alters its sub-nuclear localization and found that the JMJD2A tandem tudor domain interacts with the newly identified targets in a methyl specific manner. SET8 is a H4K20 specific mono-methyl transferase which acts preferentially on H4 integrated into nucleosomes. By employing peptide arrays we have shown that it has long recognition sequence motif covering 7 amino acids (R17H18R19K20V21L22R23). Based on the derived specificity profile, we identified only 4 potential non-histone substrate proteins. But after relaxing the specificity profile we identified several proteins and showed methylation of 22 non-histone peptides. However, apart from p53 and H4 proteins, none of the identified targets were methylated at the protein level. Celluspot analysis revealed that symmetric and asymmetric methylation on R17 of H4 tail further inhibits methylation on H4K20, while other modifications on K16 and R19 affected H4K20 methylation partially. In summary, our specificity analysis results and methylation assays demonstrate that SET8 as a highly specific histone H4 methyltransferase enzyme. The SMYD family of protein methyltransferases is a group of enzymes which are unique for having a characteristic MYND domain inserted into the catalytic SET domain. The SMYD proteins have roles in the regulation of the cell cycle and important development pathways such as heart and muscle differentiation. A member of this family, SMYD2, is an uncharacterised histone lysine methyltransferase enzyme, which has been shown to methylate both H3K4 and H3K36. In addition, it was also found to methylate one non-histone substrate (p53) and, thereby, repress its activity. Here we applied peptide arrays and derive a specificity profile for SMYD2 via two approaches: a "best target" approach using a p53 peptide as template and an unbiased random approach. Results revealed that SMYD2 possesses a strong preference for a 'LK' or 'FK' motif. With the derived sequence motif, we have identified 40 novel peptide substrates from human proteins and for 8 proteins we showed methylation at the protein level and confirmed the predicted target lysine by mutagenesis. Experiments to show cellular methylation and to understand the possible downstream consequences of methylation of some of the identified non-histone proteins are in progress