Influence of the buffer environment on the Relative Biological Effectiveness of carbon ion radiation, investigated by Scanning Force Microscopy and gel electrophoresis

This work focuses on the analysis of the influence of environment on the relative biological effectiveness (RBE) of carbon ions on molecular level. Due to the high relevance of RBE for medical applications, such as tumor therapy, and radiation protection in space, DNA damages have been investigated in order to understand the biological efficiency of heavy ion radiation. The contribution of this study to the radiobiology research consists in the analysis of plasmid DNA damages induced by carbon ion radiation in biochemical buffer environments, as well as in the calculation of the RBE of carbon ions on DNA level by mean of scanning force microscopy (SFM). In order to study the DNA damages, besides the common electrophoresis method, a new approach has been developed by using SFM. The latter method allows direct visualisation and measurement of individual DNA fragments with an accuracy of several nanometres. In addition, comparison of the results obtained by SFM and agarose gel electrophoresis methods has been performed in the present study. Sparsely ionising radiation, such as X-rays, and densely ionising radiation, such as carbon ions, have been used to irradiate plasmid DNA in trishydroxymethylaminomethane (Tris buffer) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) environments. These buffer environments exhibit different scavenging capacities for hydroxyl radical (HO0), which is produced by ionisation of water and plays the major role in the indirect DNA damage processes. Fragment distributions have been measured by SFM over a large length range, and as expected, a significantly higher degree of DNA damages was observed for increasing dose. Also a higher amount of double-strand breaks (DSBs) was observed after irradiation with carbon ions compared to X-ray irradiation. The results obtained from SFM measurements show that both types of radiation induce multiple fragmentation of the plasmid DNA in the dose range from D = 250 Gy to D = 1500 Gy. Using Tris environments at two different concentrations, a decrease of the relative biological effectiveness with the rise of Tris concentration was observed. This demonstrates the radioprotective behavior of the Tris buffer solution. In contrast, a lower scavenging capacity for all other free radicals and ions, produced by the ionisation of water, was registered in the case of HEPES buffer compared to Tris solution. This is reflected in the higher RBE values deduced from SFM and gel electrophoresis measurements after irradiation of the plasmid DNA in 20 mM HEPES environment compared to 92 mM Tris solution. These results show that HEPES and Tris environments play a major role on preventing the indirect DNA damages induced by ionising radiation and on the relative biological effectiveness of heavy ion radiation. In general, the RBE calculated from the SFM measurements presents higher values compared to gel electrophoresis data, for plasmids irradiated in all environments. Using a large set of data, obtained from the SFM measurements, it was possible to calculate the survive rate over a larger range, from 88% to 98%, while for gel electrophoresis measurements the survive rates have been calculated only for values between 96% and 99%. While the gel electrophoresis measurements provide information only about the percentage of plasmids DNA that suffered a single DSB, SFM can count the small plasmid fragments produced by multiple DSBs induced in a single plasmid. Consequently, SFM generates more detailed information regarding the amount of the induced DSBs compared to gel electrophoresis, and therefore, RBE can be calculated with more accuracy. Thus, SFM has been proven to be a more precise method to characterize on molecular level the DNA damage induced by ionizing radiations.

Protein Folding Simulations: Confinement, External Fields and Sequence Design

The present Thesis looks at the problem of protein folding using Monte Carlo and Langevin simulations, three topics in protein folding have been studied: 1) the effect of confining potential barriers, 2) the effect of a static external field and 3) the design of amino acid sequences which fold in a short time and which have a stable native state (global minimum). Regarding the first topic, we studied the confinement of a small protein of 16 amino acids known as 1NJ0 (PDB code) which has a beta-sheet structure as a native state. The confinement of proteins occurs frequently in the cell environment. Some molecules called Chaperones, present in the cytoplasm, capture the unfolded proteins in their interior and avoid the formation of aggregates and misfolded proteins. This mechanism of confinement mediated by Chaperones is not yet well understood. In the present work we considered two kinds of potential barriers which try to mimic the confinement induced by a Chaperon molecule. The first kind of potential was a purely repulsive barrier whose only effect is to create a cavity where the protein folds up correctly. The second kind of potential was a barrier which includes both attractive and repulsive effects. We performed Wang-Landau simulations to calculate the thermodynamical properties of 1NJ0. From the free energy landscape plot we found that 1NJ0 has two intermediate states in the bulk (without confinement) which are clearly separated from the native and the unfolded states. For the case of the purely repulsive barrier we found that the intermediate states get closer to each other in the free energy landscape plot and eventually they collapse into a single intermediate state. The unfolded state is more compact, compared to that in the bulk, as the size of the barrier decreases. For an attractive barrier modifications of the states (native, unfolded and intermediates) are observed depending on the degree of attraction between the protein and the walls of the barrier. The strength of the attraction is measured by the parameter $\epsilon$. A purely repulsive barrier is obtained for $\epsilon=0$ and a purely attractive barrier for $\epsilon=1$. The states are changed slightly for magnitudes of the attraction up to $\epsilon=0.4$. The disappearance of the intermediate states of 1NJ0 is already observed for $\epsilon =0.6$. A very high attractive barrier ($\epsilon \sim 1.0$) produces a completely denatured state. In the second topic of this Thesis we dealt with the interaction of a protein with an external electric field. We demonstrated by means of computer simulations, specifically by using the Wang-Landau algorithm, that the folded, unfolded, and intermediate states can be modified by means of a field. We have found that an external field can induce several modifications in the thermodynamics of these states: for relatively low magnitudes of the field ($<2.06 \times 10^8$ V/m) no major changes in the states are observed. However, for higher magnitudes than ($6.19 \times 10^8$ V/m) one observes the appearance of a new native state which exhibits a helix-like structure. In contrast, the original native state is a $\beta$-sheet structure. In the new native state all the dipoles in the backbone structure are aligned parallel to the field. The design of amino acid sequences constitutes the third topic of the present work. We have tested the Rate of Convergence criterion proposed by D. Gridnev and M. Garcia ({\it work unpublished}). We applied it to the study of off-lattice models. The Rate of Convergence criterion is used to decide if a certain sequence will fold up correctly within a relatively short time. Before the present work, the common way to decide if a certain sequence was a good/bad folder was by performing the whole dynamics until the sequence got its native state (if it existed), or by studying the curvature of the potential energy surface. There are some difficulties in the last two approaches. In the first approach, performing the complete dynamics for hundreds of sequences is a rather challenging task because of the CPU time needed. In the second approach, calculating the curvature of the potential energy surface is possible only for very smooth surfaces. The Rate of Convergence criterion seems to avoid the previous difficulties. With this criterion one does not need to perform the complete dynamics to find the good and bad sequences. Also, the criterion does not depend on the kind of force field used and therefore it can be used even for very rugged energy surfaces.

Optimization in the Technological Steps for the Fabrication of Large Area Micromirror Arrays

Micromirror arrays are a very strong candidate for future energy saving applications. Within this work, the fabrication process for these micromirror arrays has been optimized and some steps for the large area fabrication of micromirror modules were performed. At first the surface roughness of the insulation layer of silicon dioxide (SiO2) was investigated. This SiO2 thin layer was deposited on three different type of substrates i.e. silicon, glass and Polyethylene Naphthalate (PEN) substrates. The deposition techniques which has been used are Plasma Enhanced Chemical Vapor Deposition (PECVD), Physical Vapor Deposition (PVD) and Ion Beam Sputter Deposition (IBSD). The thickness of the SiO2 thin layer was kept constant at 150nm for each deposition process. The surface roughness was measured by Stylus Profilometry and Atomic Force Microscopy (AFM). It was found that the layer which was deposited by IBSD has got the minimum surface roughness value and the layer which was deposited by PECVD process has the highest surface roughness value. During the same investigation, the substrate temperature of PECVD was varied from 80° C to 300° C with the step size of 40° C and it was found that the surface roughness keeps on increasing as the substrate holder temperature increases in the PECVD process. A new insulation layer system was proposed to minimize the dielectric breakdown effect in insulation layer for micromirror arrays. The conventional bilayer system was replaced by five layer system but the total thickness of insulation layer remains the same. It was found that during the actuation of micromirror arrays structure, the dielectric breakdown effect was reduced considerably as compared to the bilayer system. In the second step the fabrication process of the micromirror arrays was successfully adapted and transferred from glass substrates to the flexible PEN substrates by optimizing the conventional process recipe. In the last section, a large module of micromirror arrays was fabricated by electrically interconnecting four 10cm×10cm micromirror modules on a glass pane having dimensions of 21cm×21cm.

Opportunities and constraints of peri-urban buffalo and dairy cattle systems in Faisalabad, Punjab, Pakistan

In the big cities of Pakistan, peri-urban dairy production plays an important role for household income generation and the supply of milk and meat to the urban population. On the other hand, milk production in general, and peri-urban dairy production in particular, faces numerous problems that have been well known for decades. Peri-urban dairy producers have been especially neglected by politicians as well as non-government-organizations (NGOs). Against this background, a study in Pakistan’s third largest city, Faisalabad (Punjab Province), was carried out with the aims of gathering basic information, determining major constraints and identifying options for improvements of the peri-urban milk production systems. For data collection, 145 peri-urban households (HH) engaged in dairy production were interviewed face to face using a structured and pretested questionnaire with an interpreter. For analyses, HH were classified into three wealth groups according to their own perception. Thus, 38 HH were poor, 95 HH well off and 12 HH rich (26.2%, 65.5% and 8.3%, respectively). The richer the respondents perceived their HH, the more frequently they were actually in possession of high value HH assets like phones, bank accounts, motorbikes, tractors and cars. Although there was no difference between the wealth groups with respect to the number of HH members (about 10, range: 1 to 23), the educational level of the HH heads differed significantly: on average, heads of poor HH had followed education for 3 years, compared to 6 years for well off HH and 8 years for rich HH. About 40% of the poor and well off HH also had off-farm incomes, while the percentage was much higher - two thirds (67%) - for the rich HH. The majority of the HH were landless (62%); the rest (55 HH) possessed agricultural land from 0.1 to 10.1 ha (average 2.8 ha), where they were growing green fodder: maize, sorghum and pearl millet in summer; berseem, sugar cane and wheat were grown in winter. Dairy animals accounted for about 60% of the herds; the number of dairy animals per HH ranged from 2 to 50 buffaloes (Nili-Ravi breed) and from 0 to 20 cows (mostly crossbred, also Sahiwal). About 37% (n=54) of the HH did not keep cattle. About three quarters of the dairy animals were lactating. The majority of the people taking care of the animals were family workers; 17.3% were hired labourers (exclusively male), employed by 11 rich and 32 well off HH; none of the poor HH employed workers, but the percentages were 33.7% for the well off and 91.7% for the rich HH. The total number of workers increased significantly with increasing wealth (poor: 2.0; well off:2.5; rich: 3.4). Overall, 69 female labourers were recorded, making up 16.8% of employed workers and one fourth of the HH’s own labourers. Apparently, their only duty was to clean the animals´ living areas; only one of them was also watering and showering the animals. Poor HH relied more on female workers than the other two groups: 27.1% of the workers of poor HH were women, but only 14.8% and 6.8% of the labour force of well off and rich HH were female. Two thirds (70%) of the HH sold milk to dhodis (middlemen) and one third (35%) to neighbours; three HH (2%) did doorstep delivery and one HH (1%) had its own shop. The 91 HH keeping both species usually sold mixed milk (97%). Clients for mixed and pure buffalo milk were dhodis (78%, respectively 59%) and neighbours (28%, respectively 47%). The highest milk prices per liter (Pakistani Rupees, 100 PKR @ 0.8 Euro) were paid by alternative clients (44 PKR; 4 HH), followed by neighbours (40 PKR, 50 HH); dhodis paid lower prices (36 PKR, 99 HH). Prices for pure buffalo and mixed milk did not differ significantly. However, HH obtaining the maximum price from the respective clients for the respective type of milk got between 20% (mixed milk, alternative clients) and 68% (mixed milk, dhodi) more than HH fetching the minimum price. Some HH (19%) reported 7% higher prices for the current summer than the preceding winter. Amount of milk sold and distance from the HH to the city center did not influence milk prices. Respondents usually named problems that directly affected their income and that were directly and constantly visible to them, such as high costs, little space and fodder shortages. Other constraints that are only influencing their income indirectly, e.g. the relatively low genetic potential of their animals due to neglected breeding as well as the short- and long-term health problems correlated with imbalanced feeding and insufficient health care, were rarely named. The same accounts for problems accompanying improper dung management (storage, disposal, burning instead of recycling) for the environment and human health. Most of the named problems are linked to each other and should be addressed within the context of the entire system. Therefore, further research should focus on systematic investigations and improvement options, taking a holistic and interdisciplinary approach instead of only working in single fields. Concerted efforts of dairy farmers, researchers, NGOs and political decision makers are necessary to create an economic, ecological and social framework that allows dairy production to serve the entire society. For this, different improvement options should be tested in terms of their impact on environment and income of the farmers, as well as feasibility and sustainability in the peri-urban zones of Faisalabad.