PhD (Food Science and Technology) / Department of Food Science and Technology / Finger millet (FM) (Eleusine coracana) grains are the staple food in Africa and India for communities with low socio-economic growth. The grains have high content of calcium, dietary fibre, carbohydrates that are rich in protein, fat, iron, phosphorus, vitamin and essential amino acids. Flours from milky cream (MC), brown and black varieties of FM were fortified with vitamin (vit) B₂ and zinc oxide (ZnO) in order to restore the nutrients that were lost during milling and sieving processes. The main aim of the study was to analyse the physical, nutritional composition and functional properties of flours from three (3) FM varieties fortified with vit B2 and ZnO. Pearl millet was used for comparison with the results obtained from this study. MC, brown and black varieties of FM flour treatments were arranged as a completely randomized design (CRD) in three (3) replicates. Physical properties: dimensional properties, colour attributes, kernel weight, bulk density (BD), true density, porosity, surface area, kernel volume, aspect ratio, sphericity and moisture content of the FM grains were analysed. Vitamins and minerals were also analysed. Functional properties: colour attributes, bulk density (BD), water absorption capacity (WAC), oil absorption capacity (OAC), swelling capacity (SC), swelling index (SI), foaming capacity (FC), foaming solubility (FS), dispersibility (Dis), viscosity (cool & hot paste), pasting properties, thermal properties and microscopic structure of FM flours were determined. Data generated were analysed using SPSS version 23.0. Moisture content of MC FM grain and flour was significantly higher (p < 0.05) than brown and black varieties but was within the specified range of <12%. This indicated the long storage shelf-life of the grains and flours. Length of brown FM grains was significantly higher (p < 0.05) when compared to MC and black varieties. MC variety was significantly higher than other varieties in width, kernel weight, bulk density, true density, aspect ratio and sphericity. The information obtained from this study will help in designing suitable equipment for planting, harvesting, storage, processing and packaging of grains. Pearl millet grain was significantly higher than FM flours in all physical properties. MC FM was significantly higher iii in L* (whiteness/ brightness), b* (yellowness/ blueness), C* (chroma), Hº (hue angle) and ZnO values compared to brown and black flours. MC raw fortified finger millet (RFFM) and instant fortified finger millet (IFFM) flours were significantly higher in vitamin B2. People prefer fortified MC flour because most of people living in South Africa use white maize as their staple food and it is similar to MC. Colour is the most important quality parameter in food processing industries and it is also attracts the consumer’s choice and preference. The vitamins and minerals that were lost during sieving, drying and milling were replaced by the two (2) fortificants added which may help to reduce the micronutrient deficiencies. Milky cream FM flours showed significantly high (p < 0.05) values in BD, WAC, OAC, SI, FC and dis. These are the fundamental physico-chemical properties that reflect the interaction between the composition and molecular structure. Length of brown FM grains was significantly higher (p < 0.05) when compared to MC and black variety. The MC variety was significantly higher than other varieties in width, kernel weight, bulk density, true density, aspect ratio and sphericity. Pearl millet was significantly higher than FM flours in all physical properties. MCFM variety was significantly higher in L* (whiteness/ brightness), b* (yellowness/ blueness), C* (chroma), and Hº (hue angle) values as compared to other FM varieties. Moisture content of MC flour was significantly higher as compared to brown and black FM flours. Pasting properties of MC raw finger millet (RFM) and RFFM flours showed significantly higher in peak viscosity (PV) (3518 cP), final viscosity (FV) (6554 cP), breakdown viscosity (BDV) (703 cP) and setback viscosity (SV) (3868 cP) as compared to other RFM and RFFM flours. Brown instant finger millet (IFM) and IFFM flours had significantly higher of PV (723 cP), trough viscosity (TV) (655 cP) and FV (983 cP). IFFM flours had significantly higher values of PV (712 cP), TV (614 cP) and FV (970 cP), respectively. The instant pearl millet (IPM) and instant fortified pearl millet (IFPM) showed significantly higher value in BDV (330 cP and 131 cP) and SV (362 cP and 346 cP), respectively. Thermal properties of black RFM was significantly higher in onset temperature (To) (74.31), peak temperature (TP) (81.94ºC) and conclusion temperature (Tc) (89.64) and RFFM starches were significantly high in To (81.80ºC), TP (84.22ºC) and TC (92.81ºC), respectively. Milky ivcream IFM starch showed significantly higher values in TO (84.57ºC), TP (87.27ºC), Tc (92.81ºC) and ΔH (7.63ºC) compared to other starches. Brown IFFM flour was significantly higher value in To (85.88ºC) and TP (87.14ºC). The starch of IFPM was significantly higher in enthalpy of gelatinisation (6.91ºC) and PHI (2.94ºC) as compared to other FM starches. Brown IFM flour showed significantly higher value in gelatinisation temperature range (13.50ºC). The microscopic structure revealed that RFM and RFFM flours had oval and smooth surfaces. RFM and RFFM flours showed positive effect after fortificants were added because the starch granules, protein bodies and protein matrix were visible and formed crystal. IFM and IFFM flours had some holes, dents and rough surfaces. Instant fortified flours showed negative effect after fortificants were added due to technological processes such as recooking and remilling of flours. Different variations observed on physical, nutritional and functional properties after adding the fortificants may affect the physical, chemical and organoleptic properties of food produced from fortified flour. There is need to fortify FM flours with ZnO which may help to replace the value lost during milling and sieving operations. It is possible that the availability of ZnO could reduce the incidence of some non-communicable diseases. / NRF

Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...

<p>QCR 20161026</p>

Worldwide, millets are regarded as significant grains; however, they are the least exploited. Millet grain is abundant in nutrients and health-beneficial phenolic compounds, making it suitable as food and feed. The diverse contents of nutrients and phenolic compounds present in finger and pearl millet are good indicators that the variety of millet available is important when selecting it for use as food or feed. The phenolic properties found in millets comprise of phenolic acids, flavonoids, and tannins, which are beneficial to human health. Research has shown that millet phenolic properties have high antioxidant activity. Phytochemicals present in millet grains have positive effects on human health by lowering the cholesterol and phytates in the body. The frantic demands on maize and its uses in multiple industries have merit the search for alternative grains, to ease the pressure. Substitution of maize with pearl and finger millets in the diets of different animals resulted in positive impact on the performance. Of late, millet grain has been incorporated in other foods and used to make traditional beverages. In Chapter 1, the topic of the research was introduced, stating the importance of the study and to motivate on the significance of millet grains. Aims and objectives were also listed. Chapter 2 presented extensive literature review on millet and their uses in human and livestock nutrition. In addition, the use of tannin as alternative feed was also reviewed. Different studies have been conducted to investigate the suitability of millet grain as an energy source, in the animal industry. However, studies on the Southern African types of millet are limited; this might be due to lack of information on their nutritional composition and their suitability as animal feed. Overall, the aim of this study was to profile the nutritional characteristics of selected millet grains in South Africa and Zimbabwe, their suitability as energy source for human and livestock and to study the effect of pearl millet type on the performance indices of Ross 308 broiler chickens. In Chapter 3, the general materials and methods used to reach the scientific conclusion for this study was summarised. In Chapter 4, physical and chemical analysis were conducted on millet grains obtained from South Africa and Zimbabwe, the results revealed that the physiochemical characteristics of the millet grain qualify it as a suitable candidate in replacing maize as an energy source. To further understand the characteristics of the millet grain, Chapter 5 analysed the phenolic compounds available in the millet grain obtained from South Africa and Zimbabwe. The results showed that the grain is endued with valuable phenolic compounds beneficial in the nutrition of both human and animals and aid as health benefit. Chapter 6 investigated the various inclusion levels of pearl millet in a bid to establish the most suitable combination for the poultry sector. Pearl millet grain used in this experiment went through different breeding improvements and selections conducted at the Grain Crop Institute in Potchefstroom, South Africa. Pearl millet grain was used as the main source of energy for Ross 308 broiler chicks for a period of 42 days with performance indices investigated. The results revealed that pearl millet can be incorporated in the diets of broiler chickens, in replacement of maize, without adversely affecting the performance. In addition, the economic justification of replacing maize with pearl millet was studied. The cost of the grains was determined and the cost per weight gain was determined. The results showed that indeed it is economically sound to replace maize with pearl millet in the diet of poultry. The quadratic function best fitting optimum treatment combination in relation to performance parameters such as body weight, body weight gain, feed conversion ratio and internal organs was also examined. In Chapter 7, the research was generally discussed to tie up the conclusions of the experiments conducted. General recommendations were also given on the compounds of millet varieties and their health benefits to both humans and animals. / Kuwo wonke umhlaba, amabele athathwa njengezinhlamvu ezibalulekile, kodwa awasetshenziswa kakhulu. Uhlamvu lwebele lunemisoco eminingi kanye nenzuzo yempilo ngama-phenolic compound, okwenza ukuthi afaneleke kakhulu njengokudla kwabantu kanye nemfuyo. Imisoco eminingi equkethwe kanye nama-phenolic compound atholakala eminweni kanye nezinhlamvu zebele ayizinkomba ezinhle zokuthi izinhlobo zamabele ezitholakalayo zibaluleke kakhulu ekukhethweni njengokudla kanye nokudla kwabantu noma imfuyo. Amaphenolic properties atholakala kumabele aqukethe ama-phenolic acids, ama-flavonids, kanye nama-tannins, ayinzuzo kakhulu empilweni yabantu. Ucwaningo luthole ukuthi ama-phenolic properties amabele anomsebenzi wezinga eliphezulu lama-antioxidant. Ama-phytochemicals atholakala kwizinhlamvu zamabele anenzuzu enhle kakhulu kwimpilo yabantu ngokwehlisa izinga le-cholesterol kanye nama-phytate emzimbeni. Ukudingeka kakhulu kombila kanye nokusetshenziswa kwawo kwizimboni eziningi, kubangele ukuthi kwenziwe ucwaningo ngezinye izinhlamvu ukwehlisa ingcindezi. Ukuthatha isikhundla sombila, sithathwa amabele kwidayethi yezilwane ezehlukene kwaba nomphumela omuhle kakhulu ngokusebenza. Kamuva nje, izinhlamvu zamabele zifakelwe kwezinye izidlo ezisetshenziswa ukwenza iziphuzo zomdabu. Kwisahluko 1, isihloko socwaningo sethulwa khona, ukuchaza ukubaluleka kocwaningo kanye nokuqikelela ngokubaluleka kwezinhlamvu zamabele. Izinhloso nezinjongo nazo zifakelwe kuhla. Isahluko 2, sethula ukubuyekezwa kwemibhalo ngamabele kanye nokusetshenziswa kwawo kubantu kanye nokudla kwemfuyo. Nangaphezu kwalokho, ukusetshenziswa kwe-tannin njengokunye ukudla kwemfuyo kuye kwabuyekezwa. Kwenziwe ucwaningo olwehlukene ukuphenyisisa ngokufaneleka kwezinhlamvu zamabele njengomthombo wamandla (we-eneji) kwimboni yezilwane. Kodwa, ucwaningo ngezinhlobo zamabele eNingizimu ne-Afrika alukenziwa ngokwanele; lokhu kungenzeka kungenxa yokuswelakala kolwazi ngemisoco equkethwe kanye nokufaneleka njengokudla kwezilwane. Kanti ngokwengamele, inhloso yalolu cwaningo bekuwukwenza iprofayili yemisoco ngezinhlamvu zamabele athile eNingizimu Afrika kanye naseZimbabwe, ukufaneleka kwawo njengomthombo wamandla (we-eneji) kubantu kanye nemfuyo, kanye nokwenza ucwaningo ngemiphumela yenhlobo yamabele ngama-performance indices wamachwane e-ROss 308. Kwisahluko 3, kusetshenziswe imetheriyali kanye namamethodi asetshenzisiwe ukufinyelela isiphetho sezesayense kulolu cwaningo, kwafinyezwa. Kwisahluko 4 kwenziwe uhlaziyo lokubambekayo kanye namakhemikhali ngezinhlamvu zamabele ngokutholakale eNingizimu Afrika kanye naseZimbabwe, imiphumela ikhombise ukugqama kwe-physiochemical kwezinhlamvu zamabele ukufaneleka kwazo njengekhandideti ekuthatheni isikhundla sombila njengomthombo wamandla. Ukuqhubeka nokuqondisisa ukuphawuleka kwezinhlamvu zamabele, iSahluko 5 sihlaziye ama-phenolic compound kwizinhlamvu zamabele aseNingizimu Afrika neZimbabwe. Imiphumela ikhombisa ukuthi uhlamvu lwamabele lunama-phenolic compound ayinzuzo ekudleni okunomsoco kubantu kanye nezilwane, kanye nokuba wusizo lwenzuzo kwimpilo. Isahluko 6 siphenyisise ngamazinga okubandakanywa kwamabele ukwenzela ukuthola ukufaneleka kwawo kumkhakha wezinkukhu. Izinhlamvu zamabele e-pearl zisetshenziswe kule ekspirimenti, eyenziwa ezigabeni ezehlukene zokuthuthukisa ukuzalisa kanye nokhetho olwenziwe ngabe-Grain Crop Institute ePotchefstroom, eNingizimu Afrika. Amabele e-pearl asetshenziswe njengomthombo omkhulu we-eneji kumachwane eRoss 308 isikhathi sezinsuku ezingu 42 kanti kwaphenyisiswa ngokusebenza kwama-indices. Imiphumela iveze ukuthi amabele epearl angafakelwa kwidayethi yamachwane, ukuthatha isikhundla sombila, ngaphandle kokuphazamisa ukusebenza. Nangaphezu kwalokho, ukufaneleka kwezomnotho ngokuthatha isikhundla zombila sithathwa ngamabele e-pearl kuye kwacwaningwa. Izindleko zezinhlamvu ziye zabekwa kanti futhi nesisindo ngezinhlamvu kuye kwabekwa. Imiphumela ikhombisa ukuthi kuyinto enhle kwezomnotho ukuthatha isikhundla sombila sithathwa ngamabele e-pearl kwidayethi yezinkukhu. Ukusebenza kwe-quadratic function kufaneleke kakhulu kwi-optimum treatment combination mayelana nama-parameter okusebenza afana nokwenyuka kwesisindo somzimba, ukuguqula i-feed conversion ratio kanye nezitho zangaphakathi nazo ziye zahlolwa. KwiSahluko 7, ucwaningo kuye kwaxoxwa ngalo ukuhlanganisa iziphetho zama-ekspirimenti enziwe. Izincomo ezinabile, ziye zanikezwa ngama-compound ezinhlobo zamabele kanye nezinzuzo zawo kwezempilo kubantu kanye nezilwane / Lefaseng ka bophara, leotša le bonwa bjalo ka mabele a bohlokwa kudu; le ge go le bjale, ke dibjalo tšeo di sa bjalwego kudu. Dithoro tša leotša di tletše ka phepo ye ntši le ditswaki tša fenoliki tšeo di nago le mohola maphelong, gomme se sa dira gore di be maleba bjalo ka dijo le phepo. Dikagare tša lona tšeo di fapafapanego le ditswaki tša fenoliki tšeo di whetšagalago ka gare ga leotša la finger le la pearl ke dilaetši tše kaone tša gore mehutahuta ya leotša yeo e hwetšagalago e bohlokwa ge e kgethwa bjalo ka sejo le phepo. Diteng tša fenoliki tšeo di hwetšwago ka agre ga leotša di na le diesiti tša fenoliki, difolabanoite, le dithaninse, tšeo di lego mohola go maphelo a batho. Dinyakišišo di laeditše gore diteng tša fenoliki tša leotša di na le mošomo wa godimo wa dihlwekišammele tšeo di bitšwago dianthioksitente. Difaethokhemikhale tšeo di lego gona ka gare ga dithoro tša leotša di na le diabe tše kaone go maphelo a batho ka go fokotša kholesterole le difaetheite mmeleng. Dinyakwa tša ka pela go lefela le mešomo ya lona ka diintastering tše ntši di dirile gore go be le nyakego ye kgolo ya dithoro tše dingwe tšeo di ka emelago lefela legato, go nolofatša kgatelelo yeo e beilwego go lefela. Go tšeela lefela legato ka leotša la pearl le la finger ka dijong tša diphoofolo tšeo di fapafapanego go feleleditše ka seabe se sekaone ka ga go šoma ga lona. Go fihla mo lebakeng le, dithoro tša leotša di tsentšwe ka dijong tše dingwe gomme tša šomišwa go dira dino tša setšo. Ka go Kgaolo ya 1, hlogotaba ya dinyakišišo e tsebagaditšwe, ya fa bohlokwa bja dinyakišišo tše le lebaka mabapi le bohlokwa bja dithoro tša leotša. Maike mišetšo le dinepo le tšona di filwe. Kgaolo ya 2 e hlagišitše tekodišišo ya dingwalwa ye e tseneletšego ka ga leotša le mešomo ya lona go phepo ya batho le ya diphoofolo. Godimo ga fao, tšhomišo ya tannin bjalo ka phepo ya boikgethelo le yona e lekodišišitšwe. Dinyakišišo tše di fapafapanego di dirilwe go nyakišiša go ba maleba ga thoro ya leotša bjalo ka methopo wa enetši, ka intastering ya diphoofolo. Le ge go le bjale, dinyakišišo tš0e di dirilwego mabapi le mehuta ya leotša ka Borwa bja Afrika ke tše nnyane; se se ka ba se bakwa ke tlhoklego ya Tshedimošo mabapi le sebiopego sa phepo ka hare ha leotša le go ba maleba ga lona bjalo ka phepo ya diphoofolo. Ka kakaretšo, maikemišetšo a dinyakišišo tše e bile go lebeledišiša dikokwane tša phepo tša dithoro tša leotša tšeo di kgethilwego ka Afrika Borwa le ka Zimbabwe, go ba maleba ga lona bjalo ka methopo wa enetši go batho le go diruiwa le go nyakišiša ka ga seabe ka ga mohuta wa leotša wa pearl go go šoma ga dipalopalo go dikgogo tša nama tša Ross 308. Ka go Kgaolo ya 3, ditlabelo le mekgwa ka kakaretšo yeo e šomišitšwego go fihlelela sephetho sa tša mahlale sa dinyakišišo tše di filwe kakaretšo. Ka go Kgaolo ya 4, tshekatsheko ya naga le ya dikhemikhale e dirilwe mabapi le dithoro tša leotša tšeo di hweditšwego ka Afrika Borwa le ka Zimbabwe, dipoelo di utollotše gore dikagare tša dikhemikhale tša thoro ya leotša di le dira le be lebele leo le loketšego go tšeela legato lefela bjalo ka methopo wa enetši. Go kwešiša go tšwela pele dikagare tša thoro ya leotša, Kgaoilo ya 5 e sekasekile diteng tša fenoliki tšeo di hwetšagalago ka gare ga thoro ya leotša leo le hwetšago ka Afrika Borwa le ka Zimbabwe. Dipoelo di laeditše gore thro ya leotša le tletše ka dikagare tša fenoliki tšeo di lego mohola go phepo ya bobedi batho le diphoofolo le gore le thuša bjalo ka kholego ya tša phepo. Kgaolo ya 6 e nyakišišitše maemo a mehutahuta a kakaretšo a leotša la pearl ka nepo ya go hwetša motswako wa maleba kudu ka lekaleng la dikgogo. Dithoro tša leotša la pearl tšeo di šomišitšwego ka mo tekodišišong ye di sepedišitšwe ka go dikaonafatšo tše di fapanego tša monontšha gomme dikgetho di dirilwe ka go Sehlongwa sa Dibjalo tša Dithoro ka Potchefstroom, ka Afrika Borwa. Dithoro tša leotša la pearl di šomišitšwe bjalo ka mothopo wa enetši go matswiana a nama a Ross 308 mo matšatšing a 42 fao go dirilwego dinyakišišo ka ga dipalopalo tša go gola ga dikgogo. Dipoelo di laeditše gore leotša la pearl le ka tsenywa ka dijong tša dikgogo tša nama, go tšeela legato lefela, ka ntle le go ama gampe go gola ga dikgogo. Godimo ga fao, lebaka la tša ekonomi la go tšeela lefela legato ka leotša la pearl le nyakišišitšwe. Theko ya dithoro e hweditšwe gomme theko ka boima bjo itšego le yona e hweditšwe. Dipoelo di laeditše gore ka nnete go tloga go kwagalago kudu go tša ekonomi go tšeela lefela legato ka leotša la pearl. Mošomo wa tekanelo wa wo o loketšego bokaone motswako wa tlhokomelo ya godimo mabapi le mahlakore a kgodišo ya dikgogo a go swana le boima bja mmele, go nona, rešio ya go fetošetša dijo le ditho tša ka gare le ona o lekodišišitšwe. Ka go Kgaolo ya 7, dinyakišišo di hlalošitšwe ka kakaretšo gore go fihlelelwe sephetho ka ga ditekodišišo tšeo di dirilwego. Ditšhišinyo ka kakaretšo le tšona di filwe mabapi le dikagare tša mehutahuta tša leotša le dikholego tša ona go tša maphelo go bobedi batho le diphoofolo / College of Agriculture and Environmental Sciences / D. Phil. Agr. (Animal Science)

This paper describes our continuing studies on stabilization of peptide structures in supersecondary conformations that are designed to mimic conformational antigenic epitopes. In this work we have used the consensus Cys2His2 zinc-finger peptide motif as a template to engineer and synthesize antigenic loop peptide segments from two protein antigens, lactate dehydrogenase C4 isozyme (LDH-C4) and human chorionic gonadotropin β subunit (β-hCG). Confirmation that the engineered peptide constructs assumed a zinc-finger conformation was obtained by absorption spectroscopy of the Co2+ complexes. The circular dichroism (CD) spectra of the free peptides show random coil conformations, while the Zn2+-complexed peptides acquired the zinc-finger motif upon titration with Zn2+, as evidenced by the appearance of absorbances indicating α-helix and some β-conformation. No peptide aggregation was observed, as these peptides were monomeric under all conditions tested. In order to examine the immunogenicity of the zinc-finger constructs, one sequence from LDH-C4 (ZFLMVF) and two sequences from β-hCG (ZF2TT3 and ZF4TT3) were selected and chimeras were synthesized to incorporate promiscuous T-cell epitopes from either tetanus toxoid or measles virus. The ZFLMVF construct was highly immunogenic in rabbits, and the ZF2TT3 and ZF4TT3 peptides were highly immunogenic in both mice and rabbits, eliciting high-titer antipeptide antibodies specific for their immunogenic sequences. However, the antibodies raised to the zinc-finger constructs showed minimal reactivity against their respective native protein antigens as determined by ELISA. This is surprising in the case of β-hCG, since the ZF2 zinc-finger peptide was an effective inhibitor of binding of anti-β-hCG-loop(38-57) antibodies to whole hCG, as assessed by a competitive inhibition radioimmunoassay. This implies that, although the cyclized 40-52 sequence from βhCG and the zinc-finger peptide ZF2 exhibit similar conformations in solution, the zinc-finger engineered loop is apparently not in a sufficiently correct conformation for antibody recognition of native hCG. Our results with the LDH-C4 zinc finger loop imply that antibody recognition of antigen involves specific side-chain interactions that must be maintained by a precise conformation.

Ubiquitination is a posttranslational modification with immense impact on a wide range of cellular processes, including proteasomal degradation, membrane dynamics, transcription, translation, cell cycle, apoptosis, DNA repair and immunity. These diverse functions stem from the various ubiquitin chain types, topologies, and attachment sites on substrate proteins. Substrate recruitment and modification on lysine, serine or threonine residues is catalyzed by ubiquitin ligases (E3s). An important E3 that decides about the fate of numerous substrates is the HECT-type ubiquitin ligase HUWE1. Depending on the substrate, HUWE1 is involved in different processes, such as cell proliferation and differentiation, DNA repair, and transcription. One of the transcription factors that is ubiquitinated by HUWE1 is the MYC interacting zinc finger protein 1 (MIZ1). MIZ1 is a BTB/POZ (Bric-à-brac, Tramtrack and Broad-Complex/Pox virus and zinc finger) zinc finger (ZF) protein that binds to DNA through its 13 C2H2-type zinc fingers and either activates or represses the transcription of target genes, including genes involved in cell cycle arrest, such as P21CIP1 (CDKN1A). The precise functions of MIZ1 depend on its interactions with the MYC-MAX heterodimer, but also its heterodimerization with other BTB-ZF proteins, such as BCL6 or NAC1. How MIZ1 interacts with HUWE1 has not been studied and, as a consequence, it has not been possible to rationally develop tools to manipulate this interaction with specificity in order to better understand the effects of the interaction on the transcriptional function of MIZ1 on target genes or processes downstream. One aspect of my research, therefore, aimed at characterizing the MIZ1-HUWE1 interaction at a structural level. I determined a crystal structure of the MIZ1-BTB-domain in complex with a peptide, referred to as ASC, derived from a C terminal region of HUWE1, previously named ‘activation segment’. The binding mode observed in this crystal structure could be validated by binding and activity assays in vitro and by cell-based co-IP experiments in the context of N-terminally truncated HUWE1 constructs. I was not able to provide unambiguous evidence for the identified binding mode in the context of full-length HUWE1, indicating that MIZ1 recognition by HUWE1 requires yet unknown regions in the cell. While the structural details of the MIZ1-HUWE1 interaction remains to be elucidated in the context of the full-length proteins, the binding mode between MIZ1BTB and ASC revealed an interesting, atypical structural feature of the BTB domain of MIZ1 that, to my knowledge, has not been described for other BTB-ZF proteins: The B3 region in MIZ1BTB is conformationally malleable, which allows for a HUWE1-ASC-peptide-mediated β-sheet extension of the upper B1/B2-strands, resulting in a mixed, 3 stranded β-sheet. Such β-sheet extension does not appear to occur in other homo- or heterodimeric BTB-ZF proteins, including MIZ1-heterodimers, since these proteins typically possess a pre-formed B3-strand in at least one subunit. Instead, BCL6 co repressor-derived peptides (SMRT and BCOR) were found to extend the lower β-sheet in BCL6BTB by binding to an adjacent ‘lateral groove’. This interaction follows a 1:1 stoichiometry, whereas the MIZ1BTB-ASC-complex shows a 2:1 stoichiometry. The crystal structure of the MIZ1BTB-ASC-complex I determined, along with comparative binding studies of ASC with monomeric, homodimeric, and heterodimeric MIZ1BTB variants, respectively, suggests that ASC selects for MIZ1BTB homodimers. The structural data I generated may serve as an entry point for the prediction of additional interaction partners of MIZ1 that also have the ability to extend the upper β-sheet of MIZ1BTB. If successful, such interaction partners and structures thereof might aid the design of peptidomimetics or small-molecule inhibitors of MIZ1 signaling. Proof-of-principle for such a structure-guided approach targeting BTB domains has been provided by small-molecule inhibitors of BCL6BTB co-repressors interactions. If a similar approach led to molecules that interfere with specific interactions of MIZ1, they would provide intriguing probes to study MIZ1 biology and may eventually allow for the development of MIZ1-directed cancer therapeutics. / Ubiquitinierung ist eine posttranslationale Modifikation mit weitreichendem Einfluss auf eine Vielzahl von zellulären Prozessen, wie proteasomale Degradation, Membrandynamik, Transkription, Translation, Zellzyklus, Apoptose, DNA-Reparatur und Immunität. Grundlage für diese Diversität ist die Möglichkeit, dass Substrate an unterschiedlichen Stellen mit verschiedenen Ubiquitin-Kettentypen modifiziert werden können. Die Substratrekrutierung und -modifikation an Lysin-, Serin oder Threonin Resten wird durch Ubiquitin-Ligasen (E3s) katalysiert. Eine wichtige Ubiquitin-Ligase, die zahlreiche Substrate reguliert, ist die HECT-Ligase HUWE1. Abhängig vom Substrat ist HUWE1 an verschiedenen Prozessen, wie der Zellproliferation und -differenzierung, DNA-Reparatur, aber auch Transkription beteiligt. Ein Transkriptionsfaktor, der von HUWE1 ubiquitiniert wird, ist MIZ1 (MYC interacting zinc finger protein 1). MIZ1 ist ein BTB/POZ (Bric-à-brac, Tramtrack and Broad-Complex/Pox Virus and Zinc finger) Zinkfinger(ZF)-Protein, das über seine 13 C2H2 Zinkfinger an DNA bindet und so die Transkription von verschiedenen Zielgenen aktivieren oder reprimieren kann. MIZ1-Zielgene sind unter anderem am Zellzyklusarrest beteiligt, wie z.B. das Gen P21CIP1 (CDKN1A). Die biologischen Funktionen von MIZ1 werden unter anderem durch seine Interaktion mit dem MYC MAX-Heterodimer, aber auch durch Heterodimerisierung mit anderen BTB ZF Proteinen, wie BCL6 oder NAC1, reguliert. Wie MIZ1 mit der HUWE1-Ligase interagiert, wurde bislang strukturell noch nicht untersucht, weshalb noch nicht gezielt kleine Moleküle zur Manipulation der Interaktion entwickelt werden konnten, um Einfluss auf die transkriptionellen Funktionen von MIZ1 oder seiner Zielgene zu nehmen. Meine Untersuchungen zielten daher unter anderem darauf ab, die MIZ1-HUWE1-Interaktion auf struktureller Ebene zu charakterisieren. Ich konnte eine Kristallstruktur der MIZ1-BTB-Domäne in Komplex mit dem HUWE1-Peptid ASC lösen, dessen Sequenz in der C-terminalen Region von HUWE1 zu finden ist und zuvor als „activation segment“ definiert wurde. Der in dieser Kristallstruktur beobachtete Bindungsmodus konnte durch Bindungs- und Aktivitätsassays in vitro und durch co-IP-Experimente in zellbasierten Assays validiert werden, jedoch nur im Zusammenhang mit N-terminal verkürzten HUWE1 Konstrukten. Es war mir nicht möglich, diesen Bindungsmodus im Kontext des HUWE1-Proteins voller Länge nachzuweisen, was darauf hindeutet, dass bei der MIZ1-Erkennung durch HUWE1 in der Zelle andere Regionen beteiligt sein könnten. Während die strukturellen Details der MIZ1-HUWE1-Interaktion im Kontext der Proteine voller Länge noch aufgeklärt werden müssen, zeigte der Bindungsmodus zwischen MIZ1BTB und ASC ein atpyisches Strukturmerkmal der BTB-Domäne von MIZ1, das meines Wissens bislang in keinem anderen BTB-ZF-Protein beschrieben wurde: Die B3-Region in MIZ1BTB zeigt eine untypische konformationelle Flexibilität, die es erlaubt, dass das HUWE1-ASC-Peptid die B1/B2-Stränge im oberen Segment von MIZ1BTB zu einem 3-strängigen β-Faltblatt erweitert. Eine solche β-Faltblatt-Erweiterung scheint in anderen homo- oder heterodimeren BTB-ZF-Proteinen, einschließlich MIZ1-Heterodimeren, nicht aufzutreten, da diese Proteine typischerweise bereits einen B3-Strang in mindestens einer Untereinheit aufweisen. Stattdessen konnte beobachtet werden, dass Peptidliganden, wie sie von den BCL6 Co-Repressoren SMRT und BCOR abgeleitet wurden, ein β-Faltblatt im unteren Segment von BCL6BTB erweitern, indem sie in der sogenannten „lateral groove“ binden, die in unmittelbarer Nähe des betreffenden β-Faltblattes lokalisiert ist. Während die Interaktion von BCL6BTB mit Co-Repressor-Peptiden eine 1:1 Stöchiometrie zeigt, beobachtete ich für den MIZ1BTB-ASC-Komplex eine 2:1 Stöchiometrie. Die Kristallstruktur des MIZ1BTB-ASC-Komplexes, zusammen mit Bindungsassays, die die Interaktion zwischen ASC und monomerem, homodimerem bzw. heterodimerem MIZ1BTB untersuchten, deuten darauf hin, dass ASC spezifisch mit MIZ1BTB-Homodimeren interagiert. Daher könnten die von mir gewonnenen Strukturinformationen dazu dienen, weitere MIZ1-Bindungspartner vorherzusagen. Falls erfolgreich, könnten die neu identifizierten Interaktionspartner und zugehörige Strukturen dazu genutzt werden, Peptidomimetika und niedermolekulare Inhibitoren zu entwickeln, die spezifische Interaktionen von MIZ1 und die zugehörigen zellulären Prozesse stören und somit als Werkzeuge zum besseren Verständnis der MIZ1 Biologie dienen könnten. Vorbild dabei können zahlreiche niedermolekulare Verbindungen sein, die zur Störung der Co-Repressor-Peptid-Bindung an BCL6BTB entwickelt wurden. Wenn es auf ähnliche Weise gelänge, spezifischen Einfluss auf die transkriptionelle Funktion von MIZ1 zu nehmen, so könnte dies von hohem therapeutischen Nutzen in der Bekämpfung verschiedener Krebsarten sein.

This master project addresses the problem of stage fright in a musical performance context. To meet my personal, uncomfortable feelings of stage fright, I decided to “prepare” my body for such situations by doing breathing exercises developed by Ilse Middendorf (The Perceptible Breath). With help and support of the Berliner Centrum für Musikermedizin at the Charité the project became a pilot study about how Ilse Middendorf's breathing exercises could minimize bodily stress of a non-wind instrument player in a stress situation. Following method was applied. I did breathing exercises over a period of six weeks. I made three recordings of the same cello piece that I know well (Josef Haydn’s Cello Concerto in D-major, 1. movement, exposition): before the six-week period, in the middle of it and at the end. The recordings represent the stress situations. The impact of breathing exercises on my cello practice and recordings were evaluated in three ways. I documented my subjective perceptions of my personal development with a questionnaire and a report. The objective results were then documented in two ways. The three recordings were sent out to professional audience members who did not know in which order recordings were done. They listened and rated them in order of their quality. Finally, I measured a series of body parameters which indicate the level of stage fright (e.g. heart rate, finger temperature, blood pressure) before and after the recordings. The results of my project are quite encouraging. On the subjective level, I felt less stress, more musicality and a new body awareness. The recordings were rated by audience members in exactly the order in which I recorded them which indicates a musical quality improvement. The body measurements showed that breathing exercises lead to a decrease in bodily stress symptoms (blood pressure and heart rate).

Acetylation of wood is a modification technique that chemically alters the wood substance and enhances several properties of wood. The basic principle is to impregnate wood with acetic anhydride to react and replace OH-groups with acetyl groups in the wood cell wall. In this way, the hygroscopicity of the modified wood is significantly reduced resulting in increased dimensional stability and durability compared with unmodified wood.The objective of this work was to study finger-jointing of acetylated Scots pine (Pinus sylvestris L.) using a conventional melamine urea formaldehyde (MUF) adhesive. Two different types of acetylated pine specimens were investigated, acetylated pine sapwood (APS) and acetylated juvenile pine (AJP), the latter originating from young forest thinning trees (ca 20-30 years). The goal was to evaluate the bending strength, i.e. modulus of rupture (MOR), of such finger-jointed samples, in particular when the acetylated wood was combined with unmodified wood, in this case, Norway spruce (Picea Abies L. Karst) (US). The finger-jointing were performed at Moelven Töreboda by applying their existing industrial procedures. In total, five different of finger jointed sample groups were prepared combining the different specimens: APS-APS, AJP-AJP, US-US, APS-US, and AJP-US. Standardized procedures were used to determine the MOR of the finger-jointed samples, both unexposed at the factory condition state and after a water-soaking-drying cycle. In addition, the experiments also included determination of the moisture content (MC), density, and modulus of elasticity (MOE) (in bending along the grain) of the individual specimens.At the unexposed state, the APS-APS samples showed the highest MOR of 63,1 MPa, while those of the AJP-AJP showed the lowest value of 42,4 MPa. The corresponding values for the US-US, AJP-US and APS-US samples was 56,7, 47,5 and 46,9 MPa, respectively. In contrast to a typical wood failure for the US-US samples, a low amount of wood failure was observed in all cases involving the acetylated wood, indicating a low adhesive anchoring in the wood substrate at the finger-joint, although a surprisingly high strength was obtained for the APS-APS samples. A significantly lower MC content of 4,9 % and a remarkably low value of 1,7 %, was found for the APS and AJP, respectively, compared with 9,2% for the US. The significantly lower MC combined with an assumed increased hydrophobicity of the acetylated wood possible causes a less effective MUF-wood bonding, or adhesion, compared with that of the unmodified wood. Possible, so-called over penetration of the MUF resin in the acetylated wood could also be an explanation for the poor wood-adhesive anchoring. The MOE of the individual APS, AJP and US specimens was 12,6, 8,3 and 11,4 GPa, respectively, indicating a significantly lower mechanical performance of AJP, and hence also of finger-joints of AJP, despite its very low MC, possible due to a higher microfibril angle in the cell walls in juvenile wood compared with mature wood. No clear correlation was found between the MOR and density of the acetylated samples.For the samples exposed to a water-soak-drying cycle, the highest MOR, and lowest reduction of 14 % compared with the unexposed state, was obtained for the US-US samples, whereas all samples involving the acetylated wood showed a distinctly higher reduction. The MOR of the AJP-AJP and AJP-US samples were reduced with 47 % and 50 %, respectively, while the MOR of the APS-APS and APS-US samples were reduced with 43 % and 23 %, respectively. It should be emphasized, however, that after the standard drying-time, which was the same for all samples, the acetylated samples, compared with the untreated ones, did not dry out to the same level as for the dry unexposed state, i.e. the acetylated samples had a high MC of ca 30-40% in these MOR tests. This high MC level could be the main reason for the dramatic strength losses. Furthermore, a less efficient wood-MUF adhesion as well as the drying under acidic conditions may also be possible causes for the reduced bending strength of the finger-jointed samples with acetylated wood.

Missing page 10. / <p> Interbedded turbidite sandstones and shales of the Sonyea Group are exposed in the Finger Lakes Stone Quarry near Ithaca, New York. In 28% of the samples studied the orientation of the grains was not significantly different from a uniform distribution. These distributions included some that were bimodal and some obtained from a bed showing cross-stratification. In 60% of the samples the grain orientations could be considered parallel to the flute marks. As the top of the massive interval was approached the grain orientation became increasingly aligned with the flute marks. A difference of 14° was found between the vector means of the flute and tool marks. </p> <p> Radiographs made of rock slabs cut from the samples used in grain orientation determinations frequently failed to show internal structures even though these were visible in the samples. A radiograph of one sample (02-08) showed cross-bedding, not visible in the sample or slab. The absence of internal structures in a radiograph do not exclude their being present. </p> / Thesis / Bachelor of Science (BSc)

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