Endo-β-mannanase is an endohydrolase enzyme responsible for the breakdown of mannan-containing polysaccharides common in the cell walls of many plants. The action of endo-β-mannanase in barley, its optimum temperature and pH for action, temporal and spatial localization, activity in the presence of hormones and sugars and its effect on the seed's mechanical strength were assayed. The development of a spectrophotometric assay for endo-β-mannanase detection was also trialed. The optimum temperature and pH for these experiments were found to be 37℃ and pH 7. Using these parameters, the endo-β-mannanase enzyme was found to be initially localized in the seed coat and moved through to the endosperm over time. The detected level of enzyme activity increased in the presence of gibberellic acid and glucose, or decreased when abscisic acid was added. Similar results were seen when the embryo was removed and the endosperm and seed coat were incubated in hormone- and sugar-containing media. The presence of exogenous endo-β-mannanase did not affect the mechanical strength of the seed but there was a strong correlation between increasing endo-β-mannanase activity and decreasing mechanical strength over time. The spectrophotometric assay for quantifying endo-β-mannanase in extracts showed promise but did not reach fruition due to unexplained sources of variation. The localization and regulation of endo-β-mannanase in barley were similar to those seen in other plants, such as tomato, lettuce and coffee. These findings have biotechnological applications within the brewery industry. By increasing the mobilization of reserves such as mannan, it is thought that the seedling can utilize this secondary carbohydrate source instead of, or at least supplementing, glucose which was mobilized from starch. This will theoretically reduce the starch and glucose lost during the malting period leaving a higher sugar content free for fermentation.

In this work phase transformations in metastable β Ti-15Mo alloy were studied using electrical resistivity measurements. The alloy was subjected to a solution treatment at a temperature higher than β- transus and quenched in water. In this condition, the microstructure of Ti-15Mo alloy consists of a metastable β - matrix and ω-phase particles. During in-situ electrical resistivity measurement in a specially designed furnace, significant temperature points which indicate phase transformations in the material were detected. The dependence of electrical resistivity on the temperature changes during heating between increasing and decreasing according to the ongoing phase transformation. The changes were observed at temperatures 225, 356 and 560 řC. A further study of these phase transformations using electrical resistivity measurements was performed on various heat treated specimens. In order to control the microstructure evolution in the material, scanning and transmission electron microscopy was used. Mechanical properties were studied using Vickers microhardness testing. The obtained results serve to identify the type and sequence of phase transformations which take place in the Ti-15Mo alloy. Powered by TCPDF (www.tcpdf.org)

Student No. 9404705H MDent Research Report School of Oral Sciences / Expression of the E-cadherin/β-catenin complex in oral squamous cell carcinoma and its correlation with histomorphology and metastasis. The immunohistochemical expression of E-cadherin and β-catenin was examined in 30 primary oral squamous cell carcinomas in patients with (n=19) and without (n=11) nodal metastasis, as confirmed on histopathological examination of the resected regional lymph nodes. The corresponding primary and nodal metastases tissue samples were available for 17 patients. The 30 primary carcinomas were histologically graded according to the invasive tumour front grading system and by conventional Broders’ criteria. None of the 30 primary carcinomas showed homogenous, membranous E-cadherin and β-catenin expression when compared to the normal oral squamous epithelium. Staining was heterogeneous in 73% (22/30) and in 77% (23/30) of the primary carcinomas stained for E-cadherin and β-catenin respectively. There was a highly significant reduction (P<0,001) of both E-cadherin and β- catenin expression with progression from the well-differentiated areas to the less differentiated tumour cells at the invasive tumour front. At the invasive tumour front, however, irrespective of the nodal status and invasive tumour front grading score, 28/30 (93%) tumours showed loss of E-cadherin expression. Loss of β-catenin expression was recorded in 22/30 (73%) cases. These findings indicate that E-cadherin and β-catenin play a key role in the loss of differentiation of tumour cells in oral squamous cell carcinoma and while they may be permissive for metastasis, in isolation, E-cadherin and β-catenin are probably not predictive of metastatic potential in oral squamous cell carcinoma.

ABSTRACT MUC1, a mucin-like transmembrane glycoprotein, is highly overexpressed and aberrantly localized in several invasive carcinomas. MUC1 is proposed to play numerous roles in the transformed behaviour of cells in which it is expressed. A number of these roles are facilitated by the interaction of MUC1 with β-catenin, a protein that is central to both cellular adhesion as well as Wnt-responsive gene transcription. The aim of this study was to investigate MUC1 expression, localization, and interaction with β-catenin, as a means of providing insight into the behaviour of human oesophageal squamous cell carcinoma. This cancer-type is exceptionally aggressive and is a major cause of cancer-related morbidity and mortality in South Africa. MUC1 is expressed and aberrantly localized in oesophageal squamous cell carcinoma cell lines, as demonstrated by RT-PCR, western blotting and indirect immunofluorescence. Moreover, evidence from coimmunoprecipitation assays shows that the MUC1 cytoplasmic tail and β-catenin form a complex both at the cell membrane and importantly, within the nucleus of these cell lines. This is the first demonstration of such a complex in the nucleus of a carcinoma derived from stratified, as opposed to simple, epithelia. Data presented here further indicates that activation of the epidermal growth factor receptor results in modulation of the association between MUC1 and β-catenin at the cell membrane. MUC1 membrane-localization, and interaction with β-catenin, may modulate cellular adhesion through steric interference of cell surface adhesion molecules as well as through sequestration of β-catenin away from adherens junctions. On the other hand, MUC1 association with β-catenin may enhance β- catenin signalling either through the stabilization of β-catenin, or as an essential functional component of the β-catenin/LEF/TCF transcription factor complex. Furthermore, results presented in this study identify oesophageal squamous cell carcinoma as a prime candidate for MUC1-specific immunotherapy. This finding is of substantial importance considering the ineffectual nature of existing therapies used in the treatment of oesophageal carcinoma.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2014-09-29T19:48:15Z No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014RuandaSilvaRafael.pdf: 903715 bytes, checksum: 324efd3857a20f7e9735070f16b1b6f9 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2014-10-06T14:07:37Z (GMT) No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014RuandaSilvaRafael.pdf: 903715 bytes, checksum: 324efd3857a20f7e9735070f16b1b6f9 (MD5) / Made available in DSpace on 2014-10-06T14:07:37Z (GMT). No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014RuandaSilvaRafael.pdf: 903715 bytes, checksum: 324efd3857a20f7e9735070f16b1b6f9 (MD5) / A enzima β – galactosidase é reconhecida por catalisar a hidrólise da lactose e possibilitar a formação de galactooligossacarídeos. O objetivo do presente trabalho foi estudar diferentes condições de imobilização de β – galactosidases de Aspergillus oryzae e Kluyveromyces lactis utilizando suporte comercial Immobead. Ambas as enzimas foram imobilizadas nos suportes tratados e não tratados com etilenodiamina e submetidas a processos de imobilização uni e multipontual. Os derivados também foram avaliados quanto ao bloqueio dos grupamentos epóxi com glicina. As análises de estabilidade ao armazenamento sob refrigeração, estabilidade térmica, ciclos de reuso para hidrólise da lactose, determinação das propriedades cinéticas e determinação de pH e temperatura ótimos foram realizadas nos derivados obtidos. Os resultados de eficiência de imobilização variaram de 30 a 50% e os valores de rendimento variaram entre 80 e 90%. Modificações químicas no suporte foram realizadas utilizando etilenodiamina com o objetivo de gerar modificações químicas no suporte, causando a rápida adsorção de enzimas e favorecendo a formação de ligações covalentes multipontuais em tempo reduzido. Verificou-se que suportes modificados com etilenodiamina imobilizaram a mesma carga de enzimas em menor tempo, quando comparados a suportes sem modificação. Entretanto, a significativa perda de atividade verificada nesses suportes durante os ciclos de reuso sugere que a superfície do suporte possa ter sido modificada em sua totalidade, dificultando a formação de ligações covalentes e permitindo a lixiviação de enzimas para o meio reacional. Derivados não bloqueados apresentaram perda considerável de atividade enzimática durante a armazenagem, indicando a ocorrência de possíveis distorções da enzima, ocasionadas pela interação de grupamentos epóxi livres. Ensaios submetidos à imobilização multipontual apresentaram melhorias em sua estabilidade térmica. Os valores de Km para as enzimas imobilizadas de K. lactis e A. oryzae foram 49,69 e 55,29 mM, valores superiores àqueles verificados para as enzimas livres (19,11 e 17,37 mM, respectivamente), indicando possíveis alterações conformacionais na estrutura da proteína, resultantes do processo de imobilização. Os resultados indicaram que derivados não tratados com etilenodiamina, submetidos à imobilização covalente multipontual e bloqueio com glicina apresentaram os resultados mais expressivos para as condições estudadas de estabilidade ao armazenamento, estabilidade térmica e ciclos de reuso para hidrólise de lactose. Esses derivados não apresentaram distorções em relação às condições ótimas de temperatura e pH quando comparadas com as respectivas enzimas livres. As β – galactosidases de A. oryzae e K. lactis submetidas à imobilização covalente multipontual no suporte Immobead posteriormente bloqueado com glicina apresentaram as melhores propriedades para futura aplicação industrial.

1 / Rongrong Zhang

<p>The beauty of the wide functionality of proteins and peptides in Nature is determined by their ability to adopt three-dimensional structures. This thesis describes artificial molecules developed to mimic secondary structures similar to those found crucial for biological activities.</p><p>In the first part of this thesis, we focused on post-translational modifications of a class of unnatural oligomers known as <i>β</i>-peptides. Through the design and synthesis of a glycosylated <i>β</i>³-peptide, the first such hybrid conjugate was reported. In this first report, a rather unstable 3₁₄-helical structure was found. Subsequently, a collection of six new glycosylated <i>β</i>³-peptides was synthesized with the aim to optimize the helical stability in water.</p><p>The ability of natural proteins, i.e. lectins, to recognize the carbohydrate residue on these unnatural peptide backbones was investigated through a biomolecular recognition study.</p><p>The second part of this thesis concerns the design of conformationally homogeneous scaffolds, which could be of importance for biomedical applications. In paper V, four- and five-membered cyclic <i>all</i>-<i>β</i>³-peptides were investigated for this purpose. In a subsequent paper, a completely different strategy was employed; herein, the ability of a single <i>β</i>²-amino acid to restrict the conformational freedom of a cyclic α-peptide was studied. </p><p>In the third part of this thesis, we synthesized and investigated the folding propensities of novel backbone modified oligomers, i.e. <i>β</i>-peptoids (<i>N</i>-substituted <i>β</i>-Ala) with α-chiral side chains.</p><p>The collective results of these studies have established the procedures required for synthesis of glycosylated <i>β</i>-peptides and deepened our understanding of the factors governing folding among such oligomers. Moreover, it was established that <i>β</i>-amino acids can be a useful tool to increase conformational stability of cyclic peptides.</p>

The beauty of the wide functionality of proteins and peptides in Nature is determined by their ability to adopt three-dimensional structures. This thesis describes artificial molecules developed to mimic secondary structures similar to those found crucial for biological activities. In the first part of this thesis, we focused on post-translational modifications of a class of unnatural oligomers known as β-peptides. Through the design and synthesis of a glycosylated β3-peptide, the first such hybrid conjugate was reported. In this first report, a rather unstable 314-helical structure was found. Subsequently, a collection of six new glycosylated β3-peptides was synthesized with the aim to optimize the helical stability in water. The ability of natural proteins, i.e. lectins, to recognize the carbohydrate residue on these unnatural peptide backbones was investigated through a biomolecular recognition study. The second part of this thesis concerns the design of conformationally homogeneous scaffolds, which could be of importance for biomedical applications. In paper V, four- and five-membered cyclic all-β3-peptides were investigated for this purpose. In a subsequent paper, a completely different strategy was employed; herein, the ability of a single β2-amino acid to restrict the conformational freedom of a cyclic α-peptide was studied. In the third part of this thesis, we synthesized and investigated the folding propensities of novel backbone modified oligomers, i.e. β-peptoids (N-substituted β-Ala) with α-chiral side chains. The collective results of these studies have established the procedures required for synthesis of glycosylated β-peptides and deepened our understanding of the factors governing folding among such oligomers. Moreover, it was established that β-amino acids can be a useful tool to increase conformational stability of cyclic peptides.

Cancer remains one of the most common causes for death in the world today. Researchers are continuously trying to improve old, and develop new, methods in order to strife this global problem. Much research is being made trying to find new specific biomarkers that can be used to detect and diagnose cancer in an early stage. One candidate protein for possible future use as a biomarker is tripeptidyl-peptidase II (TPPII) which has previously been shown to be up-regulated in Burkitt´s lymphoma. This paper focuses on the expression of TPPII on an mRNA-level to see if there is any difference between expression in human leucocytes from patients with a leukemia diagnosis and a healthy volunteer, in order to evaluate if the expression of TPPII have any future use as a biomarker. Patient samples were analyzed using real time qPCR, to study the expression of mRNA, and Western blot, in order to correlate the mRNA findings with protein expression. Three different cell lines with different characteristics regarding expression and function of TPPII were also used to validate the methods used and for comparison with the patient samples analyzed. A difference in expression of mRNA were seen between the different patient samples, both individually and between larger groups of samples with the same diagnosis, indicating a large individual variation, thus making future use in a clinical setting difficult. However, seeing as only a few samples were analyzed in this study, more research must be done in order to draw any final conclusions.

β – Tricalcium phosphate (β-TCP, Ca3(PO4)2) is a commonly used bone substitute material due to its biocompatibility and resorption. This study focused on the production of almost fully dense β-TCP ceramics with varying degrees of impurities (Ca/PO4 ratio, addition of 5% Mg). Three methods were used to produce the β-TCP ceramics, uniaxial pressing, slip-casting and isostatic pressing. In this study the isostatic pressing and sintering at 1150ºC for 20h and 15min, resulted in the densest β-TCP ceramics (97.7-99.2%). No significant differences of grain size and density could be detected between the samples produced with various compositions. These isostatically pressed samples sintered at 1150ºC were then dissolved in 0.08M aceticacid solution to simulate the in vivo resorption. It was found that the samples containing extra Mg dissolved slower. Attempts to determine the chemical composition of the grain boundaries were made without success. However, SEM observations of partly dissolved β-TCP ceramics revealed that the grain boundaries dissolved faster than the grains. The study was performed at the RMS foundation in Switzerland.