>Magister Scientiae - MSc / Diabetes Mellitus is a major global health issue, affecting over 463 million adults in the world. Metformin is the standard drug administered to most people suffering fromdiabetes; however, this medication is contraindicated in many individuals, like most of the medicines developed to combat diabetes. Many diabetic patients turn to herbal medicines due to their renowned traditional use and fewer side effects.While the beneficial effects of phytotherapy are very evident, separation of nontoxic from toxic phytochemicals is still a challenge. Phytonanotherapy is a branch in nanotechnology that seeks to find the middle ground between the fast-acting mechanism of conventional drugs which also present with long lasting or severe toxic side effects, and the slowacting mechanism of phytotherapy which presents with less severe side effects. As such, the aim of this study was to pioneer the investigation of gold nanoparticles biosynthesized using the Carprobrotus edulis fruit aqueous extract (CeFe-AuNPs) as potential treatment for diabetes mellitus. Previously optimized conditions were used to synthesize CeFe-AuNPs which were concurrently characterized using UV-Vis, dynamic light scattering, High Resolution – Transmission Electron Microscopy and Fourier Transform Infrared Spectroscopy techniques. The physichochemical stability of CeFe-AuNPs in phosphate buffer saline, 0.5 % bovine serum albumin, water and 10 % NaCl was also investigated. The effect of CeFe and CeFe-AuNPs on glucose uptake by yeast cells was investigated using 5, 10 and 25 mM glucose reactions. Acute toxicity of CeFe and CeFe- AuNPs was conducted in female Wistar rats (n = 20) and major organs were analyzed through the haematoxylin-eosin stain. The anti-diabetic effects of the CeFe (200 and 400 mg/kg) and CeFe- AuNPs (100 and 200 mg/kg) were investigated in male Wistar rats divided into seven group (n = 6). Histopathology of the pancreas, and the serum insulin were determined.

Classification can be used to predict unknown functions of proteins by using known function information. In some cases, multiple sets of data are available for classification where prediction is only part of the problem, and knowing the most reliable source for prediction is also relevant. Our goal is to develop classification techniques to find the most predictive of the multiple data sets that we have in this project. We use existing classification techniques like linear and quadratic classifications and statistical relevance measures like posterior and log p analysis in our proposed algorithm, which is able to find the data set that is expected to give the best prediction. The proposed algorithm is used on experimental readings during cell cycle of yeast and it predicts the genes that participate in cell-cycle regulation and the type of experiment that provides evidence of cell cycle involvement for any particular gene.

The Heat Shock Protein 90 (Hsp90) is an essential and highly conserved chaperone that facilitates the maturation of a wide array of client proteins, including many kinases. These clients in turn regulate a wide array of cellular processes, such as signal transduction, and transcriptional reprogramming. As a result, the activity of Hsp90 has the potential to influence physiology, which in turn may influence the ability to adapt to new environments. Previous studies using a deep mutational scanning approach, (EMPIRIC) identified multiple substitutions within a 9 amino acid substrate-binding loop of yeast Hsp90 that provides a growth advantage for yeast under elevated salinity conditions and costs of adaptation under alternate environments. These results demonstrate that genetic alterations to a small region of Hsp90 can contribute to evolutionary change and promote adaptation to specific environments. However, because Hsp90 is a large, highly dynamic and multi-functional protein the adaptive potential and evolutionary constraints of Hsp90 across diverse environments requires further investigation. In this dissertation I used a modified version of EMPIRIC to examine the impact of environmental stress on the adaptive potential, costs and evolutionary constraints for a 118 amino acid functional region of the middle domain of yeast Hsp90 under endogenous expression levels and the entire Hsp90 protein sequence under low expression levels. Endogenous Hsp90 expression levels were used to observe how environment may affect Hsp90 mutant fitness effects in nature, while low expression levels were used as a sensitive readout of Hsp90 function and fitness. In general, I found that mutations within the middle domain of Hsp90 have similar fitness effects across many environments, whereas, under low Hsp90 expression I found that the fitness effects of Hsp90 mutants differed between environments. Under individual conditions multiple variants provided a growth advantage, however these variants exhibited growth defects in other environments, indicating costs of adaptation. When comparing experimental results to 261 extant eukaryotic sequences I find that natural variants of Hsp90 support growth in all environments. I identified protein regions that are enriched in beneficial, deleterious and costly mutations that coincides with residues involved in co-chaperone-client-binding interactions, stabilization of Hsp90 client-binding interfaces, stabilization of Hsp90 interdomains and ATPase chaperone activity. In summary, this thesis uncovers the adaptive potential, costs of adaptation and evolutionary constraints of Hsp90 mutations across several environments. These results complement and extend known structural and functional information, highlighting potential adaptive mechanisms. Furthermore, this work elucidates the impact environment can have on shaping Hsp90 evolution and suggests that fluctuating environments may have played a role in the long-term evolution of Hsp90.

This thesis deals with the identification of wine yeasts isolated from the grape must using PCR-RFLP method. The yeasts were isolated from Pinot Noir grape variety must. Grapes were grown and produced in accordance with the requirements placed on organic and integrated farming. Samples were processed in the laboratory, where pure cultures of individual yeast were obtained. A commercial kit was used for yeast DNA isolation. Obtained DNA was used for further analysis. Using the polymerase chain reaction and the primers ITS1 and ITS4 a specific segment of 5.8S rDNA-ITS region was amplified. The PCR products were then detected by electrophoresis in an agarose gel, and after a subsequent purification, three restriction enzymes: HaeIII, HinfI and HhaI were subjected to restriction analysis. The DNA was digested to fragments specific for yeast species and they were detected by agarose electrophoresis. Similarity of these isolates was compared using BioNumerics program and the result is dendrogram of genetic similarity of isolated yeast. The basic chemical analysis of samples must was also performed.

The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. There is significant human nutritional demand for vegetable oil, but its use in production of renewable biomaterials and fuels has intensified the need to further increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). Both DGAT and PDAT contribute to TAG biosynthesis in an independent or overlapping manner, depending on the species. However, preferred pathway for TAG biosynthesis is not well studied in nonseed tissues such as mesocarp. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 was identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGAT1s. These data reveal that the putative DGAT1 of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For further functional analysis, the avocado DGAT1 was expressed in H1246, a TAG-deficient yeast strain and lipotoxicity rescue assays, TLC analysis, Nile Red staining were conducted. The complementation of this yeast strain confirmed enzyme activity and supported the possible role of avocado DGAT1 in TAG biosynthesis. Finally, substrate specificity of DGAT was determined by incubating microsomes with different radiolabeled substances and found that avocado DGAT1 has a preference toward oleic acid (18:1) compared to palmitic acid (16:0) while it is converting diacylglycerol (DAG) to triacylglycerol. In summary, we characterized functional DGAT1 in a basal angiosperm species, which may be metabolically engineered into crop species to produce TAG enriched in oleic acid.

No description available.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 1975 / Vita. / Bibliography: leaves 114-122. / by Marjorie C. Brandriss. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biology

The yeast cell wall provides a first barrier to the environment, confers shape and stability to the cells, and serves as a model for fungal cell wall biosynthesis and function in general. During normal growth, during mating and upon cell surface stress, new wall synthesis is induced by a conserved signaling cascade, the cell wall integrity (CWI) pathway. A signal is initiated by plasma membrane-spanning sensors and transduced through a mitogen-activated protein kinase (MAPK) cascade, which ultimately activates a transcriptional activator, Rlm1. The first part of this thesis analyses the role of this MADS-box transcription factor in the milk yeast Kluyveromyces lactis, which has not been investigated, until now. With respect to the distribution of the upstream CWI sensors, evidence for the existence of a special plasma membrane microcompartment, generally referred to as eisosomes, in the milk yeast is provided in the second part of the thesis. Regarding the transcription factor KlRlm1, its impact on the physiology of K. lactis seems to be different from its homolog in Saccharomyces cerevisiae, ScRlm1, although it clearly acts in CWI signaling, too. Thus, in contrast to the Scrlm1 mutant, a Klrlm1 deletion is sensitive, rather than hyper-resistant, towards Congo red and Calcofluor white, typical stress agents used in cell wall research. Data on cross-complementation of the two genes in the respective heterologous yeast indicate that KlRlm1 and ScRlm1 each perform their optimal function only in the native host.To investigate the impact of a Klrlm1 deletion on the transcriptional profile of K. lactis, data from total mRNA sequencing were analyzed in comparison to a wild-type strain. Many of the genes identified did not correspond to known Rlm1 target genes in S. cerevisiae, but many relate to other stress responses (e.g. KlGRE1, KlFMP16, KLLA0C05324g, KLLA0F18766g, KlUGX2) and to chitin synthesis (KlCHS1, KlSKT5 and KlYEA1), both probably connected to cell wall composition. The functions of a large group of KlRlm1 dependent genes identified here are yet uncharacterized or lack homologs in S. cerevisiae. The plasma membrane of fungi is a specialized organelle, which is ordered into several lateral domains, which we define as microcompartments, since each is composed of a special combination of proteins in their lipid environment. Such microcompartments are believed to control a variety of signaling (and transport) processes in all sorts of eukaryotic cells. Microcompartmentalization is also observed in the yeast plasma membrane, e.g. displayed by the CWI sensors in K. lactis, as shown in this thesis. Since distribution of the latter sensors is reminiscent of that of eisosomes, it was also investigated by live-cell fluorescence microscopy, how KlPil1, KlLsp1 and KlSur7 (all homologs of eisosomal proteins in S. cerevisiae) are distributed. Since they form the typical membrane patches, which are not present in deletion mutants of KlPIL1, the major structural component of eisosomes, one can conclude, that eisosomal microcompartments form in K. lactis and are composed similar to their counterparts in S. cerevisiae. The CWI sensors are excluded from these structures and form their separate microcompartments. The exact physiological function of eisosomes in fungi is still a matter of debate and future studies in K. lactis may help to address this role.

The essential open reading frame YNL152w (now called INN1) of Saccharomyces cerevisiae was previously identified in a screen for negative regulators of cell integrity signaling. Subsequent studies and data from genome-wide functional analyses suggested, that the encoded protein plays a role in cell division. This was further addressed in the thesis presented here. Functional Inn1-GFP fusions were shown to co-localize with the contractile actomyosin ring component Myo1 during cytokinesis. Mutants depleted for Inn1 failed to form a primary septum, but did not affect the dynamics of the cytokinetic actin ring (CAR). This has been attributed to the inability to couple plasma membrane ingression (hence Inn1) to CAR constriction, a phenomenon also found by Sanchez-Diaz et al. (2008). Further investigations focused on the question of how Inn1 is recruited to the bud neck and identified the cytokinetic regulators Hof1 and Cyk3, which act in concert for this purpose. Each of them contains a SH3 domain, which interacts with the proline-rich carboxy-terminal part of Inn1. Localization studies and genetic analyses indicate that Inn1 acts downstream of Hof1 and Cyk3. Either the simultaneous repression of HOF1 and CYK3 gene expression or the deletion of their SH3 domains was lethal, with a concomitant failure to localize Inn1-GFP to the division site. Overproduction of either, Hof1 or Cyk3 perturbed the dynamics of Inn1-GFP distribution, which followed that of the overproduced proteins. This atypical CAR-independent localization of Inn1 supports a presumed role of Hof1 and Cyk3 in an alternative cytokinesis pathway to form a primary septum. Since INN1 is also a multicopy suppressor of a myo1 deletion, this further supports the notion that Inn1 may be required for plasma membrane ingression, also in CAR-independent cytokinesis. Preliminary data suggest, that the protein Vrp1 is responsible for the required removal of Inn1 from the bud neck after completion of cytokinesis. The essential amino-terminal C2 domain of Inn1 may mediate plasma membrane ingression by interaction with the membrane lipid phosphatidic acid, observed in biochemical studies. Alternatively, the C2 domain has been suggested to modulate chitin synthesis in the primary septum by modulating Chs2 activity (Nishihama et al., 2009).

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第12780号 / 論生博第6号 / 新制||生||38(附属図書館) / 30763 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 豊島 文子, 教授 松本 智裕 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM