In the presence of a non-fermentable carbon source, nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae undergo a meiotic program called sporulation to form gametes called spores. While four spores are produced under standard laboratory sporulation conditions, spore number is known to be regulated by carbon availability: under carbon-depleted conditions, yeast cells package a portion of the four haploid meiotic nuclei into spores. Our lab has demonstrated that these unpackaged meiotic products undergo programmed nuclear destruction (PND) that is associated with apoptotic-like DNA fragmentation. Nevertheless, the mechanism that mediates PND remained to be elucidated. Here, I describe the execution of PND through an unusual form of autophagy that has not been documented previously in yeast. This form of autophagy is most similar to megaautophagy in plants and lysosomal membrane permeabilization in mammals. My results demonstrate further diversity in cell death programs in unicellular microbes that is potentially conserved across eukaryotes.

In the presence of a non-fermentable carbon source, nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae undergo a meiotic program called sporulation to form gametes called spores. While four spores are produced under standard laboratory sporulation conditions, spore number is known to be regulated by carbon availability: under carbon-depleted conditions, yeast cells package a portion of the four haploid meiotic nuclei into spores. Our lab has demonstrated that these unpackaged meiotic products undergo programmed nuclear destruction (PND) that is associated with apoptotic-like DNA fragmentation. Nevertheless, the mechanism that mediates PND remained to be elucidated. Here, I describe the execution of PND through an unusual form of autophagy that has not been documented previously in yeast. This form of autophagy is most similar to megaautophagy in plants and lysosomal membrane permeabilization in mammals. My results demonstrate further diversity in cell death programs in unicellular microbes that is potentially conserved across eukaryotes.

Programmed cell death (PCD) refers to a genetically controlled process causing the death of certain cells or tissues. In plants PCD is critical in normal development of for instance xylem vessels. A group of proteins called metacaspases are believed to play a pivotal role in PCD in plants. As Metacaspase 9 have been shown to be upregulated in Populus during xylem maturation this study attempted to identify genes affecting its expression in Arabidopsis thaliana by forward genetics using a reporter line with GFP fused to the promoter of Metacaspase 9 (AtMC9). Ethyl methanesulfonate seed mutagenesis was used to generate mutants resulting in eleven mutant lineages with a GFP expression pattern deviating from that of the reporter line. These mutants fell into two categories; low/no-signal mutants and ectopic expressors. Several of the low/no-signal mutants had longer roots at five to eight days after germination, a time point shown to be critical for metaxylem differentiation. Further studies of their roots would reveal whether the developing xylem is abnormal or not. Deep sequencing provided evidence for involvement of abscisic acid and polyamines in regulation of AtMC9 expression. Sequencing from a low/no-signal mutant suggests that AtMC9 expression might be affected also by disturbed lignin biosynthesis. Rescuing mutant lineages through transformations with fully functional forms of the candidate genes is the next step to experimentally validate that the candidate genes are involved in the observed changes in AtMC9 expression in each of the isolated mutants.

ABSTRCT In mammals protein tyrosine phosphorylation plays an important role in the activation of programmed cell death. However, tyrosine phosphorylation involved in cell death has not been examined in plants. These studies demonstrated that D-mannose induced cell death and DNA fragmentation in rice suspension cells. In the presence of mannose for 24 hours, tyrosine phosphorylation of two proteins, 20 kDa and 43 kDa markedly increased. After incubating 3 days, the level of phosphotyrosine accumulation declined in bands of 16 and 20 kDa. In addition, the occurrence of DNA fragmentation and two tyrosine-phosphorylated proteins, 26 kDa and 40 kDa, were detected in aged suspension-cultured cells. The expression of genes that encode mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK signalling pathway, OsMEK and OsMAPK2, are up regulated during D-mannose treatment. The results provide evidence that protein tyrosine phosphorylation as well as MEK/MAPK signalling pathway is associated with cell death in rice.

Programmed cell death (PCD) is an indispensible process in plant and animal systems that serves to eliminate cells and/or tissues and recycle nutrients from these tissues to the rest of the organism. In animals, PCD is referred to as apoptosis and is performed by caspases, a family of aspartate-specific cysteine proteinases that serve to perceive the cell death signal and execute the cell death phenotype. In 2000, Uren et al. discovered a new family of cysteine proteinases in plants called metacaspases – distant arginine/lysine-specific relatives of animal caspases – thought to be involved in plant PCD. The goal of this study was to correlate SlMCA2 expression with PCD in tomato. Polyclonal antibodies were obtained against the Type I metacaspase SlMCA2 in Solanum lycopersicum and used for Western blot analyses. BY-2 cell biolistics and in-situ hybridization were used to investigate where SlMCA2 protein and mRNA accumulate in various tissues. Results produced were replicated a minimum of three times and correlate SlMCA2 to PCD, but not initiation of PCD.

Hemoglobins (Hbs) are heme-containing proteins belonging to the globin superfamily that are ubiquitous in most living organisms including prokaryotes and eukaryotes. In addition to the first legHbs found in leguminous plants, there are another three classes of phytoglobins (Pgbs) identified in various plant species including dicots and monocots. The ability of heme groups to bind gaseous ligands such as oxygen, carbon monoxide and nitric oxide (NO) places Pgbs as multifunctional players in various processes during plant growth and development under normal or stress conditions. The objective of this project is to investigate how transcriptional manipulation of ZmPgb1.1 and ZmPgb1.2 influences somatic embryogenesis in maize (Zea mays). Suppression of either of the two genes is sufficient to induce programmed cell death (PCD) through a pathway initiated by accumulation of nitric oxide (NO) and zinc (Zn2+), and mediated by production of reactive oxygen species (ROS). The effect of the death program on the fate of the developing embryos is dependent upon the localization patterns of the two Pgbs. During somatic embryogenesis, ZmPgb1.2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, while ZmPgb1.1 transcripts extend to several embryonic domains. Suppression of ZmPgb1.2 induces PCD in the anchoring cells allowing the embryos to develop further, while suppression of ZmPgb1.1 results in massive PCD leading to embryo abortion. Cells suppressing the Pgb genes are also depleted of endogenous auxin (indole-3-acetic acid, IAA) localization established by polar auxin transport (PAT), thus suggesting a possible involvement of this plant hormone in the observed processes. Collectively, it appears that the cell specific expression of Pgbs has the capability to determine the developmental fate of embryogenic tissue during maize somatic embryogenesis through their effect on PCD. This novel regulation has implications for development and differentiation in other species.

Abstract Failure to maintain an appropriate balance between cell death and proliferation is partly due to derangements in the regulation of apoptosis. In this work, apoptosis and the expression of apoptosis regulating proteins were studied by 3' - end labeling of fragmented apoptotic DNA (TUNEL) and immunohistochemistry in a set of 147 tissue samples consisting of 44 biopsies of normal and dysplastic bronchial epithelium, and 103 non-small cell lung carcinomas. The quantity of apoptotic cells and bodies, apoptotic index (AI%), is defined as a percentage of apoptotic cells in the entire tumor cell population. Changes in the apoptotic activity were already seen in the metaplasia-dysplasia-carcinoma sequence of the lung, where the AI% increased gradually until moderate epithelial dysplasia but started to decrease after that. Thus, the AI% for invasive NSCLC (1.20 for squamous cell carcinoma and 1.24 for adenocarcinoma) was slightly lower than in premalignant bronchial epithelium (mean 1.50), but clearly higher than in normal tissue (0.20 for normal bronchial epithelium and 0.24 for lung interstitial cells). 53% of SQCCs and 50% of ACs showed p53 positive nuclei indicative of mutated p53 protein. The immunostaining of bcl-2, bax and mcl-1 revealed diffuse, cytoplasmic staining and was present in most tissues studied. No statistically significant associations between the extent of apoptosis and the expression of p53, bcl-2, bax, or mcl-1 could be found, although . The immunostaining for caspases 3, 6 and 8 was restricted to the tumor areas, reflecting increased apoptotic activity in them. The AI% was significantly higher in NSCLCs in which the single-cell staining pattern for caspase-8 was dominant (P = 0.017), whereas the expression of caspases 3 and 6 had no association with apoptosis. The number of apoptotic cells was significantly higher in NSCLC tumors with a high number of CD3+ and CD8+ T-lymphocytes (P = 0.01) and B-cells (P = 0.05). By multivariate analysis, enhanced apoptosis in NSCLC showed a 1.9-fold risk (95% CI 1.04–3.60; P = 0.04) and p53 positivity a 2.3-fold risk (95% CI 1.30–4.10; P = 0.005) for a shortened survival. Both factors appeared as independent prognostic variables. Apoptosis is clearly enhanced in premalignant and malignant lung tissue in comparison with normal tissue. Furthermore, the expression of the apoptosis-regulating genes is different in tumor tissue from that in normal tissue, and some of the changes in their expression can be seen even in the premalignant lesions of the bronchial epithelium. The expression of caspases seen only in tumor tissue implies the activation of the apoptotic mechanisms and, thus, the lowered treshold of tumor cells to undergo apoptosis. Even in the advanced stages of the disease, the immune defense is effective and the cytotoxic action of activated CD8+ T-cells clearly involves apoptosis. Based on these results it is concluded that alterations in the apoptotic activity and changes in the expression of apoptosis-regulating genes are associated with malignant transformation and growth in lung tissue.

A variety of diseases lead to the atrophy and/or death of skeletal muscle. To better understand the molecular mechanisms that mediate these processes, I have taken advantage of the intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, which undergo sequential programs of atrophy and programmed cell death at the end of metamorphosis. ISM death is mediated by changes in gene expression and numerous cell-death associated transcripts have been identified. MicroRNAs (miRs) are small (~22 nucleotide) non-coding RNAs that bind to sequences in messenger RNAs (mRNAs) and either cause translational arrest or mRNA degradation. To test the hypothesis that developmentally regulated miRs may control the stability and/or translatability of target mRNAs in the ISMs, putative mRNA targets for the test miRs have been identified and their 3’ untranslated region (UTR) have been cloned into a dual luciferase reporter plasmid. The microRNA mir-92b binds to the 3’ UTR of the Small Cytoplasmic Leucine Rich repeat Protein (SCLP) mRNA. Expression of miR-92b declines during development and SCLP expression increases with the commitment to die. I found that the miR-92b inhibits luciferase mRNA translation (spectrophotometric plate assays), but does not lead to transcript degradation (quantitative polymerase chain reaction; qPCR). miR-92 plays a survival role in several mammalian tissues and is repressed in two types of cardiomyopathy. Consequently, understanding how miRs regulate mRNA translation and stability may provide a better understanding of the regulation of muscle atrophy and death as well as provide novel tools for diagnostics or therapeutics.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21303号 / 医博第4392号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM