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Ghrelin is a 28 amino acid peptide hormone with a unique octanoic acid modification that has an extensive range of physiological effects, including stimulation of growth hormone (GH) release, appetite regulation, and modulation of reproductive functions. The cognate receptor for ghrelin is the growth hormone secretagogue receptor (GHS-R), a G protein-coupled receptor with two documented isoforms, the functional GHS-R type 1a and the C-terminally truncated GHS-R type 1b. Several ghrelin variants have also been identified in addition to the n-octanoylated form of ghrelin. In our laboratory, we have identified a novel exon 3-deleted preproghrelin variant that retains sequence for the mature ghrelin hormone and also encodes a novel C-terminal peptide (designated as C-terminal 3 peptide). There is emerging evidence to suggest that the ghrelin axis, encompassing ghrelin, several ghrelin variants and both forms of the GHS-R, is implicated in tumour growth. The objective of this project is to investigate the role of the ghrelin axis in hormone-dependent cancer and to further characterise the expression and function of the novel exon 3-deleted preproghrelin isoform. Hormone-dependent cancers, including prostate and breast cancers, are significant causes of morbidity and mortality in the Western world. Improved diagnoses and treatments earlier in the progression of the disease are urgently required to improve patient outcomes. Growth factors play an integral role in prostate and breast cancer, particularly in the emergence of aggressive, hormone-refractory disease that is resistant to standard therapies. We have previously identified ghrelin as being a novel growth factor for prostate cancer cells in vitro and have hypothesised that this may be extended to other hormone-dependent cancer types including breast cancer. In the current study, techniques including real-time quantitative RT-PCR, Western blot analysis and immunohistochemistry have been used to determine and quantitate ghrelin, exon 3-deleted preproghrelin and GHS-R expression in prostate and breast cancer. Ghrelin and exon 3-deleted preproghrelin are highly expressed in prostate cancer tissues compared to expression levels in normal prostate glands. Similarly, breast carcinoma specimens display greater immunoreactivity for ghrelin and exon 3-deleted preproghrelin than normal breast tissues. Expression of the exon 3-deleted preproghrelin mRNA isoform is upregulated in the oestrogen-independent, highly malignant MDA-MB-435 breast cancer cell line compared to the non-tumourigenic MCF-10A breast epithelial cell line, suggesting that augmented transcription of the isoform is associated with an increased malignant potential in breast cancer. The functional GHS-R type 1a is expressed in normal breast tissue and breast cancer specimens and cell lines. In contrast, the truncated GHS-R type 1b isoform is exclusively expressed in breast carcinoma. These data suggest that GHS-R type 1b, ghrelin and exon 3-deleted preproghrelin display potential as novel diagnostic markers for prostate and breast cancer. These studies have been the first to demonstrate that ghrelin may have an important role in cell proliferation in breast and prostate cancer. Functional assays demonstrated that (10nM) ghrelin stimulated proliferation in the LNCaP prostate cancer cell lines (45.0 ± 1.7% above control, P &lt0.01) and rapidly activated the ERK 1/2 mitogen-activated kinase (MAPK) pathway in both PC3 and LNCaP cell lines. It does not, however, protect these cells from chemically-induced apoptosis. The MAPK inhibitors PD98059 and U0126 blocked ghrelin-induced MAPK activation, as well as cell proliferation, in both cell lines. Prostate cancer cells secrete mature ghrelin in vitro, and may therefore stimulate MAPK pathways in an autocrine manner. Ghrelin also appears to act as a growth factor in breast cancer cell proliferation, as the growth of MDA-MB-435 and MDA-MB-231 breast cancer cell lines is significantly increased by ghrelin treatment. Our findings suggest that the ghrelin axis could provide an important new target for adjunctive therapies for both breast and prostate cancer. The C-terminal 3 peptide derived from exon 3-deleted preproghrelin may be an important new component of the ghrelin axis and studies into its function are currently in progress. Although it did not induce MAPK cascades or stimulate proliferation in prostate or breast cancer cell lines, the discovery of a murine counterpart, exon 4-deleted preproghrelin, indicates that it is highly conserved. Exon 4-deleted preproghrelin is expressed in all mouse tissues examined, with stomach being the predominant site of synthesis. Other components of the ghrelin axis were also found to be present in a wide-range of mouse tissues including brain, ovary and prostate. This comprehensive report has paved the way for future work with in vivo mouse models of cancer. This study has provided a substantial basis for the further evaluation of ghrelin, exon 3-deleted preproghrelin and the GHS-R type 1b as novel diagnostic/prognostic markers for prostate and breast cancer and supports the rationale for targeting the ghrelin axis for treatment of these tumours. Keywords: Ghrelin, exon 3-deleted preproghrelin, GHS-R, growth factors, MAPK, ERK 1/2, hormone-dependent cancer, prostate, breast, diagnostic/prognostic marker, therapeutic targets.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21661号 / 医博第4467号 / 新制||医||1035(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 篠原 隆司, 教授 小川 修, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Baxa is a pro-apoptotic member of the Bcl-2 protein family that regulates a key point in the control of apoptosis. The bax RNA undergoes a complex pattern of RNA splicing, with eight splice isoforms known to date. The next most abundant isoform to Baxa is Baxp, which has a unique carboxyl-terminal sequence and consequently lacks the transmembrane domain of Baxa. This study characterized Baxp as part of a larger project aimed at deducing the role of cellular localization in Bax protein function. A transient transfection assay was designed to determine the cell death activity of a protein in adherent cells. Baxp induced cell death to a greater extent than Baxa when transiently expressed in NIH 3T3 cells. The levels of Baxp expression were always low, regardless of the cell type or transfection method used. Additionally, Baxp adopted a conformation in which amino acids 13 to 19 are accessible to the monoclonal antibody 6A7. In Baxa this epitope is only exposed in the membrane-bound activated conformation that is associated with accessibility of the BH3 domain of Baxa that mediates protein-protein interactions among Bcl-2 family members. Although the mechanism remains elusive, Baxp was identified as a potent inducer of cell death. / Thesis / Master of Science (MSc)

In most eukaryotes, alternative splicing is an important regulatory mechanism of gene expression that results in a single gene coding for multiple protein isoforms, thus largely increases the diversity of the proteome. RNA-seq is widely used for genome-wide splicing isoform quantification, and several effective and powerful methods have been developed for splicing analysis with RNA-seq data. However, it remains problematic for genes with low coverages or large number of isoforms. These difficulties may in principle be ameliorated by exploiting correlations encoded in the structured data sources. This thesis contributes to developments of Bayesian methods for splicing analysis by leveraging additional information in multiple datasets with structured prior distributions. First, we developed DICEseq, the first isoform quantification method tailored to time-series RNA-seq experiments. DICEseq explicitly models the correlations between experiments at different time points to aid the quantification of isoforms across experiments. Numerical experiments on both simulated and real datasets show that DICEseq yields more accurate results than state-of-the-art methods, an advantage that can become considerable at low coverage levels. Furthermore, DICEseq permits to quantify the trade-off between temporal sampling of RNA and depth of sequencing, frequently an important choice when planning experiments. Second, we developed BRIE (Bayesian Regression for Isoform Estimation), a Bayesian hierarchical model which resolves the difficulties in splicing analysis in single-cell RNA-seq (scRNA-seq) data by learning an informative prior distribution from sequence features. This method combines the quantification and imputation for splicing analysis via a Bayesian way, which is particularly useful in scRNA-seq data due to its extreme low coverages and high technical noises. We validated BRIE on several scRNA-seq data sets, showing that BRIE yields reproducible estimates of exon inclusion ratios in single cells. Third, we provided an effective tool by using Bayes factor to sensitively detect differential splicing between different single cells. When applying BRIE to a few real datasets, we found interesting heterogeneity patterns in splicing events across cell population, for example alternative exons in DNMT3B. In summary, this thesis proposes structured Bayesian methods to integrate multiple datasets to improve splicing analysis and study its biological functions.

Orientador: Augusto Ducati Luchessi / Resumo: O fator de início de tradução de eucariotos 5A (eIF5A) é uma proteína altamente conservada em archaeas e eucariotos. Caracteriza-se por ser a única proteína conhecida a apresentar o resíduo de aminoácido hipusina, produzido a partir de uma modificação pós-traducional denominada hipusinação, a qual é essencial para sua atividade. Atualmente eIF5A é correlacionada com o início, elongação e término da tradução, além disso, sua função têm sido associada com diferentes processos celulares, fisiológicos e patológicos. Em humanos, é predito que eIF5A seja gerada a partir de cinco variantes transcricionais (A, B, C, D e X5). As variantes B, C, D e X5 codificam a proteína canônica isoforma B de 17 kDa, a qual vêm sendo estudada nas últimas décadas. Entretanto, é previsto que a variante A codifique uma isoforma alternativa (isoforma A) de 20 kDa, que tem uma sequência adicional de 30 aminoácidos na extremidade N-terminal. Neste contexto, o presente estudo teve como objetivo caracterizar eventos moleculares inéditos envolvendo a isoforma A de eIF5A1 humana. Neste estudo, mostramos que a isoforma A é produzida em células HeLa, a qual foi mostrada ser passível de hipusinação. Análises de bioinformática mostraram que a isoforma A possui alta probabilidade de direcionamento para mitocôndria. Assim, técnicas de fracionamento subcelular mostraram que a isoforma A co-purifica com a mitocôndria, diferentemente da isoforma B. Análises de silenciamento gênico em células HeLa, utilizando moléculas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein in archaeas and eukaryotes. It is characterized by being the only protein known to present the amino acid residue hypusine, produced from a post-translational modification called hypusination, which is essential for its activity. Currently eIF5A is correlated with the beginning, elongation and termination of translation, and its function has been associated with different cellular, physiological and pathological processes. In humans, eIF5A is predicted to be generated from five transcriptional variants (A, B, C, D and X5). Variants B, C, D and X5 encode the 17 kDa canonical protein B, which has been studied in recent decades. However, variant A is expected to encode an alternative 20 kDa isoform (A isoform), which has an additional 30 amino acid sequence in the N-terminal region. In this context, the present study aimed to characterize unpublished molecular events involving the human eIF5A1 isoform A. In this study, we showed that in HeLa cells, isoform A was detected, which was shown to be subject to hypusination. Bioinformatics analysis showed that isoform A has a high probability of targeting mitochondria. Thus, subcellular fractionation techniques have shown that isoform A co-purifies with mitochondria, unlike isoform B. Analysis of gene silencing in HeLa cells using eIF5A1 isoform A-specific siRNA molecules showed that protein depletion is capable of cause apoptosis in cells, as well as... (Complete abstract click electronic access below) / Doutor

Two experiments were used to evaluate the potential interaction of fat source and vitamin E (VE) in heavy slaughter weight pigs. In Experiment 1, a total of 64 individually-fed pigs (28.41 ± 0.83 kg) were randomly assigned to 8 dietary treatments in a 4×2 factorial arrangement. Fat treatments included cornstarch (CS), tallow (TW), corn-oil (CO), and coconut-oil (CN). VE treatments were dietary α-tocopheryl acetate (ATA) at 11 and 200 ppm. In Experiment 2, a total of 72 individually fed pigs (28.55 ± 1.16 kg) were randomly assigned to 12 dietary treatments in a 2 × 6 factorial arrangement. Fat treatments were TW and CO. VE treatments included four levels of ATA (11, 40, 100, and 200 ppm) and two levels of mixed tocopherols (primarily γ-tocopherol; 40 and 100 ppm). VE deposition, growth performance, and meat quality were measured in both experiments. In both experiments, interaction between fat sources and VE were detected (P < 0.01) on plasma VE concentration, which increased (P < 0.01) with time and with increasing dietary VE, but increased faster (P < 0.05) in pigs fed with CN and TW compared to pigs fed CS and CO. Compared to CO, more saturated dietary fat sources (CN and TW) led to firmer belly (P < 0.01), which had more (P < 0.01) SFA and MUFA while less (P < 0.05) Feed/Gain in Phase 4 and Phase 5. In Experiment 2, increasing dietary ATA increased overall ADG (linear, P = 0.02), with an interaction (P < 0.05) with fat sources on cumulative ADG during Phase 1-4, wherein pigs fed CO, but not TW, had increased ADG with increasing dietary ATA. Increasing dietary ATA increased (quadratic, P < 0.05) liver SOD activity, and decreased (quadratic, P < 0.05) liver MDA content. The oxidative stability of loin was improved (P < 0.01) when dietary ATA increased over 40 ppm. In summary, both dietary fat source and VE supplementation affected the response measures.

Renin is an enzyme that catalyzes the rate-limiting step in the production of angiotensin peptides, and is thus a key regulator of processes controlled by angiotensin such as blood pressure, hydromineral balance, and metabolism. Our laboratory and others have previously identified a novel isoform of renin (icRen) which, as a result of the utilization of an alternate first exon, lacks the signal peptide and first third of the pro-segment of classical secreted renin (sRen). This alternate icRen isoform thus remains within the cytoplasm of the cell, but is constitutively active. Here, we report that while sRen is the predominant form of renin expressed in most tissues during development, icRen is the predominant form of renin within the adult brain. Thus, we hypothesized that sRen and icRen play distinct physiological roles in adult mice. To examine this hypothesis, we have utilized the Cre-LoxP system to selectively delete either isoform globally or within selected cell types such as neurons and glia. We have successfully developed a "sRen-flox" model, in which endogenous mouse sRen isoform can be selectively deleted, while not affecting endogenous icRen production. Breeding these mice against the E2A-Cre, Nestin-Cre, and GFAP-Cre mouse lines resulted in global-, neuronal-, and glial-specific knockouts of sRen, respectively. Physiological characterization of resulting mice has uncovered postnatal lethality, hypotension, renal atrophy, vascular dysfunction and decreased body weight and white adipose in the global knockouts. Depletion of sRen from only neuronal or glial cells does not appear to alter any of these phenotypes at baseline. From these data, we conclude that while peripheral sRen is of primary importance to blood pressure regulation, hydromineral balance, and metabolism, central expression of this isoform is unimportant. Further, comparison of our results to published findings from global total renin knockout models indirectly supports a role for icRen in the brain. We are currently in the process of generating icRen-flox and subsequent knockout mice, which will be useful models to directly analyze the physiological role(s) of icRen.

Histone deacetylase (HDAC) inhibition has recently emerged as a novel therapy for cancer treatment. However, currently approved histone deacetylase inhibitors (HDACi) are pan-inhibitors thus inhibiting all 11 zinc dependent HDAC isoforms including those not involved in tumorigenesis. These inhibitors are also associated with various side effects including a potentially fatal cardiotoxicity. To address these issues, isoform selective HDACi were designed and synthesized. The use of 3-hydroxy-pyridin-2-thione (3HPT) as zinc chelation group resulted in small molecules devoid of HDAC1 inhibition but active against HDAC6 and/or 8. Selected 3HPT containing HDACi displayed anticancer activity against various cancer cell lines including DU145, LNCaP and Jurkat. Surprisingly, the lead-compounds were very potent against Jurkat Jγ cells which are resistant to SAHA-induced apoptosis. HDACi were also targeted to cancer cells using folic or pteroic acids as targeting groups. Incorporation of the folic acid into the HDACi pharmacophoric model resulted in inhibitors selective for HDAC6, whereas pteroic-based HDACi inhibited both HDAC1 and 6. Only the pteroic-based inhibitors displayed anticancer activities against folate receptor overexpressing tumors such KB and HeLa. Furthermore, cell-based studies established the inhibition of HDAC1 as the basis for the anticancer activities of the pteroic-based HDACi.