Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition. We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing. By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.

Cultivable land is a limited resource and agriculture contributes to some of the most serious environmental problems facing the planet like, global warming, eutrophication and loss of biodiversity. The specialized, industrial agriculture with monoculture and extensive input of external energy, commercial fertilizer, and chemical pesticides are all essentially unsustainable. An adaptation to other methods of production is necessary to preserve the environment for future generations. The aim of this study is to describe the factors that distinguish sustainable from unsustainable agriculture. The results will be used to examine if and how the European Union (EU) works in order to create a more sustainable agricultural industry and to analyze the preconditions for such development. We have used a method based on abduction and we have a hermeneutic and ecological economic approach. The empirical survey is of a qualitative nature and is based on four interviews and literature studies. The results of this study shows that a sustainable agriculture should be build on ecological premises, which is easier to achieve in small-scale, functional, integrated and cycle-based farming. Modern farming’s dependence of fossil fuel and pesticides and the excess use of land must cease. Furthermore, there is an urgent need to decrease our consumption of meat products. EU’s agricultural policy is based on a worldview that relies on production optimization and is growth-oriented and these approaches must be changed. Certain positive changes have occurred within EU, but the decision-making process is slow and bureaucratic. Financially strong lobbyists have too much influence and power, compared to the rest of society. This makes it difficult to work for change and attain actual sustainability.

This thesis presents a practical approach to reduce the overall testing time in a spherical near-field (SNF) antenna measurement environment. The premise of this work is that the acquisition time is mostly dominated by the mechanical movement and the processing electronic. Moreover, it is assumed that the transformation time to go from the near-field domain to the far-field domain (NF-FF transform) is small compared to the acquisition time. Thus this operation can be done repeatedly while the acquisition is on-going without significantly affecting the overall test time. This situation allows to continuously evaluate the far-field (FF) of the antenna under test (AUT), so that certain decision functions based on the radiation pattern of the antenna can be monitored. Such decision functions are based on the antenna specification, such as the gain, the side lobe level, etc. We do not proceed with a complete scan of the measurement sphere but effectively allow the probe to follow a directed path under control of an acquisition rule, so that the sampled near-field (NF) datapoints constitute an acquisition map on the sphere. The acquisition can then be terminated based on decision function values, allowing the smallest amount of data needed to ensure accurate determination of the AUT performance measures.

ATP dependent chromatin remodelers use the energy from ATP hydrolysis to move, disassemble or alter the composition of nucleosomes. Though all remodelers share a conserved ATP hydrolysis and DNA translocase domain, their biochemical actions and in-vivo characteristics differ because of their subunits and accessory domains in the catalytic subunit that regulate its activity. Understanding how these domains contribute to remodeler regulation in terms of substrate interaction and regulation of the catalytic subunit is therefore important to understanding what causes a remodeler to behave differently, and what are the mechanistic underpinnings of such behavior. In this study we have addressed these questions using the SWI/SNF remodeler from budding yeast (Saccharomyces cerevisiae) to explore how different remodelers compare to SWI/SNF in terms of nucleosome interaction. Using a chemical based histone – remodeler photo-crosslinking and labeling approach, we show that different remodelers contact nucleosomes in patterns unique to their functions, and even remodelers that belong to the same family interact with nucleosomes in a unique manner to accomplish their respective remodeling results. In addition we delineate the functions of the AT hook motifs in the catalytic subunit of SWI/SNF using in-vitro and in-vivo techniques. We demonstrate the necessity of the regulatory action of the motif in the context of SWI/SNF remodeling due to its requirement for efficient ATP hydrolysis by the catalytic domain and therefore efficient remodeling. We also demonstrate for the first time that SWI/SNF in yeast is involved in transcriptional repression with evidence that the AT hook alters SWI/SNF activity at particular genomic regions. Regulation of SWI/SNF activity is an increasingly important topic of study, with mutations that cause SWI/SNF dysfunction being implicated in a large number of cancers and neurological diseases. We attempt to find out the biochemical implications of mutations in the catalytic, SnAC and AT hook motifs with respect to SWI/SNF activity. Taken together, this study provides an insight into some of the different mechanisms in which remodelers are regulated using budding yeast as a model system.

Thyroid Tumorigenesis is typically a well understood process, with well delineated oncogenic factors. Follicular and papillary thyroid cancers are typically survivable, with 5-year survival rates being >95% for Stage I-III of both cancer types. Anaplastic thyroid cancer, in contrast, lacks this prognosis, and is the most lethal of all endocrine-related cancers. The median survival time after a diagnosis is generally between 6-8 months, with a 5-year survival rate of <10%. Current treatment for anaplastic thyroid cancers routinely meet roadblocks, as resistance is quickly developed. Even non-discriminatory kinase inactivators, such as sorafenib, which are generally considered a drug of last resort, are unable to effect survival rates. As such, there is a clear need for further investigation of the causes of anaplastic thyroid cancer mechanisms. Previous work in the Carr lab revealed a novel regulatory pathway of an oncogene that is associated with several other endocrine-related cancers, as well as other non-endocrine-related cancers. Specifically, the Runt-related transcription factor 2 (Runx2) was found to be suppressed via direct binding of the thyroid hormone receptor beta 1 isoform (TRß1) to its proximal promotor. Runx2 was previously shown to be associated with increasing malignancy, with Runx2 occurring at low-levels in indolent cell lines, whilst occurring at high-levels in more malignant cell lines. TRß1, conversely, exhibited the opposite relationship. Endogenous levels of TRß1 were found to be high in indolent cell lines and were depleted in malignant cell lines. These findings were further confirmed via tissue microarrays. Restoration of TRß1 in malignant cell lines diminished Runx2 mRNA and protein levels, which was corroborated by evidence from electrophoretic mobility-shift assays, and chromatin immunoprecipitations that TRß1 was able to directly bind Runx2 promotor 1. Current studies have investigated the nuclear protein profile that associates with TRß1 to alter Runx2 transcription. Through EMSA-to-Mass Spectrometry methodologies, as well as novel DNA pulldown techniques, binding partners have been elucidated. Findings have also been confirmed via classical immunoprecipitations. Specifically, our findings show that TRß1 complexes with the brahma-related gene 1 (BRG1) protein, the nuclear co-repressor (NCOR), and BRG1-associated protein 60 (BAF60). BRG1 functions by preferentially recruiting histone deacetylases (HDAC), with BRG1 and the HDAC’s acting to alter chromatin, and thus transcription. Future studies aim at examining whether other proteins complex with TRß1 to alter Runx2 transcription, and whether these complexes are altered in aggressive cell lines.

This thesis presents a practical approach to reduce the overall testing time in a spherical near-field (SNF) antenna measurement environment. The premise of this work is that the acquisition time is mostly dominated by the mechanical movement and the processing electronic. Moreover, it is assumed that the transformation time to go from the near-field domain to the far-field domain (NF-FF transform) is small compared to the acquisition time. Thus this operation can be done repeatedly while the acquisition is on-going without significantly affecting the overall test time. This situation allows to continuously evaluate the far-field (FF) of the antenna under test (AUT), so that certain decision functions based on the radiation pattern of the antenna can be monitored. Such decision functions are based on the antenna specification, such as the gain, the side lobe level, etc. We do not proceed with a complete scan of the measurement sphere but effectively allow the probe to follow a directed path under control of an acquisition rule, so that the sampled near-field (NF) datapoints constitute an acquisition map on the sphere. The acquisition can then be terminated based on decision function values, allowing the smallest amount of data needed to ensure accurate determination of the AUT performance measures.

<p>In eukaryotic systems, the genetic material of the cell –DNA– is packed into a protein-dense structure called chromatin. Chromatin structure is critical for preservation of the genetic material as well as coordination of vital processes such as DNA replication, transcription and DNA damage repair. The fundamental repeating unit of chromatin is nucleosome which is composed of an octamer of small alkaline proteins called histones and the DNA wrapped around this octamer. The nucleosomes are then packed into higher-order structures leading to formation of 3D chromatin architecture. The chromatin is a dynamic structure; the spacing between nucleosomes, or the folding of the larger chromatin segments is subjected to alterations during embryonic development, tissue specifications or <i>simply during any event that require gene expression changes</i>. Failure in proper regulation of chromatin structure has been associated with embryonic defects and disease such as cancer. </p> <p>This work has focused on a class of ATP-dependent chromatin remodeling complexes known as switch/sucrose-non-fermentable (SWI/SNF) or BRG-associated factors (BAF) complex. This family of complexes act on chromatin and alter its physical structure by mobilizing histones or nucleosome particles through the activity of its ATPase –BRG1 or BRM, enabling more accessible DNA for the other factors such as transcription factors to localize and recruit transcription machinery. In particular, we discovered and biochemically defined a novel version of this family of chromatin complexes that we named as GLTSCR1/1L-BAF (GBAF). GLTSCR1 and GLTSCR1L are two uncharacterized paralogous proteins that have been identified as BRG1-interacting proteins. Biochemically surveying the essence of this interaction, we realized that these proteins incorporates into a previously unknown SWI/SNF family complex that lacks well-characterized SWI/SNF subunits such as ARID1/2, BAF170, BAF47; instead, uniquely comprise GLTSCR1/1L and bromodomain-containing protein BRD9. Focusing on the GLTSCR1 subunit, we observed that its absence is well-tolerated by many different cell types except slight growth retardation by prostate cancer cells. Expanding the cohort of prostate cancer cells, we realized that not the paralogous subunits GLTSCR1 or GLTSCR1L but unique and non-redundant subunit BRD9 is the major GBAF-dependence in prostate cancer cells. We observed that especially the androgen-receptor positive cell lines have severe growth defects upon <i>BRD9 </i>knockdown or inhibition. <i>In vivo, </i>we showed that xenografts with <i>BRD9 </i>knockdown prostate cancer cells (LNCaP) have smaller tumor size. We demonstrated that BRD9 inhibition can block the expression of androgen-receptor targets. Similarly, <i>BRD9 </i>knockdown and treatment with antiandrogen drug (enzalutamide) has overlapping transcriptional effects. Mechanistically, we showed that BRD9 interacts with AR and it colocalizes with AR in subset of AR -binding sites. Surprisingly, we realized that BRD9 depletion has similar transcriptional and phenotypic effects as BET protein inhibitors. BET protein family contains 4 bromodomain containing proteins (BRD2, BRD3, BRD4, BRDT). These proteins were previously shown to be critical for AR-dependent gene expression. We detected interaction between BRD9 and BRD2/4. We demonstrated that BRD4 and BRD9 had shared binding sites on genome, a fraction of which are co-bound by AR. At particular target sites we showed that BRD9 localization is dependent on BET proteins, but not the other way around. Taking together, we provided some evidences that GBAF targeting through BRD9 can be a novel therapeutic approach for prostate cancer. Growing body of reports suggested that current therapy options targeting the androgen receptor is failing due to acquired resistance. Therefore, targeting the AR pathways via its coregulators such as BET proteins or SWI/SNF complexes can serve as potent alternative approaches. Further research is needed to elucidate the roles of GBAF and BET proteins in androgen receptor independent prostate cancer cells, which are still responsive to GBAF or BET manipulations although to a lesser extent.</p>

No description available.

<div> <p>Mammalian embryos undergo a dramatic amount of epigenetic remodeling during the first week of development to establish the correct epigenetic status to support the developmental program. SWI/SNF chromatin remodeling complexes are multi-subunits complexes and utilize energy from ATP hydrolysis to modify chromatin structure non-covalently. The collection of subunits determines the identity of a given SWI/SNF chromatin-remodeling complex, directs its activity, and dictate where that complex will act. The aims of this study were to 1) determine the requirement of SNF5, a SWI/SNF core subunit found in BAF and PBAF complexes during preimplantation porcine embryo development, 2) determine the requirement of BRD7, a PBAF complex-specific subunit during preimplantation porcine embryo development, and 3) investigate the role of <i>CDH1</i>, a downstream gene regulated by ARID1A, another subunit found exclusively in BAF complexes, in cleavage stage porcine embryos. Our results indicate that the differential requirement for each subunit during early embryo development. Depletion of different subunits results in embryo arrest at distinct developmental stage. Together, our data suggest the SWI/SNF chromatin remodeling complexes are necessary for proper porcine embryo development and this requirement is associated with the composition of the complex.</p> </div> <br>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21300号 / 医博第4389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 戸井 雅和, 教授 小川 修, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM