A massificação dos estudos da medicina translacional permite aos pesquisadores que usufruam de fontes de dados das mais diversas áreas. Uma área de suma importância e a bioinformatica, que agrega o alta capacidade de processamento computacional disponível atualmente, com a infindável quantidade de dados gerada por métodos de sequenciamento de ultima geração, para entregar aos pesquisadores uma quantidade rica de dados para serem analisados. Apesar da disponibilidade desses dados, a expertise necessária para analisa-los dificulta que profissionais com pouco conhecimento em bioinformatica, estatística e ciência da computação possam realizar pesquisas e analises com estes dados. Dada esta situação, este trabalho consistiu em criar uma ferramenta que tira proveito da integração de múltiplas bases de dados proporcionada pelo framework IPTrans, permitindo que usuários da área biomédica realizem analises com os dados contidos nessas bases. Com base em outras ferramentas existentes e em um levantamento de requisitos junto a potenciais usuários, foram identificadas as funcionalidades mais importantes e assim foi projetada e implementada a IPTrans Advanced Analysis Tool (IPTrans A2Tool). Esta ferramenta permite que usuários façam analises de expressão diferencial mais comuns como heatmaps, volcano plots, consenso de agrupamentos e blox-plot. Além disso, a ferramenta proporciona um algoritmo de mineração de dados baseado na extração de regras de associação entre dados clínicos e biomoleculares, que permite ao usuário descobrir novas associações entre a expressão dos genes dados clínicos e fenotípicos. Adicionalmente a este trabalho, foi criado também o BioBank Warden, um sistema de controle de dados clínicos e amostras biomoleculares, que foi utilizado como uma das fontes de dados para o IPTrans A2Tool. Este sistema permite que usuários adicionem informações clinicas de pacientes e também das amostras extraídas para a realização de estudos. Uma avaliação preliminar de usabilidade, realizada junto a profissionais da área biomédica, mostrou que as ferramentas possuem potencial para serem utilizadas no contexto da medicina translacional. / The great number of translational medicine studies allows researchers to make benefit of data sources from various fields. An area of great importance is bioinformatics, which combines the high computational processing capabilities found nowadays with the endless amount of data generated by next-generation sequencing methods, to give researchers a rich amount of data to be analyzed. Despite the availability of such data, the expertise required to analyze it makes difficult for professionals with little knowledge in bioinformatics, statistics or computer science, to conduct research and analysis on this data. Given this situation, this work was intended to create a tool that takes advantage of multiple databases integration capabilities provided by IPTrans and that allows users to perform analysis on the data contained in these databases. To accomplish that other tools were studied in order to observe which features our framework should aggregate and thus was created the IPTrans A2Tool (IPTrans Advanced Analysis Tool). This tool allows users to perform differential expression analysis and generate output as heatmaps, volcano plots, consensus clustering and blox-plots. In addition, the tool provides an association rule extraction algorithm between clinical and biomolecular data, allowing the user to discover hidden associations between the expression of analyzed genes and clinical data. As a by-product of this work was also created the BioBank Warden a clinical data and biomolecular samples management system that was used as one of the data sources for IPTrans A2Tool. This system allows users to add patients clinical information and also of samples taken for carrying out studies. In addition, the system provides a strong research group and project permission management that ensures only authorized people to have access to patients data.

Lysine methylation is a post-translational modification that regulates a large array of functionally diverse processes that are vital for cellular function. The role of methylation is best characterized on histone proteins due to their high concentration in the cell, but alongside histone modifications, lower abundance non-histone methylation is emerging as a prevalent and functionally diverse regulator of cellular processes. The direct biological impact of non-histone lysine methylation is less well understood because they are difficult to detect. The dynamic concentration range of the proteome masks their signal during proteomic analysis which impedes the detection of these low abundance methylated proteins. Increasing the concentration of proteins bearing methylation is required for improved discovery. This requires enriching the post-translational modification with a capturing reagent prior to analysis. This thesis details an optimized method for using the supramolecular host p-sulfonatocalix[4]arene as a stationary phase methyllysine enrichment reagent for real-life cell-extracted proteins. Prior to the optimizations described in this thesis, cell-derived peptide extracts were not retained within an early generation upper-rim modified calixarene column. But with the new protocols detailed in this thesis, proteins extracted from both cultured prostate cancer cells and industrially sourced brewer’s yeast were successfully retained by a lower-rim modified calixarene column. Thousands of methylated proteins with diverse functions and cellular localization were discovered using this method. Detection of low abundance methylated proteins will aid our discovery of all cellular methylation marks, which in turn, will help delineate their biological functions. / Graduate / 2020-11-30

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22118号 / 医博第4531号 / 新制||医||1039(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 松田 道行, 教授 小柳 義夫, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated. Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond.

G proteins regulate various physiological processes by way of transducing a wide variety of signals ranging from hormonal to sensory stimuli. Malfunctions in G protein signaling lead to numerous diseases. G protein signaling begins with binding of a ligand to a G protein-coupled receptor resulting in a conformational change that leads to the exchange of a GDP for a GTP on G α. The GTP bound α subunit dissociates for its stable Gβγ dimer partner. G α-GTP and Gβγ control the activity of effector enzymes and ion channels that ultimately orchestrate the cellular response to stimulus. Current reports have shown phosducin-like protein (PhLP1) as a co-chaperone with the chaperonin-containing tailless complex polypeptide-1 (CCT) in the assembly of Gβγ dimer. However, the studies did not address the role of PhLP1 and CCT in the translation and eventual assembly of Gβγ dimer. The data presented in Chapter 2 shows a co-translational assembly of Gβγ dimer regulated by PhLP1 and CCT. Chapter 3 discusses the role of PhLP2A and PhLP3 in CCT-mediated assembly of actin and tubulin in mammalian cells. PhLP2 and PhLP3 are members of the phosducin gene family that interact with CCT. Several studies in yeast suggest that PhLP2 promotes CCT-dependent β-actin folding while PhLP3 enhances β-tubulin folding. However, human PhLP2 has been shown to inhibit β-actin folding, indicating that PhLP2 and possibly PhLP3 have very different functions in humans than they do in yeast. As a result, this study investigates in depth the role of PhLP2 and PhLP3 in CCT-dependent β-actin and β-tubulin folding in human cells.

Neuroblastoma (NB) is the most common extracranial solid tumour in childhood. NB is thought to arise from the failed differentiation of neural crest progenitor cells that would normally form tissues of the adrenal gland and sympathetic nervous system. These neural crest progenitors then uncontrollably proliferate forming a tumour. Despite aggressive surgery and chemotherapy, the cure rate of high-risk NB patients remains below 30%. Our laboratory has shown that human NB tumour specimens and high-risk patient derived cell lines express pannexin 1 (PANX1), and that treatment with the PANX1 channel blockers carbenoxolone or probenecid constitute reduce NB progression in vitro and in vivo. PANX1 is a glycoprotein that forms single membrane channels best known to serve as conduits for ATP release. Interestingly, while PANX1 was also detected in control neurons by western blotting, its banding pattern was strikingly different as a band at around 50 kDa was found in all NB cell lines, but not in neurons. Using shRNA targeting PANX1 and deglycosylation enzymes, I have shown that this band corresponds to a PANX1 glycosylated species. PANX1 has been reported to be phosphorylated in NB at amino acid Y10. PANX1 is also predicted to be glycosylated at N255. In order to study the role of these post-translational modifications, myc-tagged Y10F and N255A PANX1 mutants were engineered by site-directed mutagenesis. Immunolocalization and cell surface biotinylation assays suggest that the localization both mutants at the cell surface is reduced compared to that of myc-PANX1. Dye uptake assays revealed that myc-Y10F has significantly reduced channel activity. Expression of myc-Y10F and myc-N255A in NB cells inhibited cell proliferation and decreased metastatic potential in vitro. Further analysis of NB tumour specimens revealed that there is a missense mutation in PANX1 resulting in the formation of truncated peptide (amino acid 1-99). Interestingly, I have found that when co-expressed with myc-PANX1, PANX11-99, reduced PANX1 channel activity. Taken together, these findings indicate that phosphorylation on Y10 and glycosylation on N255 regulate PANX1 channel activity and exacerbate NB malignancy, while the expression of PANX11-99 in NB may be beneficial.

The first report of polyphosphates (polyP) was in 1890 by L. Liberman and since then, polyP’s role in biology has been explored. PolyPs are chains of phosphoanhydride-linked inorganic phosphates ranging from 3-1000s of units in length. These chains are implicated in many cellular pathways including blood clotting, bacterial virulence, and neuroproteotoxic disease. Given the diversity of polyP, they make an excellent candidate in the development of novel therapeutics. In yeast, polyP is synthesized by the vacuolar transporter chaperone (VTC) complex as a translocation event into the vacuole lumen. In 2015, polyP chains were found to act as a post-translational modification termed polyphosphorylation on yeast proteins (Nsr1 and Top1). This modification occurs non-enzymatically on lysine residues within poly-acidic, serine, and lysine (PASK) motifs and can only be detected via electrophoretic mobility shift on NuPAGE gels. We have since expanded the pool of yeast polyphosphorylated substrates to 25, with an enrichment of proteins with roles related to RNA biology. Additionally, we were the first group to demonstrate polyphosphorylation of 6 human proteins by expressing E. coli PPK1 in HEK293T cells. We next focused on elaborating how polyP is being regulated via the VTC complex by assessing which protein trafficking pathways are critical for VTC localization at the vacuole membrane. We found the adaptor protein 3 (AP-3) complex is responsible for localizing Vtc5 subunit to the vacuole membrane and in AP-3 mutants, Vtc5 becomes mislocalized to the vacuole lumen and degraded. Vtc5 degradation, upon AP-3 mutation, is mediated by the endosomal sorting complex required for transport (ESCRT) complex. The loss of polyP in AP-3 mutants is imparted by Vtc5 mislocalization. In humans, mutations in AP-3 cause a rare genetic disorder termed Hermansky-Pudlak Syndrome (HPS) which has a wide range of symptoms. These include defects in polyP accumulation in platelets, likely related to a loss of polyP. We expect that our work using yeast will provide a framework for understanding fundamental aspects of polyP biology related to HPS and other health conditions.

The molecular target of epigenetic signaling is chromatin. Histones are extensively post-translationally modified (PTM), and many of these individual modifications have been studied in depth. As PTMs occur at multiple positions within histones, the degree to which these modifications might influence each other remains one of the major challenges of chromatin biology. Although major discoveries in understanding the complex repertoire of histone modifications were achieved using reductionist experimental systems with synthetic histone peptides, they do not explain the role of putative PTM cross-talks in a chromatin context. However, generating chromatin substrates of defined modification status has proved to be a technically challenging task. In this thesis, I first demonstrate our work on establishing a novel approach to produce libraries of modified nucleosomes. We employed protein trans-splicing and sortase-mediated ligation strategies to incorporate chemical modifications on histone tails of ‘ligation-ready’ nucleosomes. Subsequently, the ‘designer’ nucleosome libraries were used for testing the binding of heterochromatin protein 1 (HP1) and elucidated the previously uncharacterized crosstalk of H3K9me2 and S28ph marks. Further investigations explained the mechanism of this crosstalk and highlighted the importance of developing chemical biology tools for elucidating complex chromatin signaling. Second, I describe our reconstitution systems for the assembly of semisynthetic recombinant chromatin carrying methylation marks on DNA and distinct modifications on histones, e.g. H3K9me3. I aimed to understand the mechanisms of the interplay between chromatin and one of the DNA maintenance methylation factors, UHRF1. I showed that UHRF1 strongly interacts with nucleosomes containing linker DNA. However, it exerts only residual enzymatic activity in this context. Based on functional H3 ubiquitylation assays in vitro, I found that hemi-methylated nucleosomes stimulate enzymatic activity of UHRF1, suggesting that the protein’s chromatin targeting and activation are a two-step process. The positioning of hemi-methylated CpG on nucleosome regulates UHRF1 target selectivity. Further, mutational analysis revealed that the PHD domain of the factor is indispensable for H3 binding and that its SRA domain is required for catalytic activation. Overall, our work adds a new layer of positional complexity to the me½CpG-dependent regulation of UHRF1 and expands the current model of DNA methylation maintenance.

Noninvasive brain-computer interfaces (BCIs) use scalp-recorded electrical activity from the brain to control an application. Over the past 20 years, research demonstrating that BCIs can provide communication and control to individuals with severe motor impairment has increased almost exponentially. Although considerable effort has been dedicated to offline analysis for improving signal detection and translation, far less effort has been made to conduct online studies with target populations. Thus, there remains a great need for both long-term and translational BCI studies that include individuals with disabilities in their own homes. Completing these studies is the only sure means to answer questions about BCI utility and reliability. Here we suggest an algorithm for candidate selection for electroencephalographic (EEG)-based BCI home studies. This algorithm takes into account BCI end-users and their environment and should assist in study design and substantially improve subject retention rates, thereby improving the overall efficacy of BCI home studies. It is the result of a workshop at the Fifth International BCI Meeting that allowed us to leverage the expertise of multiple research laboratories and people from multiple backgrounds in BCI research.

Behavioral contrast occurs when a change in reinforcement rate in one context causes behavior to change in the opposite direction in another context. Positive contrast occurs when a decrease in the rate of reinforcement in one context results in an increase in behavior in another context. Negative contrast occurs when an increase in the rate of reinforcement in one context results in a decrease in behavior in another context. Research with nonhumans has found that positive contrast is more reliably produced than negative contrast. Research with nonhumans has also found that positive contrast is influenced to a larger degree by changes in reinforcement rate in the following context (vs. in the preceding context); however, results regarding negative contrast and the influence of preceding versus following contexts have been mixed. Finally, within-session contrast effects have been demonstrated in nonhumans. Relative to the entire environmental context, the largest change in behavior occurs immediately prior to (anticipatory contrast) or immediately following (local contrast) the change in reinforcement rate. Behavioral contrast has applied implications, in that practitioners may only be able to implement interventions in one context, which may result in concomitant worsening of behavior in other contexts. Few studies with humans have compared positive and negative contrast, and none have separated preceding- and following schedule effects or have systematically investigated within-session contrast. The purpose of this study was to investigate these effects in humans in a translational arrangement. Positive contrast was found in five of six cases, while negative contrast was found in only three of six. The effect of the following schedule was larger with positive contrast, but the effect of the preceding schedule was larger with negative contrast. There were no systematic within-session effects characteristic of anticipatory or local effects.