Skeletal muscle demonstrates a remarkable malleability and can adjust its metabolic and contractile properties in response to changes in environmental stimuli. The proteome represents a highly dynamic and versatile entity that coordinates the adaptive response of skeletal muscle through adjustments in individual protein abundance, modulated by changes in the synthesis and degradation rate of proteins. Previously research relating to protein turnover has been largely limited to average synthesis rates of protein mixtures e.g. from whole/ subfractions of muscle homogenates. This project utilises dynamic proteome profiling which combines deuterium oxide labelling and proteomic techniques with computational biology, to investigate muscle protein dynamics at the individual protein level. This work used 3 in vivo human and rodent exercise models to studying the complexity of dynamic proteome adaptation. Identifying that exercise induced adaptation can occur on the level of protein turnover, independent of classical changes in protein abundance. Which seems of particular importance in counteracting proteostatic stress (i.e. obesity and disease). Subsequently the need to investigate the relationship between protein dynamics and proteome remodelling resulted in the observation that changes in protein abundance do not follow a simple linear trajectory of adaptation. Also, protein specific synthesis rates seemingly undergo time-dependent adaptations when explaining remodelling in the context of an animal model of programmable resistance training. Finally, this thesis provides new evidence to demonstrate that human exercise training results in unique alterations in the rates of protein synthesis and degradation that is training mode and status dependent despite networks of proteins resulting in the same abundance changes. This work raises potential questions as to the extent of exercise induced adaptation explained by modulation of protein synthesis, particularly in the context of endurance training. Whereby changes in degradation are implicated as a key driver of endurance exercise induced proteome remodelling. This work represents some of the first of its kind to present novel details as to the potential roles of protein synthesis, degradation, and turnover rate in modulating differences in skeletal muscle health and function induced by exercise. / Thesis / Candidate in Philosophy

Eukaryotic cells are highly heterogeneous. These cells are arranged into different compartments, carrying out separate functions and facilitating biological processes. Proteins are the effector biomolecules targeted to subcellular locations that help fulfill specific tasks in living organisms. Spatial proteomics can help unravel molecularly how protein abundance and localization are altered in cells, which is not feasible in traditional bulk-scale proteomics. To achieve this, our lab has developed a miniaturized sample processing platform called nanoPOTS, reduced separation columns' inner diameter to increase ionization efficiency and concentrate analytes for mass spectrometers and optimized data acquisition modes for increasing proteome coverage in spatial and single-cell proteomics and applying these techniques to studying protein dynamics in various biological samples and conditions.This dissertation details the extension of our techniques to other limited biological samples. We expanded the nanoPOTS sample processing workflow to formalin-fixed, paraffin-embedded tissues (FFPE). By optimizing extraction solvents, times, and temperatures, we obtained the highest proteome coverage in FFPE tissues compared to fresh frozen tissues. Our observations revealed an average of 1312 and 3184 high-confidence master proteins in 50 – 200 µm square cut regions of a 10 µm thick FFPE-preserved mouse liver tissue, achieving 88% of the proteome coverage compared to that obtained from fresh frozen tissues of the equivalent size. We then characterized our fully automated sample preparation and analysis workflow, autoPOTS, for FFPE spatial proteomics. We applied the optimized nanoPOTS sample preparation condition to analyze normal, precancerous, and cancerous lesions of FFPE-preserved pancreatic ductal adenocarcinoma (PDAC) human samples, achieving an average coverage of 3000 proteins from 200 µm squares of each cell type. We identified some highly expressed proteins using differential analysis for cancerous lesions. We also optimized microLIFE, a cellenONE software add-on instrument, to detect and isolate low-input bacteria samples using Escherichia coli (E coli). We collected proteomic data using both Wide Window data Acquisition and Data-Independent Acquisition. On average, we identified 800 and 1300 proteins in WWA and DIA, respectively. We applied microLIFE to identify proteins involved in Salmonella pathogenicity island-I (SPI) impacted by oxygen availability in their growth medium and observed 50% and above average of difference classes of SPI compared with bulk-scale proteomics. This novel software can enable low-input spatial proteomics.

京都大学 / 新制・課程博士 / 博士(ゲノム医学) / 甲第25211号 / 医博JD第3号 / 新制||医JD||1(附属図書館) / 京都大学大学院医学研究科ゲノム医学国際連携専攻 / (主査)教授 村川 泰裕, 教授 平井 豊博, 教授 William Foulkes (ﾏｷﾞﾙ大学), 准教授 George Thanassoulis (ﾏｷﾞﾙ大学), 教授 Krzysztof Kiryluk (ｺﾛﾝﾋﾞｱ大学) / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

In the United States, breast cancer is the most commonly diagnosed cancer and is the second most common cause of cancer-related deaths among women. Among the various subtypes of breast cancer, 25-30% of diagnoses present themselves as human epidermal growth factor receptor 2 positive (HER-2+). HER-2 is a protein receptor located on the cell surface that interacts with other proteins and signaling molecules to translate extracellular signals into cellular process such as cell growth and replication. However, in breast cancer, there is a drastic increase in the number of HER-2 proteins on the cell surface, that causes excessive cell growth and proliferation, and ultimately tumor formation. The most frequent treatment of HER-2+ breast cancers includes the use of a single agent inhibitor that directly blocks the HER-2 protein to prevent over-signaling and cell growth. However, after continuous use, breast cancer cells develop drug resistance, as other proteins such as the insulin-like growth factor 1 receptor (IGF-1R) and the protein kinase B (AKT) can also interfere and cause cell growth and replication. In this study, we propose that the use of a multi-agent treatment targeting the HER-2, IGF-1R, and AKT proteins will be more effective than a single-agent treatment of HER-2 alone. Through protein analysis by mass spectrometry, we intend to illuminate the different cellular responses to both treatment types. The results indicate that the single drug treatment targeting Her-2 appears to increase processes related cellular repair, while the multi-drug treatment indicates an increase in processes related to programmed cell death; both treatments appear to block the transmission of protein signaling. / MS / In the United States, breast cancer is the most commonly diagnosed cancer and is the second most common cause of cancer-related deaths among women. Among the various subtypes of breast cancer, 25-30% of diagnoses present themselves as human epidermal growth factor receptor 2 positive (HER-2+). HER-2 is a protein receptor located on the cell surface that interacts with other proteins and signaling molecules to translate extracellular signals into cellular process such as cell growth and replication. However, in breast cancer, there is a drastic increase in the number of HER-2 proteins on the cell surface, that causes excessive cell growth and proliferation, and ultimately tumor formation. The most frequent treatment of HER-2+ breast cancers includes the use of a single agent inhibitor that directly blocks the HER-2 protein to prevent over-signaling and cell growth. However, after continuous use, breast cancer cells develop drug resistance, as other proteins such as the insulin-like growth factor 1 receptor (IGF-1R) and the protein kinase B (AKT) can also interfere and cause cell growth and replication. In this study, we propose that the use of a multi-agent treatment targeting the HER-2, IGF-1R, and AKT proteins will be more effective than a single-agent treatment of HER-2 alone. Through protein analysis by mass spectrometry, we intend to illuminate the different cellular responses to both treatment types. The results indicate that the single drug treatment targeting Her-2 appears to increase processes related cellular repair, while the multi-drug treatment indicates an increase in processes related to programmed cell death; both treatments appear to block the transmission of protein signaling.

Yes / Purpose: Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopatholigical features. Our aim, in this pilot study, was to investigate the intra- and inter-patient protein composition of nipple secretions and the implications for their use as liquid biopsies. Experimental design: Matched pairs of NAF (n=15) were characterised for physicochemical properties and SDS PAGE. Four pairs were selected for semi-quantitative proteomic profiling and trypsin-digested peptides analysed using 2D LC Orbitrap Fusion mass spectrometry. The resulting data was subject to bioinformatics analysis and statistical evaluation for functional significance. Results: A total of 1990 unique proteins were identified many of which are established cancer associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals was observed. Conclusions: This was the most complete proteomic study of NAF to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort. / Proteomics research was supported by Yorkshire Cancer Research projects, BPP047 and B381PA.

Yes / There has been tremendous progress in detection of breast cancer in postmenopausal women, resulting in two-thirds of women surviving more than 20 years after treatment. However, breast cancer remains the leading cause of cancerrelated deaths in premenopausal women. Breast cancer is increasing in younger women due to changes in life-style as well as those at high risk as carriers of mutations in high-penetrance genes. Premenopausal women with breast cancer are more likely to be diagnosed with aggressive tumours and therefore have a lower survival rate. Mammography plays an important role in detecting breast cancer in postmenopausal women, but is considerably less sensitive in younger women. Imaging techniques, such as contrast-enhanced MRI improve sensitivity, but as with all imaging approaches, cannot differentiate between benign and malignant growths. Hence, current well-established detection methods are falling short of providing adequate safety, convenience, sensitivity and specificity for premenopausal women on a global level, necessitating the exploration of new methods. In order to detect and prevent the disease in high risk women as early as possible, methods that require more frequent monitoring need to be developed. The emergence of “omics” strategies over the last 20 years, enabling the characterisation and understanding of breast cancer at the molecular level, are providing the potential for long term, longitudinal monitoring of the disease. Tissue and serum biomarkers for breast cancer stratification, diagnosis and predictive outcome have emerged, but have not successfully translated into clinical screening for early detection of the disease. The use of breast-specific liquid biopsies, such as nipple aspirate fluid (NAF), a natural secretion produced by breast epithelial cells, can be collected non-invasively for biomarker profiling. As we move towards an age of active surveillance, home-based liquid biopsy collection kits are increasingly being applied and these could provide a paradigm shift where NAF biomarker profiling is used for routine breast health monitoring. The current status of established and newly emerging imaging techniques for early detection of breast cancer and the potential for alternative biomarker screening of liquid biopsies, particularly those applied to high-risk, premenopausal women, will be reviewed. / Proteomics research was supported by Yorkshire Cancer Research projects, BPP047 and B381PA, and co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation projects ΥΓΕΙΑ/ΒΙΟΣ/0311(ΒΙΕ/07) and NEKYP/0311/17.

Yes / Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates β-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis. / Cancer Research UK (CRUK Beatson Institute C596/A17196, CRUK Glasgow Centre C596/A18076 and S.Z. C596/A12935)

No / Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but <22% in common with an adenoma patient. In invasive carcinoma patients, several cellular and extracellular proteins that were significantly increased (Periostin, Small breast epithelial mucin) or decreased (Kinectin) have previously been associated with breast cancer, thereby supporting this approach for a larger disease-stage characterization effort.

Concussion is the most common form of brain injury, comprising over 80% of traumatic brain injuries (TBIs) occurring in the United States and around the world. While many individuals are able to fully recover in the weeks following a concussion, an estimated 50% of patients go on to suffer from persistent symptoms that may range from months to years in duration. Among the most common complaints of those with persistent symptoms is sadness or depression, and significantly elevated rates of suicide have been reported in this population. Females are more likely to develop persistent symptoms and have reported higher rates of neuropsychiatric symptoms than males following injury. Altered glutamatergic neurotransmission has been implicated as a possible cause of depression following concussion due to similarities in glutamatergic changes that occur following concussion and during depression, independently of brain injury. Excitotoxicity is known to occur following TBI, resulting in neuronal death, and dysfunction in the cells that survive. Glutamatergic dysfunction occurring in regions such as the hippocampus, prefrontal cortex or amygdala, may be a driver of depressed mood or major depressive disorder (MDD) in concussed individuals as these regions, among others, have previously been linked to MDD. There are currently no approved pharmacological treatments for TBI symptoms. Therefore, gaining insight into chronic pathophysiology underlying TBI symptoms, including depression, is essential to support the development of therapeutic approaches for patients. The glutamate system represents a promising avenue of investigation in the context of chronic TBI pathophysiology. Using a clinically-relevant rodent model of concussion, this work sought to elucidate chronic glutamatergic changes occurring in the brain in association with the development of depression-like behavior following injury. Delayed-onset deficits in social and self-care behaviors were observed in association with region-specific changes in N-methyl-D-aspartate (NMDA) receptor and glutamate transporter expression in injured male animals. Females responded differently to injury, showing disinhibition and compulsive behaviors in conjunction with upregulation of glutamatergic signaling proteins. Further, this work aimed to investigate chronic sex-specific responses to TBI. Direct comparisons of behavioral changes in injured males and females demonstrated differences in both the emergence and nature of behavioral deficits. Examinations of hippocampal subregions showed deeper specificity in expressional changes in glutamatergic markers with both region- and sex-specific alterations observed at a chronic time point. Additionally, proteomic analysis was employed to evaluate widespread protein-level changes in the injured frontal cortex, and results revealed significant dysregulation in pathways involved in excitatory neurotransmission and calcium signaling. However, the dysregulated proteins within these pathways differed in a sex-dependent manner, indicating a sexual dimorphism in chronic TBI pathophysiology. Potential drug targets were also identified for investigation in future studies. The fundamental work presented in these studies provides strong evidence of dynamic, sex-specific modifications in the glutamate system in association with chronic TBI deficits in a translational model, ultimately providing a foundation for future development of therapeutic options to improve the lives of patients suffering with persistent symptoms. / Doctor of Philosophy / Concussion is the most common form of brain injury, comprising over 80% of traumatic brain injuries (TBIs) occurring in the United States and around the world. While many individuals are able to fully recover in the weeks following a concussion, an estimated 50% of patients go on to suffer from persistent symptoms that may range from months to years in duration. Among the most common complaints of those with persistent symptoms is sadness or depression, and significantly elevated rates of suicide have been reported in this population. Females are also more likely to develop persistent concussion symptoms and tend to experience more severe symptoms than males, but the reasons for this are not known. Changes in important signaling proteins, which allow neurons to properly communicate with one another, have been observed in the brains of concussed patients. Changes in the same proteins have been reported in depressed patients, leading to the belief that these signaling proteins may be involved in the development of depression after a brain injury. These proteins are involved in glutamate signaling, and glutamate is the main excitatory neurotransmitter in the brain. Excitatory neurotransmission is responsible for activating receptors which can lead to the firing of neurons, hence its importance in neuron communication. There are currently no approved treatments for TBI symptoms. Therefore, gaining insight into the chronic biological changes underlying TBI symptoms, including depression, is essential to support the development of therapies for patients. Using a rodent model of concussion, this work identified long-term changes in important glutamate signaling proteins in injured animals which were observed alongside depression-like behaviors. Injured male rodents were less social and were less motivated to take care of themselves, similar to humans with major depressive disorder (MDD). Female rodents showed different changes in depression-like behaviors and also developed behaviors similar to humans with obsessive compulsive disorder (OCD). This work demonstrated the development of sex-specific symptoms in injured animals, and these were accompanied by different changes in key glutamate signaling proteins, which suggests problems in neuron communication due to injury. These protein changes were detected in regions of the brain linked to MDD. The fundamental work presented in these studies provides strong evidence of dynamic, sex-specific modifications in the glutamate system in association with chronic TBI symptoms, ultimately providing a foundation for future development of therapeutic options to improve the lives of patients suffering with persistent symptoms.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, with a five-year survival rate of only 13%. Patients are most commonly diagnosed in late stages, once the tumor has metastasized to distal organs, limiting treatment options. For PDAC patients, metastases most often go to the liver. However, there has been limited studies profiling matching human primary tumors and liver metastases because of the difficulty in procuring such samples. To understand the system in which primary tumors spread to the liver, an omics approach is most appropriate. Proteins are the final effectors of the cell, making mass spectrometry-based proteomics an extremely useful tool in understanding the metastatic landscape of PDAC. Here, we present a novel study that offers a comprehensive proteomic characterization of matching human primary PDAC tumors and liver metastases. Using mass spectrometry-based proteomics, we were able to elucidate immune signatures on both the global proteomic and phosphoproteomic level in PDAC, as well as. In addition, this work can serve as a useful resource to the community to help generate more hypotheses and follow-up experiments. Finally, we demonstrate the versatility and potential of mass spectrometry as a tool to probe various biological systems by offering a systematic comparison of the two main mass spectrometry acquisition types used in bottom-up proteomics. We found that data-dependent acquisition (DDA) and data-independent acquisition (DIA) can produce comparable results. However, DDA, in combination with isobaric labeling, disproportionally suffers from ratio compression, and DIA’s incompatibility with isobaric labeling for multiplexing can be a big drawback when working with limited sample amounts.