published_or_final_version / Botany / Doctoral / Doctor of Philosophy

Li Ka Shing Prizes for best MPhil thesis in the Faculties ofDentistry, Engineering, Medicine and Science, 2006-2007 / published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Down syndrome (DS), caused by an extra chromosome 21, affects 1 in 750 live births, and is characterized by cognitive impairment as well as several congenital defects. Currently, little is known about the molecular pathogenesis of DS and no direct genotype-phenotype relationship has yet been confirmed. The current screening test for DS subjects many women to undergo invasive procedures such as amniocentesis due to suboptimal sensitivity and specificity. Therefore, this study aimed to discover novel biomarkers to improve screening tests, and to discovery dysregulated molecular pathways in DS-affected fetus to better understand pathogenesis. To achieve this objective, proteomic analyses of amniotic fluid (AF) and amniotic fluid cells (amniocytes) were performed using mass spectrometry (MS), which allows discovery of a large number of proteins in complex biological samples. Since AF contains the most information of the developing fetus, we first generated the most comprehensive list of proteins present in AF by using high resolution MS. We then performed quantitative analyses of proteins from AF as well as amniocytes to reveal novel biomarkers and clues to altered molecular mechanisms of DS. Comparison between the proteome of AF from unaffected and DS-affected pregnancies allowed selection of 60 candidate biomarkers based on spectral counting. Two candidates, APP and TNC-C, were verified by immunoassays to show two-fold increase in AF from DS-pregnancies. Additionally, CPA4, MUC13, CEL, DPP4 and MMP2 were verified to be differentially expressed in trisomy 21-AF via selected reaction monitoring assays using triple-quadruple mass spectrometer. Amniocytes from DS-affected and unaffected fetuses were also quantitatively analyzed by using Stable Isotope Labelling of Amino acids in Cell culture technique. Over 4900 proteins were identified from amniocyte lysate and supernatant by LTQ-Orbitrap mass spectrometer, and 85% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labelled amino acids. Proteins that consistently showed aberrant expression from affected amniocytes have been selected for further verification and molecular network analyses since they may play a role in DS pathogenesis.

Thesis advisor: Eranthie Weerapana / Functional amino acids that play critical roles in catalysis and regulation are known to display elevated nucleophilicity and can be selectively targeted for covalent modification by reactive electrophiles. Chemical-proteomic platforms, such as activity-based protein profiling (ABPP), exploit this reactivity by utilizing chemical probes to covalently modify active-site residues to inform on the functional state of enzymes within complex proteomes. These and other applications rely on the availability of a diverse array of electrophiles and detailed knowledge of the reactivity and amino-acid specificity of these groups. The sulfonyl fluoride activity-based probe (ABP) DAS1 was discovered to label and inhibit both serine proteases and glutathione S-transferases (GSTs). In the case of GSTs, DAS1 covalently bound to a tyrosine residue, despite predicted serine reactivity. Investigation of potential aryl halide electrophiles for ABPP found that chloronitrobenzene RB2 and dichlorotriazine RB7 covalently modify cysteine and lysine residues in target proteins. Applying an existing ABP, iodoacetamide alkyne (IA-alkyne), demonstrated the ability of ABPP to discover novel reactive residues in short open reading frame (sORF)-encoded peptides, as well as previously unannotated cysteine residues on glycolysis enzymes. These studies illustrate the development and characterization of novel electrophiles and demonstrate the application of ABPs to interrogate biological systems. Looking further ahead, the novel electrophiles also provide new tools for the development of covalent inhibitors for treatment of disease. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The allocation of energetic resources into competing physiological systems is crucial for the survival and fitness of an organism. Maintenance of active growth and effective immune function is energetically costly and therefore, trade-offs should have evolved to optimise the allocation of resources into these systems according to physiological status. While recent studies have begun to elucidate the mechanisms by which cross-talk exists between these two systems, more work is needed to characterise their interactions. A high-throughput proteomics approach was developed to investigate the molecular mechanisms underpinning fast growth and molecular cross-talk between growth and immune function. This approach revealed unique routes to fast growth in two different growth-accelerated coho salmon strains, including increased protein synthesis in fish overexpressing growth hormone (GH) through a focussed up-regulation of translation machinery. Conversely, selectively-bred fish showed more complex alterations in multiple metabolic pathways potentially underlying increased growth and domestication. Additionally, the liver proteome response of rainbow trout to a bacterial challenge unveiled host defence and immune proteins upregulated during the acute phase response (APR), along with candidate proteins involved in re-allocation of energetic resources during an immune response. Additionally, novel genetic expansions of salmonid complement C3 proteins upregulated during bacterial challenge were identified and characterised. Finally, the AMP-activated kinase (AMPK) system was investigated due to its role in managing energetic status, making it an ideal system to investigate crosstalk between growth and immunity. Novel salmonid-specific genetic expansions in AMPK subunits (α, β and γ) were demonstrated and their mRNA level expression analysed in fast-growing fish strains exposed to immune stimuli, where an increase in expression of several subunits was observed for fish overexpressing GH. However, a significant downregulation in expression of several AMPK subunit genes occurred in response to immune stimuli. Overall, this project provides insights into links between growth and immune function in salmonids, which are relevant to the aquaculture industry, where the aim is to maximise fish growth while retaining strong immunocompetence.

The work presented in this thesis aims at unravelling human chromatin composition by quantitative proteomics to outline the functional and structural changes occurring during the life of human cells. Chromatin is the structure formed by proteins and RNAs interacting with the genetic material. At present, chromatin is not well defined. It is not easy to investigate either the composition of its constituent proteins or how this arrangement changes. We set out to analyse the chromatin composition changes occurring during the cell cycle. Our procedure couples a SILAC mass spectrometry-based approach with a newly developed biochemical chromatin purification method, which involves fixation of proteins to DNA. By testing two different fixation times (5 and 10 minutes) and three phases of the cell cycle (G1/S, G2, M), we quantified ~3000 proteins providing a broad picture of the global changes on chromatin protein composition. Surprisingly, chromatin seems to be occupied by many unexpected proteins (40%) that appear to be increased during mitosis. We hypothesized that these unexpected proteins come into contact with DNA during mitosis when the nuclear envelope breaks down and the highly negatively charged DNA can be found in proximity to extra nuclear proteins. We used Pulse-SILAC technique that allows to distinguish newly synthesized proteins to test this possibility. By comparing in a single cell cycle and during G0 arrest the incorporation of new proteins into chromatin with their synthesis in the cytoplasm and in the whole cell, we could not find a different behaviour for the unexpected proteins as result of mitosis. Despite the efforts in tracking down the origin of these unexpected proteins, it is still uncertain whether their presence on chromatin is the result of a biological process or, in part, a drawback of the purification methods adopted. However, proving their genuine presence on chromatin will be important to elucidate how chromatin functions.

Proteomic profiling studies of schizophrenia have the potential to shed further light on this debilitating and poorly understood condition which affects up to 1% of the world’s population. However, recent studies suggest that the field of proteomics in general has been hindered by poor application of bioinformatic strategies, contributing to the failure of many findings to validate. In the context of schizophrenia research, there is therefore a need for a more robust application and integration of existing statistical approaches to proteomic datasets, as well as the development of new methodologies to offer solutions to current challenges. The aims of this thesis were multi-fold. Many studies have stipulated the need for new diagnostic and prognostic strategies to aid psychiatrists, particularly in predicting disease conversion from the prodromal phase. Proteomic data from serum samples was used to investigate the potential for statistical models based on biomarker panels to offer a new and clinically relevant approach. Models were trained based on either differential protein (chapter 3) or peptide (chapter 4) expression levels between schizophrenia patients and controls, as measured through multiplex immunoassay or targeted mass spectrometry technologies. In chapter 4, an SVM model based on 21 peptides was identified that is both highly sensitive and specific as a diagnostic and prognostic tool in symptomatic individuals. Furthermore, in recent years, few preclinical innovations have been made in schizophrenia research in either in vitro or in vivo studies, resulting in a standstill in the development of treatments. In chapters 5 and 6 of this thesis, proteomic information from a novel cellular model of schizophrenia was analyzed. In chapter 5, cell signalling alterations in vitro were identified which may underpin dysfunctional microglial activation in at least a subgroup of patients, thus representing new drug targets in the CNS. Subsequent analysis identified compounds which have the potential to ameliorate the observed changes. Lastly, in chapters 7 and 8, a novel systems biology methodology was developed for the functional comparison of proteomic changes in brain tissue from existing preclinical rodent models of psychiatric disorders to those in human post-mortem samples, providing a new means of overcoming some of the translational hurdles of preclinical psychiatric research. The application of different bioinformatic strategies to a range of proteomic datasets in this thesis has yielded a number of findings which have enhanced the understanding of schizophrenia pathophysiology and provide a platform for future studies towards the goal of improving outcomes for patients affected by this disorder.

Poverty stricken areas of the world are affected by Neglected Tropical Diseases, with an estimated 1 billion sufferers. As well as inadequate living conditions and healthcare, there has been very little pharmaceutical incentive to tackle these diseases. As a result, the diseases are still spreading. Drugs available on the market suffer from poor efficacy, high toxicity, increasing resistance and inappropriate dosing for rural treatment. The nature of many NTDs prevents the use of vaccinations. Therefore, more efficacious and safe treatments are sought after. The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited in a number of drugs and drug targets. The same cannot be said for the kinetoplastids. Drug discovery programmes have focused on targeting enzymes of the folate metabolism with very little clinical success. Despite showing significant inhibition of the parasitic enzymes, potency is seen to decrease in cellular and animal models. Understanding how the folate pathway operates in these organisms could provide insight into where and how anti-folate compounds bind. This information could then be used to facilitate better drug treatments for the kinetoplastids. This thesis describes a number of approaches undertaken to better understand folate metabolism in kinetoplastids. Clinical and literature anti-folate compounds were immobilized onto resins, followed by chemical proteomics, utilizing novel techniques (iTRAQ), to allow for target identification. Using competition studies, specific and non-specific targets were identified in parasitic lysate (T. brucei and L. major) for each anti-folate compound. This method was further exploited by creating a folate resin (Folate beads). The resin had the potential to pull down 9 proteins from the “folate-ome”. In future studies, the resin can be used to enrich for the folate proteins in kinetoplastids and related organisms. Alongside the studies of the folate proteins, it was also desired to study proteins involved in the essential pterin pathway. This pathway has not been extensively studied in kintoplastids, with the exception of PTR1 (by-pass protein for DHFR). The failure to synthesise pterin derivatives for bead coupling led to a fragment screening campaign being carried out on QDPR in leishmania major. Working through a triage workflow, two moderately potent fragments were identified, showing inhibition against LmQDPR. Through structure-free optimization strategies, greater than 100 optimized fragments were synthesised in a bid to understand SAR. Although this work remains incomplete, LmQDPR has been successfully crystalized with 23 hit fragments, which are awaiting further biophysical analysis to understand binding.

Analysis of complex protein samples is demanding due to the wide dynamic range of expression levels and the limited detection range of technology. Proteomics relies heavily on the development of new fractionation strategies to help reduce complexity, and overcome the technological and biological challenges associated with proteome analysis. Here, the development of a prototype instrument named ??Microflow MF10?? was explored to enrich for particular classes of proteins. The MF10 was found to have a number of advantages over commercially available fractionation systems. Due to the reduced separation electrode distance, fractionation was rapid, occuring within ~0.125 kVH over 2-6 fractions under native conditions but longer under denaturing conditions. As low as 2 ng peptide could be fractionated with recovery for downstream analysis achievable. The ability to alter protein charge by changing the pH (acidic (pI 3.6) to basic environments (pI 10.4)) allows selection of proteins based on charge/mobility, size, shape, buffer ionic strength, pH and field strength. Proteins <10 kDa are also not routinely analysed because current technology is unable to cater for this region of the proteome. Peptide enrichment using the MF10 was achieved using a 7-protein/peptide standard mix (1-25 kDa), to the 1-5 kDa fraction with simultaneous fractionation of the higher mass protein standards. Plasma was also used to enrich for the peptidome (< 5 kDa) in the presence of the proteome. Enrichment of 73 proteins inclusive of 22 proteins in the 1-25 kDa fraction was achieved compared to a total equivalent of 42 proteins from unfractionated plasma. Rare samples (≤ 106 cells) from stem cell populations or derived clinically are challenging due to the absolute limits in protein copy number and abundance. CD34+ haematopoietic stem cells and CD4+/CD8+ T-cells were used to develop fractionation methods and elucidate the cell differentiation process. MF10 fractionation and analysis by SDS-PAGE and LC-MS/MS revealed 24 differentially expressed proteins between the 3 cell populations, which may be involved in cell differentiation. To quantify these expression differences, iTRAQ with 2-D LC-MS/MS was applied. This study has highlighted the challenges associated with samples of limited quantity. It has been successful in understanding the effects of various conditions on the electrophoretic mobility of proteins, which in proteomics, has remained largely unexplored.