Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular cells, such as endothelial cells (ECs). Hereditary (H) PAH is mainly caused by ―loss-of-function‖ mutations in the gene coding for the bone morphogenetic protein type II receptor (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. The hypothesis of this thesis was that BMPR2 mutations produce an imbalance in EC protein expression and/or activity that is integrally related to the development of abnormalities in lung vascular function and structure in HPAH. Patient-specific blood-outgrowth endothelial cells (BOECs) expanded ex vivo from peripheral blood mononuclear cells from patients with HPAH and healthy subjects were used to examine the consequences of BMPR2 mutations on the BOEC protein expression profile as well as on their functionality. Functional analyses of the BOECs revealed that HPAH-derived BOECs are more susceptible to apoptosis and more proliferative compared with healthy controls. Protein isolates of BOECs from patients with HPAH and from healthy subjects were subjected to 2-D gel electrophoresis and stained for total proteins and phosphoproteins, and to a quantitative computerassisted analysis. Differentially regulated proteins were identified by mass spectrometry (LC-MS/MS). Of the 416 total proteins detected under basal conditions, 11 were significantly downregulated in HPAH-derived BOECs and 11, including the translationally controlled tumor protein (TCTP), were upregulated. TCTP has previously been shown to be involved in systemic arteriolar remodeling, inflammation and growth. Therefore, the potential role of TCTP in PAH was studied in vivo in the SU5416 rat model of severe angioproliferative PAH. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. Similarly, abundant TCTP immunostaining was also seen in human PAH lung sections, again associated with complex vascular lesions. In BOECs, TCTP was found to participate in cell growth and survival. These data suggest that TCTP could play an important role in PAH by mediating pro-survival and growth signaling in vascular cells, contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

In this investigation a number of post-genomic technologies have be applied to study the dermatological disorders of psoriasis and keloid disease. In spite of considerable research focus on these diseases the pathogenesis remains unclear and currently no cure is available however, both diseases are manageable by drug intervention. It is common place that patients who are suffering from skin disorders are diagnosed and the extent of the disease assessed by a dermatologist which may be subjective due to human error. The availability and application of methods to screen patients and quantify the level of disease or response to treatment has obvious benefits in disease management. The work has incorporated a two-pronged approach combining the spectroscopic analysis of excised tissue samples and the phenotypic profiling of a rapidly proliferating cell line in response to drug intervention. The initial analysis of psoriatic skin samples by MALDI-MS provided poor results which remain relatively unexplained; however similar problems have been observed by other research groups. In a complementary approach the HaCaT cell line was exposed to increasing concentrations of three anti-psoriatic drugs namely dithranol, methotrexate and ciclosporin and the cells profiled using both metabolomic and proteomic methods. A number of metabolic pathways were highlighted including glycolysis and the TCA cycle. This has resulted in a selection of potential biomarkers which could be investigated in further work. In a small follow on study a collection of plasma samples from patients undergoing methotrexate treatment were analysed. The level of patient metadata and the number of samples was relatively limiting however, a subset of metabolites were significantly altered between responders and non-responders and with further validation could be potential biomarkers of successful treatment. The analysis of excised keloid samples was conducted using FT-IR microspectroscopy where it was possible to successfully discriminate between keloid and normal tissue. The use of imaging FTIR illustrated the complex cellular composition within a keloid scar, with increased lipid, amide and phosphate levels being observed. These measurable variations could, in the future, be incorporated into surgical procedures to allow targeted excision ensuring all keloid areas are removed. Finally a SERS-based analysis was conducted to investigate the possibility of probing dynamic enzymatic processes. This was successful and with the use of varying reporter molecules could be a beneficial tool for the analysis of metabolic processes.This project has successfully used a number of bio-analytical techniques to investigate dermatological problems. While the ultimate goal would be the application of a single analytical technique to provide answers to biological questions, it has been found that a number of complimentary techniques and statistical data handling approaches can provide a valuable insight into the problems posed.

Adhesion complexes (ACs) are large macromolecular complexes of integrins and associated proteins that connect the actin cytoskeleton to the extracellular matrix. In migrating cells, ACs are highly dynamic -- forming and maturing at the cell front and disassembling at the cell rear. The turnover of ACs enables and localises the necessary traction forces required for cell migration. There is evidence for the spatiotemporal recruitment of specific proteins during AC maturation or disassembly; however, a holistic understanding of the compositional changes to ACs during these states is lacking. To this end, we sought to characterise the dynamic changes that occur at ACs during turnover using a mass spectrometry (MS)-based proteomics approach. A major challenge in studying AC turnover is the desynchronised nature of AC formation, maturation and disassembly within a population of cells. Therefore a nocodazole-washout assay was used to synchronise microtubule-induced AC maturation and disassembly. To study the dynamics of AC turnover by MS, an AC isolation method was optimised for use with the nocodazole-washout assay. Subsequently, the maturation of ACs by the loss of microtubules was studied by MS-based proteomics, and it was found that this resulted in the overall accumulation of adhesion proteins, and also the conversion of fibrillar adhesions to focal adhesions. Studying the dynamic process of AC disassembly requires a sensitive MS quantification method; as such, label-free quantitative methods were compared, and it was found that LC-MS peak ion intensity quantification performed better than spectral counting. Using optimised methodologies for isolation of ACs and MS quantification, the dynamics of AC disassembly was analysed over the course of the nocodazole-washout assay. It was found that in general, microtubules were enriched around ACs, whereas many structural AC proteins decreased over time. In summary, we have optimised methods for the study of ACs by MS-based proteomics, and applied these methods to the study of AC turnover.

Orientadores: Iscia Teresinha Lopes Cendes, André Schwambach Vieira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T00:14:14Z (GMT). No. of bitstreams: 1 MoratodoCanto_Amanda_M.pdf: 2477839 bytes, checksum: f2e9db3a96d271cc686d6d66697b0e59 (MD5) Previous issue date: 2015 / Resumo: O uso de modelos animais para estudo de doenças humanas é importante para o entendimento dos mecanismos fisiopatológicos destas doenças. Particularmente, os modelos que reproduzem a Epilepsia do Lobo Temporal (ELT) em roedores, apresentam uma epileptogenicidade similar à encontrada em tecidos "epilépticos" humanos quando estudados ex vivo. A ELT afeta cerca de 40% dos pacientes adultos, e é caracterizada clinicamente por um desenvolvimento progressivo de crises epilépticas com foco no lobo temporal. Pacientes que apresentam ELT normalmente não respondem aos tratamentos. Destas, a Epilepsia do Lobo Temporal Mesial (ELTM) e a mais comum e se caracteriza pelo acometimento das estruturas mesiais do lobo temporal, como o caso da Esclerose Hipocampal. A proteômica dispõe de ferramentas poderosas que nos permitem elucidar mecanismos biológicos complexos, encontrar proteínas alteradas em todo o organismo e descrever padrões de expressões proteicas em diferentes condições fisiológicas e patológicas. Portanto, é relevante analisar esse padrão de expressão no hipocampo de modelos animais de ELTM usando técnicas de proteômica, a fim de gerar informações que nos auxiliem no entendimento dos mecanismos envolvidos na epileptogênese desses modelos. No presente estudo, as proteínas identificadas podem nos indicar novas vias envolvidas com a epileptogênese. Além disso, nossos dados demonstram que uma complexidade molecular adicional pode ser observada quando analisamos as diferentes sub-regiões do hipocampo separadamente. Portanto, acreditamos que a integração dos dados de proteômica com dados obtidos por outras "ômicas" podem gerar dados ainda mais informativos sobre esses processos neuronais / Abstract: Studies about human diseases using animal models are really important to our understanding about the physiopathology mechanisms from those diseases. Particularly, the models that reproduce the Temporal Lobe Epilepsy (TLE) in rodents, presents epileptogenicity similar to that found in ex vivo human tissues. The TLE affects around 40% of the adult patients and it is clinic characterized by a progressive development of seizures with temporal lobe focus, caused by an unbalance between the excitatory and inhibitory neurotransmission. Patients who present that type of epilepsy normally don¿t respond well to the treatments. Of this type of epilepsy, the Mesial Temporal Lobe Epilepsy (MTLE) is the most common one and it is characterized by the commitment of the mesial temporal lobe structures, such as in the Hipocampal Sclerosis. To realize these studies the proteomics has many powerful tools that allow us to elucidate complex biological mechanisms, to find altered proteins in the whole organism and describe protein expression patterns in different physiological and pathological conditions. Therefore, it¿s relevant to study this protein expression pattern in the hippocampus of animal models of MTLE using proteomics techniques, searching for informative data that lead us to the understanding of the involved mechanisms in the epileptogenicity. In this study we identified proteins that can indicate new pathways involved in the epileptogenesis processes. Furthermore, our data demonstrate that additional molecular complexity could be observed as hippocampal subfields were analyzed separately. We believe that the further integration of the proteomic data with other "omics" approaches could generate even more informative data about those neuronal processes / Mestrado / Fisiopatologia Médica / Mestra em Ciências

With a global growing demand in food production, agricultural output must increase accordingly. An increased use of saline soils and brackish water would contribute to the required increase in world food production. Abiotic stresses, such as salinity and drought, are also major limiters of crop growth globally - most crops are relatively salt sensitive and are significantly affected when exposed to salt in the range of 50 to 200 mM NaCl. Genomic resources from plants that naturally thrive in highly saline environments have the potential to be valuable in the generation of salt tolerant crops; however, these resources have been largely unexplored. Salicornia bigelovii is a plant native to Mexico and the United States that grows in salt marshes and coastal regions. It can thrive in environments with salt concentrations higher than seawater. In contrast to most crops, S. bigelovii is able to accumulate very high concentrations (in the order of 1.5 M) of Na+ and Cl- in its photosynthetically active succulent shoots. Part of this tolerance is likely to include the storage of Na+ in the vacuoles of the shoots, making S. bigelovii a good model for understanding mechanisms of Na+ compartmentalization in the vacuoles and a good resource for gene discovery. In this research project, phenotypic, genomic, transcriptomic, and proteomic approaches have been used for the identification of candidate genes involved in salinity tolerance in S. bigelovii. The genomes and transcriptomes of three Salicornia species have been sequenced. This information has been used to support the characterization of the salt-induced transcriptome of S. bigelovii shoots and the salt-induced proteome of various organellar membrane enriched fractions from S. bigelovii shoots, which led to the creation of organellar membrane proteomes. Yeast spot assays at different salt concentrations revealed several proteins increasing or decreasing yeast salt tolerance. This work aims to create the basis for Salicornia research by providing a genome, transcriptomes, and organellar proteomes, contributing to salinity tolerance research overall. We identified a set of candidate genes for salinity tolerance with the aim of shedding some light on the mechanisms by which this plant thrives in highly saline environments.

Cerebral malaria (CM) is a severe neurological complication of malaria infection that results from interrelated pathologies. Despite extensive research efforts, the mechanism of the disease is not completely understood. Clinical studies, postmortem analysis, and animal models have been the main research arenas in CM. In this thesis, shotgun proteomics approach was used to further understand the pathology of human and experimental CM. The mechanism by which CM turns fatal is yet to be identified. A clinical proteomics study was conducted on pooled plasma samples from children with reversible or fatal CM from the Gambia. The results show that depletion of coagulation factors and increased levels of circulating proteasomes are associated with fatal pediatric CM. This data suggests that the ongoing coagulation during CM might be a disseminated intravascular coagulation state that eventually causes depletion of the coagulation factors leading to petechial hemorrhages. In addition, the mechanism(s) by which blood transfusion benefits CM in children was investigated. To that end, the concentration and multimerization pattern of von-willebrand factor, and the concentration of haptoglobin in the plasma of children with CM who received blood transfusions were measured. In addition to clinical studies, experimental cerebral malaria (ECM) in mice has been long used as a model for the disease. A shotgun proteomics workflow was optimized to identify the proteomic signature of the brain tissue of mice with ECM.Because of the utmost importance of membrane proteins in the pathology of the disease, sample fractionation and filter aided sample preparation were used to recover them. The proteomic signature of the brains of mice infected with P. berghei ANKA that developed neurological syndrome, mice infected with P. berghei NK56 that developed severe malaria but without neurological signs, and non-infected mice, were compared to identify CM specific proteins. Among the differentially expressed proteins in mice that developed neurological signs, coagulation and acute-phase proteins were enriched. The data are in accordance with data from the clinical study. Taken together, the results support the role of coagulation and platelets adhesion in the pathogenesis of the disease.

Global climate change imposes physiological constraints on marine ecosystems that can alter the distribution of intertidal organisms. In one such instance, the native cold-adapted mussel Mytilus trossulus is being replaced along its southern range by the invasive warm-adapted Mytilus galloprovincialis. These blue mussels occur throughout rocky intertidal zones where they are subjected to greatly varying environmental conditions known to induce oxidative stress. We hypothesize that while under acute stress, related Mytilus congeners undergo a shift in redox potential from NADH-fueled respiratory pathways to pathways producing NADPH as a way to decrease the production of reactive oxygen species (ROS) and provide reducing equivalents to detoxify ROS. Additionally, we hypothesize that sirtuins (SIRT; a family of NAD-dependent deacetylases) might be involved in the regulation of this metabolic transition. To test the latter, a discovery approach will be used to analyze the proteomic response of M. galloprovincialis and M. trossulus to the pro-oxidant menadione, and sirtuin-inhibitors nicotinamide and suramin. Menadione can induce oxidative stress by increasing endogenous peroxide and superoxide radicals, while suramin and nicotinamde both inhibit sirtuin activity. Organisms were exposed to these compounds in filtered seawater for 8 h, followed by a 24.5 h recovery period under constant aeration. A multivariate analysis utilizing 2D-gel electrophoresis and protein identification via mass spectrometry showed that 18% and 17% of all identified protein spots detected demonstrated changes in abundance in M. galloprovincialis and M. trossulus, respectively. Using matrix-assisted laser desorption ionization (MALDI) tandem time-of-light mass spectrometry, we were able to identify 32-41% of proteins, depending on the species. The two Mytilus congeners showed the greatest differences in changes of protein abundance for oxidative stress proteins (including NADP-dependent isocitrate dehydrogenase). Both congeners showed similar effects in response to simultaneous sirtuin inhibition and MIOS for proteins involved in protein degradation (proteasome), cytoskeletal modifications (actin and tubulin), proteins regulating actin filament growth (F-actin capping protein), amino acid metabolism and stress signaling (G-proteins, small G-proteins and MAPK). Results indicate that protein acetylation plays an important role in the oxidative stress response of M. galloprovincialis. More specifically this suggests that sirtuins play an important role in regulating the general stress response in M. galloprovincialis and thus contribute to the greater stress resistance of this species. Furthermore, changes in the abundance of several molecular chaperones suggest a greater effect of sirtuins in regulating the cellular response to heat stress, which could in part explain why this species is more heat-tolerant than the native M. trossulus.

The National Cancer Institution reported over 80,000 diagnoses of bladder cancer (BCa) in the United States in 2018. Despite these numbers, minimal research toward developing new diagnostic techniques and treatment options are underway. Evidence suggests a significant increase in non-specific a-naphthyl acetateesterase levels in BCa patient’s urine. There has been little research focused on identification of the esterase present. It is also suggested that elevated oxidative stress resulting in production of reactive oxygen species (ROS) is common in tumorigenic bladder cells as a result of increased metabolic activity. Oxidized protein hydrolase (OPH) is an 80kD serine protease, previously found to be elevated in many other types of cancer. OPH degrades proteins damaged by ROS and also exhibits a highly specific esterase activity toward (AcApNA) N-acetyl-alanyl-p-nitroanilide and ANAA (α-naphthyl N-acetylalaninate) containing substrate. Investigation of OPH expression in BCa could result in development of new diagnostic techniques and possible application toward prodrugs targeting cells with elevated ROS and/or OPH. Due to lack of commercial OPH, a positive control for this protein is needed for testing. To do this E. coli(BL-21 DE-3) were cultured and inserted with pET-21a (+) plasmids containing a human OPH gene insert prior to a His7 tag. After being selectively grown on ampicillin media, the bacteria were induced by IPTG and digested using lysozyme. The soluble rOPH suspended in the supernatant was separated from the pellet by centrifugation and further purified using Ni-NTA resin chromatography columns specific for the His7 tag sequence. The UM-UC-3 bladder cancer cell line, commonly used in published research to screen efficiency of chemotherapeutics, were cultured in accordance to ATCC. These cells were then compared against none tumorigenic bladder cancer cells and rOPH in a series of tests. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) were transferred for western blot analysis using antibodies specific for human OPH to investigate the expression levels present in cells. Native-PAGE electrophoresis showed OPH esterase activity across these cells using S-ANAA substrate as a specific esterase colorimetric stain. With these results, possible treatment options can be investigated with use of novel prodrug chemotherapy specifically targeting OPH in BCa cells, ultimately leading to apoptosis in effected cells. These events may also lead to possible biomarkers used for easier and earlier diagnosis of BCa across various spectrums.

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.