Chronic liver diseases, which includes alcoholic liver disease (ALD), are consistently among the top 15 leading causes of death in the United States. ALD is characterized by progression from a normal liver to fatty liver disease (hepatic steatosis), which can lead to cirrhosis, alcoholic hepatitis, and liver failure. We have identified a novel role of phosphohistidine signaling, mediated through phosphohistidine phosphatase 1 (PHPT1), in the onset of hepatic steatosis. We have identified PHPT1 as a target of selective oxidation following acute ethanol exposure as well as being downregulated following chronic ethanol exposure. We mapped the oxidative modification site and developed a mass-spectrometry based phosphohistidine phosphatase assay to determine the impact of PHPT1 oxidative modification during acute ethanol exposure. To further understand the role of PHPT1 and phosphohistidine signaling during chronic ethanol exposure, we have developed PHPT1 overexpression and knockout mouse models. These mouse models were characterized using mass spectrometry-based proteomics. They were then utilized in a 10-day chronic ethanol plus binge model to determine the impact of PHPT1 expression on the onset of ethanol-induced hepatic steatosis. In addition, advanced mass spectrometry-based phenotypic characterization was performed on the treated liver tissues to determine the key regulators and canonical pathways influencing phosphohistidine signaling during chronic ethanol exposure. We have evidence to suggest that PHPT1 overexpression plays a protective role in the onset of hepatic steatosis, the PHPT1 heterozygous model is more susceptible to liver damage, and the complete knockout model is embryonically lethal. Additionally, we have identified novel pathways and regulators involved in phosphohistidine signaling during the development of ethanol-induced hepatic steatosis.

PAIVA, Ana Luiza Sobral. Respostas bioquímicas do feijão-de-corda [Vigna unguiculata L. (Walp.)] ao estresse salino e infecção pelo vírus do mosaico severo do caupi (CPSMV) reveladas pela proteômica quantitativa livre de marcação. 2015. 200 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2015. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-08T12:56:09Z No. of bitstreams: 1 2015_dis_alspaiva.pdf: 4225070 bytes, checksum: 0559261a4c594b2649cdb60e4563c1fc (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:16:37Z (GMT) No. of bitstreams: 1 2015_dis_alspaiva.pdf: 4225070 bytes, checksum: 0559261a4c594b2649cdb60e4563c1fc (MD5) / Made available in DSpace on 2016-08-02T20:16:37Z (GMT). No. of bitstreams: 1 2015_dis_alspaiva.pdf: 4225070 bytes, checksum: 0559261a4c594b2649cdb60e4563c1fc (MD5) Previous issue date: 2015 / As sessile organisms, plants are exposed to a plethora of environmental stresses to which they must respond to maintain efficient growth and survival. Therefore, in order to improve our understanding on the complex mechanisms involved in the cowpea response to salt stress and to a compatible interaction with the cowpea severe mosaic virus (CPSMV), we used a label-free quantitative proteomic approach to identify the salt and virus responsive proteins in the leaves of the Pitiuba (CE-31) cultivar. The proteins extracted from the leaves (control and treated) 2 and 6 days post-treatment only with salt (DPS), only infected with CPSMV (DPV) or both of them (DPSV) were analyzed using mass spectrometry. At 2 DPS, 350 proteins with at least two-fold differences in abundance, in comparison with controls, were differentially accumulated in the leaves of the salt-treated (80% up and 20% down-accumulated), 281 at 2DPV (25% up and 75% down-accumulated) and 321 at 2 DPSV (45% up and 55% down-accumulated) plants. At 6 DPS, 350 proteins were differentially accumulated in the leaves of the salt-treated (90% up and 10% down-accumulated), 225 at 6 DPV (80% up and 20% down-accumulated) and 315 at 6 DPSV (94% up and 6% down-accumulated) plants. The qualitative analysis showed biochemical differences when the cowpea plants were challenged concurrently with both stresses. To cope with salinity, cowpea increased the abundance of proteins directly involved with the salt tolerance mechanisms. The results indicated that the CPSMV induce the down-accumulating of several proteins to invade and spread in host at early infection period (2 DPV), but at 6 DPV plant can induce accumulation of diverse proteins related with defense, although these strategies can’t avoid the negatives effects of disease. When exposed simultaneously to salt/CPSMV stresses, a balance in protein accumulation involved in many biological process. This is the first work employing this approach in cowpea and providing evidences of the plant biochemical mechanisms involved in the responses of cowpea to these stresses. / Como organismos sésseis, as plantas são expostas a uma variedade de estresses ambientais aos quais devem responder para sobreviverem e se desenvolverem. A fim de melhorar a nossa compreensão sobre os mecanismos complexos envolvidos na resposta do feijão-de-corda ao estresse salino e na interação compatível com o vírus do mosaico severo do caupi (CPSMV), foi utilizada uma abordagem proteômica quantitativa, livre de marcação, para identificar proteínas, responsivas a essess estresses em folhas de feijão-de-corda, cv. CE-31. As proteínas extraídas a partir de folhas primárias, 2 e 6 dias após o tratamento só com o sal (DPS), somente infectadas (DPV), ou sob ação combinada dos dois (DPSV) foram analisadas, usando espectrometria de massas e comparadas com grupo controle. No 2° DPS, foram identificadas 350 proteínas diferencialmente acumuladas (80% aumentaram em abundância e 20% diminuíram), no 2° DPV 281 (25% aumentaram em abundância e 75% diminuíram) e no 2° DPSV 321 (45% aumentaram em abundância e 55% diminuíram). Já no 6° DPS, foram identificadas 350 proteínas diferencialmente acumuladas (90% mostraram aumento em abundância e 10% diminuição), no 6° DPV 225 (80% aumentaram em abundância e 20% diminuíram) e no 6° DPSV 315 proteínas(94% aumentaram em abundância e 6% diminuíram). Para lidar com a salinidade, o cv. CE-31 aumentou a abundância de proteínas envolvidas diretamente com os mecanismos de tolerância ao sal. Em relação à infecção da planta pelo CPSMV, os resultados obtidos indicaram que o vírus induz redução na abundância de várias proteínas nos tempos iniciais de infecção, provavelmente favorecendo a invasão e propagação na planta, mas, no 6° DPSV, a planta recupera sua capacidade de acionar mecanismos de defesa, embora esses já não sejam mais efetivos para evitar o estabelecimento da doença viral. Durante exposição simultânea da planta ao sal e ao vírus, ocorreu um equilíbrio entre o aumento e diminuição em abundância de proteínas envolvidas em diversos processos metabólicos. Esse trabalho é pioneiro nessa abordagem em feijão-de-corda e fornece evidências dos mecanismos bioquímicos envolvidos nas resposta da planta a esses estresses.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As sessile organisms, plants are exposed to a plethora of environmental stresses to which they must respond to maintain efficient growth and survival. Therefore, in order to improve our understanding on the complex mechanisms involved in the cowpea response to salt stress and to a compatible interaction with the cowpea severe mosaic virus (CPSMV), we used a label-free quantitative proteomic approach to identify the salt and virus responsive proteins in the leaves of the Pitiuba (CE-31) cultivar. The proteins extracted from the leaves (control and treated) 2 and 6 days post-treatment only with salt (DPS), only infected with CPSMV (DPV) or both of them (DPSV) were analyzed using mass spectrometry. At 2 DPS, 350 proteins with at least two-fold differences in abundance, in comparison with controls, were differentially accumulated in the leaves of the salt-treated (80% up and 20% down-accumulated), 281 at 2DPV (25% up and 75% down-accumulated) and 321 at 2 DPSV (45% up and 55% down-accumulated) plants. At 6 DPS, 350 proteins were differentially accumulated in the leaves of the salt-treated (90% up and 10% down-accumulated), 225 at 6 DPV (80% up and 20% down-accumulated) and 315 at 6 DPSV (94% up and 6% down-accumulated) plants. The qualitative analysis showed biochemical differences when the cowpea plants were challenged concurrently with both stresses. To cope with salinity, cowpea increased the abundance of proteins directly involved with the salt tolerance mechanisms. The results indicated that the CPSMV induce the down-accumulating of several proteins to invade and spread in host at early infection period (2 DPV), but at 6 DPV plant can induce accumulation of diverse proteins related with defense, although these strategies canât avoid the negatives effects of disease. When exposed simultaneously to salt/CPSMV stresses, a balance in protein accumulation involved in many biological process. This is the first work employing this approach in cowpea and providing evidences of the plant biochemical mechanisms involved in the responses of cowpea to these stresses. / Como organismos sÃsseis, as plantas sÃo expostas a uma variedade de estresses ambientais aos quais devem responder para sobreviverem e se desenvolverem. A fim de melhorar a nossa compreensÃo sobre os mecanismos complexos envolvidos na resposta do feijÃo-de-corda ao estresse salino e na interaÃÃo compatÃvel com o vÃrus do mosaico severo do caupi (CPSMV), foi utilizada uma abordagem proteÃmica quantitativa, livre de marcaÃÃo, para identificar proteÃnas, responsivas a essess estresses em folhas de feijÃo-de-corda, cv. CE-31. As proteÃnas extraÃdas a partir de folhas primÃrias, 2 e 6 dias apÃs o tratamento sà com o sal (DPS), somente infectadas (DPV), ou sob aÃÃo combinada dos dois (DPSV) foram analisadas, usando espectrometria de massas e comparadas com grupo controle. No 2 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (80% aumentaram em abundÃncia e 20% diminuÃram), no 2 DPV 281 (25% aumentaram em abundÃncia e 75% diminuÃram) e no 2 DPSV 321 (45% aumentaram em abundÃncia e 55% diminuÃram). Jà no 6 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (90% mostraram aumento em abundÃncia e 10% diminuiÃÃo), no 6 DPV 225 (80% aumentaram em abundÃncia e 20% diminuÃram) e no 6 DPSV 315 proteÃnas(94% aumentaram em abundÃncia e 6% diminuÃram). Para lidar com a salinidade, o cv. CE-31 aumentou a abundÃncia de proteÃnas envolvidas diretamente com os mecanismos de tolerÃncia ao sal. Em relaÃÃo à infecÃÃo da planta pelo CPSMV, os resultados obtidos indicaram que o vÃrus induz reduÃÃo na abundÃncia de vÃrias proteÃnas nos tempos iniciais de infecÃÃo, provavelmente favorecendo a invasÃo e propagaÃÃo na planta, mas, no 6 DPSV, a planta recupera sua capacidade de acionar mecanismos de defesa, embora esses jà nÃo sejam mais efetivos para evitar o estabelecimento da doenÃa viral. Durante exposiÃÃo simultÃnea da planta ao sal e ao vÃrus, ocorreu um equilÃbrio entre o aumento e diminuiÃÃo em abundÃncia de proteÃnas envolvidas em diversos processos metabÃlicos. Esse trabalho à pioneiro nessa abordagem em feijÃo-de-corda e fornece evidÃncias dos mecanismos bioquÃmicos envolvidos nas resposta da planta a esses estresses.

Este estudo trata das proteínas relacionadas ao desenvolvimento e à formação da parede celular vegetal de cana-de-açúcar, com o objetivo de auxiliar no desenvolvimento de novas tecnologias para a produção de etanol celulósico a partir do bagaço de cana. Com isso, as proteínas de parede celular de entrenós e folhas de plantas com 4 meses de idade em dois estádios de desenvolvimento, juvenil e maduro, foram identificadas. Para extração foi utilizado o método não destrutivo por infiltração a vácuo utilizando dois sais, 0,2 M de CaCl2 e 2 M de LiCl seguido de centrifugação. As amostras complexas foram digeridas, fracionadas, sequenciadas por LC-MSE . Os peptídeos foram processados utilizando o ProteinLynx 2.5 e comparados com a base de dados de ESTs traduzidos de cana e sorgo. A anotação das proteínas foi realizada com base no programa PFAM e dividas em classes funcionais. Apenas as proteínas que apareceram em pelo menos duas das três repetições biológicas foram utilizadas na análise principal. Para prever a localização subcelular das proteínas selecionadas utilizaram-se os softwares: SignalP, TargetP, Predotar e TMHMM. Apenas aquelas proteínas que foram preditas para serem secretadas por dois ou mais programas foram consideradas como proteínas de parede celular (PPC). Ao todo, 543 proteínas foram consideradas como PPC: 205 em entrenós jovens, 143 em entrenós maduros, 124 em folhas jovens e 71 em folhas maduras. Dentre essas proteínas, 365 foram consideradas diferentes, e caracterizadas em dez classes funcionais. A análise estatística compreendeu a análise de PCA e PLS-DA, havendo diferença estatística entre os tratamentos analisados. Neste trabalho, foram encontradas 66 glicosil-hidrolases e 39 peroxidases, sendo 14 e 11 exclusivas de tecidos juvenis, respectivamente. Essas proteínas são conhecidas por terem funções relacionadas à quebra e ao remodelamento dos polissacarídeos da parede celular vegetal, e, portanto, foram indicadas neste estudo como alvo de pesquisas futuras que utilizem as próprias enzimas da planta para otimização da produção do etanol celulósico.Individualmente, este estudo foi o que mais identificou PPCs dentre a literatura existente, além de ter sido pioneiro na utilização da análise quantitativa para PPC. / This study provides information about the proteins of the cell wall of sugarcane at diferente stages of development and formation. The aim of this study is to assist in the development of new technologies for the production of cellulosic ethanol from sugarcane bagasse. Cell wall proteins from 4-month-old internodes and leaves of sugarcane in two developmental stages, juvenile and mature, have been identified. Protein extraction was performed with a non-destructive method by using vacuum infiltration with two salts, 0.2 M CaCl2 and 2 M LiCl, followed by centrifugation. Complex samples were digested, fractionated and sequenced by LC-MSE. Peptides were processed by ProteinLynx 2.5 and compared to the translated sugarcane and sorghum ESTs database. The annotation of the proteins was performed using PFAM and the functional classification was according the one used in other related studies. Only the proteins that appeared in at least two of the three biological replicates were used in the main analysis. In order to predict the subcellular localization of these proteins, SignalP, TargetP, TMHMM and Predotar softwares were used. Only those proteins that were predicted to be secreted by two or more programs were considered as cell wall proteins (PPS). Altogether, 543 proteins were classified as PPC: 205 inimmature internodes, 143 in mature internodes, 124 in young leaves and 71 in matured leaves. Among these proteins, 365 were considered different, and divided into ten functional classes. Statistical analysis was made with PCA and PLSDA, confirming that there were statistical differences among the treatments. In this work, 66 glycoside hydrolases and 39 peroxidases c identified, being 14 and 11 unique to young tissues, respectively. These proteins have their function related to plant cell wall polysaccharides breakdown and remodeling, and, therewith, the glycoside hydrolases and peroxidases found in this study were indicated to be the target of future research using the plant\'s own enzymes to optimize the cellulosic ethanol production. Individually, this study was the one that most identified PPC among the existing literature, and is a pioneer in the use of quantitative analysis for PPCs.

Many diseases are caused by proteins or peptides folding incorrectly and aggregating into fibrils or plaques, including Alzheimer’s disease, Parkinson's disease and type II diabetes. Amyloid formation in the human pancreas occurs via the aggregation of a 37 amino acid peptide called amylin or IAPP which is shown to be toxic to pancreatic β cells. Amylin (IAPP) aggregation initiates a large number of events, leading ultimately to cell death. However the exact cytotoxic action of human IAPP and also the underlying molecular events leading from amylin (IAPP) aggregation to β cell death is still unknown. The toxic effect of human amylin (IAPP) is thought to involve changes in the expression of several genes and proteins. Further transcriptional and proteomics studies in this field can therefore facilitate the identifications of new targets whose expression are affected by amylin (IAPP). These information could be further used to construct an integrated model of the signalling and regulatory pathways through which amylin (IAPP) interacts with cellular metabolism.To investigate the effects of amylin (IAPP) aggregation on the islets proteome in this study, rat Rin-5F cell line, reported as a model of pancreatic β cell, was used. MTT assay was initially performed to determine the effect of IAPP on the cell viability at different time points. The isolated proteins form the untreated and IAPP treated Rin-5F cells were then fractionated by off gel electrophoresis and analysed by quantitative label free LC- MS/MS approach.Label free quantification of IAPP treated Rin-5F cells has identified the altered expression of many proteins, some of which were previously suggested in the literature to be involved in the pathogenesis of type 2 diabetes. These proteins were map to several pathways (including glycolysis and proteasome) whose expressions were significantly affected upon amylin (IAPP) exposure. The IAPP responsive proteins were also structured into a well connected network. Some of the hub proteins identified in this network were greatly affected as the result of IAPP treatments of RIN-5F cells. Our data therefore revealed the effect of IAPP on several proteins and pathways that might be important in the pathogenesis of type 2 diabetes.

No description available.

The mapping of the refractive index of cells has been extensively studied since 1950s. This optical parameter constitutes a key biophysical property strongly correlated to fundamental cell parameters such, e.g., intracellular mass distribution and protein concentration. Experimental studies evidence that the cell refractive index (Refractive Index) provides critical insights to understand diverse cellular structures and interpret pathological states, including diverse stages of diseases. However, measuring the refractive indices of biological specimens satisfying clinical requirements is currently challenging, since there is a lack of spectral signatures of sub-cellular components in the visible range due to their transparent nature. Designing methods capable of extracting visible fingerprints of cellular components remains attracting large research interests. In this work, I have contributed to this project by fabricating and characterizing a black nanostructured membrane that dynamically interacts with cancerous cells and furnishes label-free structural color generation by exploiting the inherent contrast mechanisms of them. Thus, adequately meeting morphology differentiation to assist in biomedical research. I have tested the system with HCT116 colorectal cancer cells. In addition, this special membrane allows refractive index recovery and cell thickness mapping with commonly available bright-field microscopy equipment. Therefore, it is of considerable clinical importance to allow the generation of qualitative information about cell morphology to add in medicine and biophysics research.

Mass spectrometry is the primary technology of proteomics. For the analysis of complex proteomes, protein identities and quantities are inferred from their peptides that are generated by cleaving all proteins with the endopeptidase trypsin. But there is one major disadvantage that is due to biophysical differences, different peptides cause different intensities. Miscellaneous approaches have been developed to circumvent this problem based on the chemical or metabolic introduction of heavy stable isotopes. This enables to monitor protein abundance differences of two or more samples on the same tryptic peptides that differ in mass only. Absolute quantification can be achieved similar by spiking-in synthetic isotopical labeled counterparts of a sample’s tryptic peptides. However, labeling technics suffer from high prices, introduced biases, need for extensive manual control, laborious implementation and implementation restrictions. Therefore, a multiplicity of label-free approaches have been developed that profit from instrumental improvements targeting reliability of identifications and reproducibility of quantitative values. No extensive systematic comparison of label-free quantitative parameters has been published so far presumably because of the laborious implementation. An analysis of primary label-free parameters and associated normalization methods is presented here that compares dynamic and linear ranges and accuracies in the estimation of protein amounts. This facilitated the establishment of label-free procedures addressing three fundamental questions in proteomics: what is a sample’s composition, are proteins that share a specific property enriched and what are the differences between two (or more) samples. A new mathematic model is presented that defines and elucidates enrichment. The procedures were applied first to analyze and compare stem cell plasma membrane proteomes. This is an ambitious model for proteomics because of only small amounts of arduous to analyze, partial hydrophobic proteins in a complex proteomic and chemical background. It is of scientific relevance, as membrane proteins are the cell’s communication interface that enable cell type specific processes and hence can be used to define, isolate and quantify those. The success of cell surface proteome enrichment, the quantitative composition of the proteome and the proteomic difference between stem cells isolated from the dental pulp and cultivated in different media is shown. Secondly, the procedures were applied to the analysis of transient protein networks that assemble onto proteo-liposomes in a newly designed recruitment assay that fully recapitulates membrane sorting as seen in vivo. All transmembrane proteins need to be trafficked to other organelles’ membranes by vesicular trafficking. Sorting signals within the cytosolic regions of the protein cargos trigger the formation of trafficking complexes around those. The transient membrane complexes additionally recognize organelle or organelle-domain specific membrane lipids, such as phosphatidylinositol phosphates. Different trafficking ways are characterized by different trafficking complexes. The elucidation of trafficking complexes that form around a transmembrane protein of interest discloses its trafficking routes and involved signaling processes. The synthetic proteo-liposomes were prepared from chemically defined lipids and heterologous expressed cytosolic domains of type I or type II membrane receptors. The proteomic analyses of such samples are challenging because of huge proteomic backgrounds of proteins binding to the liposomes irrespective of the receptor and relatively small amounts and numbers of receptor-specific binders. Though the basic idea is to elucidate sorting machineries and study membrane trafficking processes, such experiments are untargeted and miscellaneous discoveries were achieved. We elucidated that the apical determinant crumbs 2 is a cargo of the retromer complex. This revealed a fameless level of control for the establishment of cell polarity. We found retromer along with the adapter complexes AP 4 and AP 5 trafficking the beta amyloid precursor protein APP. This confirmed recent publications and yielded new insights. Moreover, many more proteins and complexes appeared to associate with the cytosolic part of APP (AICD) in a membrane context-dependent or -independent manner. Among those, some were so far unknown to interact with AICD, like mTORC1 and the PIKFyve complex.

In this work a Raman flow cytometer is presented. It is a whole new microfluidic device that takes advantage of basic principles of Raman spectroscopy and fluorescent flow cytometry mixed together in a system of particularly shaped channels. These are indeed composed by specific shape and sizes – thanks to which cells can flow one-by-one – and a trap by means of which cells are trapped in order to perform Raman analysis on single ones in a constant and passive way. In this sense the microfluidic device promotes a fast method to look for single cells in a whole multicellular sample. It is a label-free analysis and this means that, on the contrary of what happens with fluorescent flow cytometry, the sample does not need to undergo any particular time-consuming pretreatment before being analyzed. Moreover it gives a complete information about the biochemical content of the sample thanks to the involvement of Raman spectroscopy as method of analysis. Many thought about a device like this, but eventually it is the first one being designed, fabricated and tested. The materials involved in the production of the Raman flow cytometer are chosen wisely. In particular the chip – the most important component of the device – is multilayered, being composed by a slide of calcium fluoride (which gives a negligible signal in Raman analyses), a photosensitive resist containing a pattern with channels and another slide of calcium fluoride in order for the channels to be sealed on both sides. The chip is, in turn, connected to gaskets and external frames. Several fabrication processes are followed to ultimately get the complete Raman flow cytometer and experiments on red blood cells demonstrate its validity in this field.

No description available.