Leishmaniosen gehören zu den tropischen Krankheiten und bedrohen geschätzte 350 Millionen Menschen in 88 Ländern weltweit. Die schwerste Form, viszerale Leishmaniose, betrifft die ärmsten Bevölkerungsschichten und ist die Ursache für circa 50 000 Todesfälle pro Jahr. Es wird angenommen, dass die Entwicklung eines Impfstoffs möglich ist, aber trotz aller Bemühungen, steht derzeit noch kein Impfstoff zur Verfügung. Im Rahmen dieser Arbeit wurde ein Impfstoff gegen viszerale Leishmaniose entwickelt und in vivo auf pre-klinischer Ebene getestet. Des Weiteren wurden rekombinante Membranvesikel konstruiert, um ein Boostreagenz zu erhalten. Die Herstellung sowohl des rekombinanten Salmonellenimpfstoffs als auch der Membranvesikel sollte, trotz des geringen Handelspreis, ökonomisch praktikabel sein, was besonders wichtig ist für Menschen in den betroffenen Entwicklungsländern. Der erste Schritt war die Auswahl neuartiger Antigenkandidaten aus einem Proteomics Datensatz, in dem beide Leishmania Lebensformen verglichen wurden. Der Schwerpunkt wurde auf abundante, hypothetische Proteine gelegt, die sowohl in Pro- als auch Amastigoten identifiziert wurden, in Leishmanienarten hochkonserviert sind aber gleichzeitig keine Sequenzhomologien zu humanen und murinen Proteinen besitzen. Diese Antigene wurden in unterschiedlicher Menge auf der Oberfläche und im Cytoplasma von S. typhimurium SL3261 und auch auf Membranvesikeln exprimiert. Impfstämme wurden selektiert in Hinsicht auf ihre bakterielle Fitness und Antigenexpression. Es konnte gezeigt werden, dass LinJ08.1140-, LinJ23.0410-exprimierende Impfstämme oder eine Mischung dieser in der Lage waren besonders anfällige BALB/c Mäuse vor L. major und wichtiger L. donovani Infektion zu schützen. Analyse der humoralen Immunantwort deutet darauf hin, dass der Impfschutz das Ergbnis einer TH1 Antwort war. Erste Schritte zur Aufklärung struktureller und funktioneller Eigenschaften von LinJ08.1140 wurden unternommen. Es wird allgemein angenommen, dass antigenspezifische CD4+ und CD8+ T-Zellen am Schutz beteiligt sind. Daher wurde für LinJ08.1140 potentielle MHC-I Epitope mit Hilfe von bioinformatischen Programmen vorhergesagt. Zusätzlich deuten Fluoreszenz-färbungen mit antigenspezifischen Antikörpern in L. major Promastigoten darauf hin, dass LinJ08.1140 eine Rolle bei der Zellteilung spielt. / Leishmaniasis is a neglected tropical disease and currently an estimated 350 million people in 88 countries around the world are at risk. Its most severe form, visceral leishmaniasis, affects the poorest people in a population and causes an estimated 50 000 deaths every year. Vaccination is thought to be feasible but despite all efforts, no vaccine is yet available. Vaccines will mainly be targeted for people in developing countries such as India, thus focus has to be placed on affordability. In this thesis a vaccine against visceral leishmaniasis was designed and evaluated in vivo at pre-clinical level. Furthermore, recombinant outer membrane vesicles were developed in an attempt to create a booster reagent. Both, the recombinant salmonella vaccine and the preparation of outer membrane vesicles should be commercially viable, and can still be sold at low prices, which is crucial for people in developing countries. First, novel antigen candidates were selected using proteomics data comparing leishmania life stages. Abundant and hypothetic proteins, which have been identified in both parasite life stages and have high sequence homology throughout Leishmania species while lacking homologues in human and mouse, were selected. These antigens were differentially expressed on the surface or in the cytosol of S. typhimurium SL3261 and in the form of outer membrane vesicles. A two step procedure was developed to select optimised vaccine strains based on bacterial fitness and antigen expression. Selected salmonella strains expressing LinJ08.1140, LinJ23.0410 or an admixture of these strains are shown to protect susceptible BALB/c mice by reducing visceralisation of L. major and more importantly L. donovani infections. Analysis of vaccine specific antibody responses suggests that protection resulted from induction of a TH1 response. First steps were undertaken towards resolving functional and structural properties of the most protective antigen LinJ08.1140. Putative MHC-I epitopes of antigen LinJ08.1140 were predicted using bioinformatics since antigen-specific CD4+ and CD8+ T cells are believed to be required. In addition, immunofluorescent staining of LinJ08.1140 in L. major promastigotes suggested a functional role for this antigen in parasite cell division, since especially dividing cells emmited a strong fluorescence signal.

The sensitivity, speed, and reproducibility of modern mass spectrometers enable in-depth new functional looks into the cellular proteome. Thousands of proteins can be detected in a single sample. In Co-Fractionation Mass Spectrometry (CF-MS) method, the input sample is fractionated by any biochemical method of choice. The reduced complexity of each fractionated sample leads to better proteome coverage. The separation profiles provide functional information on the proteins. This application has been used to predict organelle localization based on co-purification with marker proteins. More recently, CF-MS is being used to measure the apparent masses and determine the localization of soluble or membrane-associated protein complexes. This Ph.D. dissertation focuses on the extension of the boundary of CF-MS application to learn how protein complex evolution and protein complex composition have been accomplished. In the first part of this dissertation, the data will be presented on the degree to which variation in protein oligomerization across plant species is present, how proteomics in phylogenetic analysis (phyloproteomics/evolutionary proteomics) helps understand the evolutionary changes, and how oligomerization drives neofunctionalization during plant evolution. The latter part will describe that CF-MS coupled with multiple orthogonal chromatographic separations increases the resolving power of the profiling technique, enabling the composition of protein complexes to be predicted in the subaleurone layers of rice endosperm. Lots of novel protein complexes involved in RNA binding protein, translation, and the tissue-species metabolism will be discussed.

<p>Home cage aggression in male laboratory mice continues to challenge preclinical researchers. It reduces animal welfare and can alter research parameters, potentially reducing the validity and reliability of study data. While simply reducing aggression would be beneficial, promoting socio-positive, affiliative behaviors would greatly improve mouse welfare as mice are a social species. Mice also use olfaction to communicate, so this sensory modality could be used as a tool to improve social interactions in the home cage. A scoping review of the literature on how mammalian odor signals impact same sex social behavior found that studies are dominated by rodent subjects, treatments from urine, and aggression measures (Chapter 1). As a whole, urine treatments had a variable effect on aggression. This review highlights that treatments from non-urinary sources are not often tested, and affiliative behavior is rarely measured.</p> <p>One murine odor source worth exploring is found in used nesting material. Mice build complex nests for insulation and it has been speculated that the nest holds odor signals that appease home cage aggression, particularly aggression triggered by cage cleaning. It has been suggested that the nest contains secretions from plantar sweat glands, but the chemical content of neither nesting material nor plantar sweat have been examined. The main goals of this dissertation are to identify the odors stored in used nesting material, determine the sources of those odors, and test them for a behavioral role.</p> <p>Samples of used nesting material were collected from cages of group housed male mice. Further, plantar sweat, saliva, and urine were collected from the dominant and subordinate mouse in each cage as plausible odor sources. All samples were analyzed for protein and volatile organic compound content. Home cage aggression and affiliative behavior were also recorded to compare to odor profiles. Protein profiles showed that used nesting material contains a variety of proteins that primarily originate from plantar sweat, saliva, and urine sources (Chapter 2). A large proportion of these proteins contain messages about individual identity and bind volatile compounds that further contribute to identity cues. This suggests that the nest aids in maintaining a familiar odor environment. Analysis of volatile content showed that small compounds in the nest are also traced back to plantar sweat, saliva, and urine sources (Chapter 3). Few of the compounds have a known behavior role. However, one compound detected in nest, sweat, and saliva samples had a negative correlation with home cage aggression and three compounds (two from sweat and one from urine) had a positive correlation with affiliative behaviors, making them potential candidates for controlled studies on social behavior.</p> <p>Before testing the four candidate compounds, a challenge from the correlation study needed to be addressed. Body fluid samples were collected from individual mice based on social status, as this factor impacts production of known murine pheromones. Further, aggression is typically directed from a dominant to a subordinate mouse for territorial reasons. An aggression appeasement signal is likely to be produced by a subordinate to mitigate the dominant mouse’s perceived threat. Data from the correlation study showed no odor profile differences based on social status, and the pheromones that are known to vary with social status did not differ between dominant and subordinate mice. Therefore, Chapter 4 assesses the convergent validity of several dominance measures. Over one week, home cage interactions were observed in group housed male mice. For every aggression occurrence, the aggressor and target mouse was recorded to calculate individual dominance rankings in each cage. Then, individual mice were evaluated for the following measures known to correlate with dominance: levels of urinary darcin (a murine pheromone); scores from three rounds of the tube test; and ratio of preputial gland weight to body length. Postmortem wounding was also compared. Results showed that urinary darcin and preputial gland ratio have strong convergent validity with dominance ranking based on home cage aggression.</p> <p>Finally, the four candidate compounds (identified in Chapter 3) were developed into treatment solutions to assess their effect on home cage social behavior (Chapter 5). Cages of group housed male mice were randomly assigned one of five treatments (four compounds + control) and home cage aggression and affiliative behavior were recorded for one week. Postmortem wounding was recorded as a secondary aggression measure and social stress was measured through fecal corticosterone metabolites from each cage’s dominant and subordinate mouse (rank based on preputial gland ratio). Treatment did not predict changes in most measures. This may be due to limitations in application or from the original correlation study, which are further discussed.</p> <p>Although the final study showed null results, future research is still warranted to fine tune application methods and gain a better understanding of how odor signals impact interactions other than aggression. The relationship between olfaction and affiliative behaviors is largely unexamined and this dissertation is a first step in filling that gap.</p>

<p dir="ltr">Newborn animals undergo a lot of early-life stress that heavily impact on their long-term growth, performance, and welfare. Typically, the stress would indirectly interfere with the capacity of these neonates to utilize dietary nutrients and consequently impact tissue growth and development. In piglets, weaning is a stressful situation characterized by disruption of intestinal epithelial cell development which causes poor digestion of solid feed and a negative impact on absorption of nutrients especially in the post-gastric region. In addition, weaning in piglets could cause an increase in cellular assault by reactive oxygen species thereby potentially causing gut leakiness and paracellular loss of nutrients along the intestinal tract. In broiler chickens, access to feed may take up to 72 h following hatching which may affect their gut development as well as their gut microbiota. After the first feed ingestion, there is a sharp increase in the gut microbiota which triggers an increase in the development of the immune system as well as the gut. There is continuous attention on the strategies and nutritional interventions to mitigate or ameliorate the adverse effects of early life stressors in these food animals, especially in broiler chickens and piglets. In the studies described in this dissertation, myo-inositol (purely supplemented or phytase-induced) and luteolin were tested as nutritional strategies to mitigate the effects of early-life stressors on growth and the potential mechanisms by which myo-inositol and luteolin regulate growth were investigated.</p><p dir="ltr">In study I, the effect of myo-inositol on growth in 128 postweaning piglets fed protein-deficient corn-soy diets was tested. There were 4 dietary treatments in a randomized complete block design with body weight as the blocking factor. The treatments consisted of 1 positive control (PC) diet formulated to meet all the nutrient requirements of the piglets with a 20% crude protein (CP); the remaining 3 diets were the negative control (NC) diets with a 3% reduction in CP, a 2 g/kg myo-inositol supplemented negative control diet (NC+INO), and phytase (3,000 FTU/kg) supplemented negative control (NC+PHY) diet. The results showed that phytase enhanced the apparent total tract digestibility (ATTD) of P in the weanling pigs. Myo-inositol supplementation in a protein-deficient diet improved (P < 0.05) porcine plasma myo-inositol concentration while an in vitro myo-inositol incubation with intestinal epithelial cells increased the expression of genes that encode for Claudin-1, Claudin-3, Claudin-4, ZO-1, NaPiIIb, GLUT2, and SLC7A2. The in vitro analysis of tight junction integrity in the IPEC-J2 cells indicated by the transepithelial electrical resistance and FITC-Dextran permeability showed an enhancement in response to myo-inositol treatment. Although the in vivo study found that myo-inositol did not improve growth performance or ATTD, the in vitro myo-inositol enhanced markers of gut health and function.</p><p dir="ltr">In study II, the effect of myo-inositol on the growth of broiler chickens was tested. In this study, there were 6 experimental treatments based on two dietary protein levels (PC and NC) and three supplement types (BASAL, INO, and PHY) resulting in a 2 x 3 factorial arrangement in a completely randomized design. A total of 384 broiler chickens comprising 6 treatments with eight replicates per treatment and 8 birds per replicate were used. The birds were fed a common starter diet for the initial 7 days after they arrived at the poultry unit followed by a 14-day trial. The protein-deficient diet decreased the feed efficiency of the birds. Phytase addition increased (P < 0.05) the apparent ileal digestibility (AID) and ATTD of P and Ca in both PC and NC groups. The jejunal gut morphology was enhanced by supplemental phytase as indicated by an increase in villus height and the ratio of the villus height-to-crypt depth, coupled with an increase in serum myo-inositol concentration caused by both myo-inositol and phytase. In conclusion, myo-inositol showed a differential influence on growth performance, nutrient digestibility, and gut morphology.</p><p dir="ltr">In study III, the effects of luteolin on weanling pigs and IPEC-J2 cells were examined. A total of 48 piglets were randomly allotted to two dietary treatments consisting of a control group and a luteolin (LUT)-supplemented dietary group for a 4-week trial. A weekly assessment of the growth performance and expression of specific proteins in the jejunal mucosa was performed. In each dietary group, 8 piglets were slaughtered at weeks 1, 2, and 4 postweaning to collect blood, jejunal and ileal mucosa, and tissues. Luteolin supplementation numerically improved the ADG and G:F of the pigs. Luteolin feeding altered the jejunal and ileal gut morphology with increased villi height (P < 0.05) and villus height-to-crypt depth ratio (VCR, P < 0.05) in the jejunum and decreased crypt depth in the ileum. The effect of luteolin on IPEC-J2 global proteome and phosphor-proteome showed that luteolin could potentially improve intestinal barrier integrity by enhancing the abundance of proteins important in cell growth and survival. </p><p dir="ltr">In summary, dietary supplementation with myo-inositol and luteolin could regulate growth and nutrient digestibility in broiler chickens and weanling pigs by enhancing the integrity of intestinal cells and facilitating the expression of nutrient transporters that are significant in the uptake of nutrients across the lining of the gastrointestinal tract. Phytase supplementation improves the P release from phytate in the diets thereby alleviating its loss.</p>

Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer. The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided. Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD. In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies. / <p>QC 20170302</p>

The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

O crescimento, desenvolvimento e manutenção do tecido ósseo são processos altamente regulados. Diversas proteínas como hormônios, fatores de crescimento e citocinas estão envolvidas nestes processos e exercem atividade direta sobre células osteoblástica e osteoclástica, atuando em sua diferenciação e ativação metabólica. O processo de regeneração óssea é iniciado por fatores estimuladores locais como as proteínas morfogenética óssea (BMP Bone Morphogenetic Proteins). As BMPs são um produto do metabolismo dos osteoblastos, odontoblastos e de várias células tumorais, sendo armazenadas na forma de concentrados no osso, dentina e em células neoplásicas do osteossarcoma e de certos tumores odontogênicos, tais como: fibroma cementificante, cementoblastoma benigno, dentinoma, fibroma odontogênico e odontoma. Esclarecer os mecanismos que controlam a remodelação óssea é uma questão bastante relevante. Nesse sentido, as células-tronco mesenquimais têm despertado grande interesse devido ao seu potencial envolvimento no processo de reparo tissular. A obtenção de osteoblastos funcionais a partir de células-tronco mesenquimais tem sido utilizada na engenharia de tecidos e terapia celular. Desse modo, no presente trabalho foi realizada uma análise proteômica das proteínas envolvidas nas diversas fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea de rato Wistar e humana, no sentido de obter maiores informações sobre a diferenciação celular e a biologia do tecido ósseo. Células-tronco mesenquimais obtidas de medula óssea foram cultivadas em meio osteogênico por diferentes períodos para obter células em diversas fases da diferenciação osteoblástica. Para análise proteômica foram utilizadas ferramentas como a estratégia de shotgun proteomics e quantificação relativa (iTRAQ - Isobaric Tag for Relative and Absolute Quantitation) para separação de proteínas e a espectrometria de massas para a identificação e quantificação relativa de proteínas e peptídeos. Neste contexto, os nossos resultados nos levam a concluir que: as CTMs de medula óssea de rato Wistar expressam genes que estão envolvidos na diferenciação osteogênica quando estimuladas in vitro formando matriz óssea no período de 14 dias, ou seja, o fator estimulante no microambiente é de fundamental importância; as CTMs de medula óssea humana apresentaram resultados semelhantes com as CTMs de ratos em nível genômico durante a diferenciação osteogênica, entretanto quando estimuladas in vitro formaram a matriz óssea no período de 21 dias; utilizando duas abordagens proteômicas, foi possível identificar proteínas importantes que estão envolvidas no processo de diferenciação. Mas cabe salientar que, embora tenham sido detectados genes que parecem envolvidos no processo de diferenciação, isso não teve reflexo no proteoma dessas células nos períodos de 7 e 14 dias da indução de diferenciação à osteogênese, o que indica que a maior parte da funcionalidade dessas células quanto aos outros processos biológicos estão preservados, como por exemplo a proliferação celular permaneceu sem grandes alterações. Isso indica que manipulações de isolamento, cultivo e indução da diferenciação dessas células não afetaram o proteoma, com aspectos positivos para a utilização de células-tronco mesenquimais em terapia celular. Do ponto de vista metodológico, esse trabalho abre perspectivas da utilização de estratégias proteômicas baseadas na marcação por isóbaros em combinação com separação de proteínas por eletroforese unidimensional SDS-PAGE para a análise de amostras biologicamente complexas e de quantidades limitadas de obtenção como células-tronco mesenquimais. O estudo da expressão de proteínas durante as fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea deve refletir seu estado funcional e contribuir para o entendimento das diversas vias envolvidas no processo de diferenciação. / The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy. Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins. In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of mesenchymal stem cells in cell therapy. From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as mesenchymal stem cells. The study of protein expression during stages of osteoblast differentiation of mesenchymal stem cells from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

Cellular defense and metabolism are important biological processes in living cells. In this study, these two biological processes were investigated in two selected disease models: diabetes mellitus (DM) and severe acute respiratory syndrome associated coronavirus (SARS-CoV) infection by two-dimensional gel electrophoresis (2DE) coupled with Matrix-Assisted Laser Desorption Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)-based proteomic approaches. The major findings are summarized as follows: / Our results on DM investigation can help to better understand the pathophysiological changes in patients with DM and the pathogenesis of hyperglycemia-caused complications. Data obtained from SARS-CoV studies provided novel insights into the molecular basis of the host cell response upon viral infection. / Protein profile of streptozotocin (STZ)-induced diabetic animal tissues, including mice liver, kidney and eye, and rats sera, indicated that DM has an impaired cellular defense system. These include the impairment in reactive oxygen species scavenging and the impairment in activation of complement system and innate immunity, and the enhancement in blood coagulation reaction. Our results also demonstrated that glycolysis and gluconeogenesis did not alter significantly in the liver of STZ-diabetic mice, while fatty acid oxidation and TCA cycle were attenuated under the same conditions. Moreover, we also detected other abnormal metabolism in aldehyde and amino acid, especially glutamate metabolism and the urea cycle. Abnormalities were also detected in lipid transport and metabolism. Besides, protein profile of mouse liver c37 cells indicated that high glucose may induce apoptosis in these cells, and this apoptotic effect may be mediated via the mitochondrial pathway. Furthermore, the proteomic results from the in vivo and in vitro diabetic models have prompted us to look for glucose responsive element on the promoters of these up-regulated hepatic genes. We found that the mouse aldolase 2 gene has glucose responsiveness in c37 cells treated with high glucose by semi-quantitative RT-PCR and promoter transfection assay. Finally, protein profile of Vero E6 cells strongly implicated that SARS-CoV can induce anti-apoptosis. This effect may be mediated via the mitochondrial pathway. Our data also suggested that the anti-apoptotic activity may be required for viral replication at the early stage of infection. While under the condition of long-term infection, this may be needed for viral survival. / Zhong Mingqi. / "October 2005." / Advisers: Sai Ming Ngai; Hon Ki Cheng. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6217. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 223-248). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. 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