Made available in DSpace on 2016-03-07T19:20:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-11-05. Added 1 bitstream(s) on 2016-03-07T19:24:50Z : No. of bitstreams: 1 000859360.pdf: 1034814 bytes, checksum: c0a4def8be0c2f45d09cbd7c089d88da (MD5) Bitstreams deleted on 2016-03-28T13:37:51Z: 000859360.pdf,. Added 1 bitstream(s) on 2016-03-28T13:38:52Z : No. of bitstreams: 1 000859360.pdf: 1034815 bytes, checksum: 0e2a44e2cca5e564a6825b47d8322e41 (MD5) / O fipronil é um inseticida amplamente utilizado para controlar pragas nas plantações e em animais. Existem relatos de intoxicação devido à exposição de fipronil em mamíferos e o fígado é um alvo potencial. No presente estudo, foram avaliados os efeitos dos metabólitos do fipronil, fipronil dessulfinil e fipronil sulfona sobre a bioenergética, produção de espécies reativas de oxigênio e efluxo de cálcio em mitocôndrias isoladas de fígado de rato. O fipronil dessulfinil (5 a 25 μM) inibiu o estado 3 da respiração em mitocôndrias energizadas com glutamato mais malato, substratos do complexo I da cadeia respiratória, e diminuiu o potencial de membrana mitocondrial, resultando em inibição da síntese de adenosina trifosfato. O metabólito dessulfinil também causou desacoplamento nas mitocôndrias energizadas com succinato e estimulou o efluxo de cálcio. O metabólito sulfona também atuou como inibidor e desacoplador mitocondrial e estimulou o efluxo de cálcio, porém em menores concentrações (0,5 a 5 μM). Os efeitos dos metabólitos do fipronil na bioenergética mitocondrial e homeostase do cálcio podem estar relacionados com os efeitos tóxicos do inseticida no fígado / Fipronil is an insecticide extensively used to control pests in crops and animals. There are relates of poisoning due to exposure of fipronil in mammals and the liver is a potential target. In this study, we evaluated the effects of metabolites of fipronil, fipronildesulfinyl and fipronilsulfone on the bioenergetics, reactive oxygen species production and calcium efflux from mitochondria isolated from rat liver. Desulfinyl (5 to 25 μM) inhibited state-3 respiration in mitochondria energized with glutamate plus malate, substrates of complex I of the respiratory chain, and decreased the mitochondrial membrane potential resulting in inhibition of. Synthesis. Desulfinyl also caused uncoupling in succinate-energized mitochondria and calcium efflux. The metabolitesulfone also acted as mitochondrial inhibitors and uncouplers and caused calcium efflux, but inlower concentrations (0.5 to 5 μM). These effects of metabolites of fipronil on mitochondrial bioenergetics and calcium homeostasis may be related to toxic effects of the insecticide in the liver

Orientador: Fábio Erminio Mingatto / Banca: Daniel Nicodemo / Banca: Sérgio Diniz Garcia / Resumo: O fipronil é um inseticida amplamente utilizado para controlar pragas nas plantações e em animais. Existem relatos de intoxicação devido à exposição de fipronil em mamíferos e o fígado é um alvo potencial. No presente estudo, foram avaliados os efeitos dos metabólitos do fipronil, fipronil dessulfinil e fipronil sulfona sobre a bioenergética, produção de espécies reativas de oxigênio e efluxo de cálcio em mitocôndrias isoladas de fígado de rato. O fipronil dessulfinil (5 a 25 μM) inibiu o estado 3 da respiração em mitocôndrias energizadas com glutamato mais malato, substratos do complexo I da cadeia respiratória, e diminuiu o potencial de membrana mitocondrial, resultando em inibição da síntese de adenosina trifosfato. O metabólito dessulfinil também causou desacoplamento nas mitocôndrias energizadas com succinato e estimulou o efluxo de cálcio. O metabólito sulfona também atuou como inibidor e desacoplador mitocondrial e estimulou o efluxo de cálcio, porém em menores concentrações (0,5 a 5 μM). Os efeitos dos metabólitos do fipronil na bioenergética mitocondrial e homeostase do cálcio podem estar relacionados com os efeitos tóxicos do inseticida no fígado / Abstract: Fipronil is an insecticide extensively used to control pests in crops and animals. There are relates of poisoning due to exposure of fipronil in mammals and the liver is a potential target. In this study, we evaluated the effects of metabolites of fipronil, fipronildesulfinyl and fipronilsulfone on the bioenergetics, reactive oxygen species production and calcium efflux from mitochondria isolated from rat liver. Desulfinyl (5 to 25 μM) inhibited state-3 respiration in mitochondria energized with glutamate plus malate, substrates of complex I of the respiratory chain, and decreased the mitochondrial membrane potential resulting in inhibition of. Synthesis. Desulfinyl also caused uncoupling in succinate-energized mitochondria and calcium efflux. The metabolitesulfone also acted as mitochondrial inhibitors and uncouplers and caused calcium efflux, but inlower concentrations (0.5 to 5 μM). These effects of metabolites of fipronil on mitochondrial bioenergetics and calcium homeostasis may be related to toxic effects of the insecticide in the liver / Mestre

Mitosis is a highly regulated process by which a cell duplicates and distributes its chromosomal DNA into two identical daughter cells equally. Equal distribution of the chromosomes is crucial for accurate propagation of genetic information. This is essential for maintaining viability and preventing genomic instability that can potentially lead to cancer. In order to avoid unequal distribution of chromosomes, cells employ a surveillance mechanism called the spindle assembly checkpoint (SAC). The SAC is an inhibitory signalling network, which delays segregation of chromosomes, until they have stably attached to spindle microtubules through their multi-protein platforms, known as kinetochores. The main target of the SAC is the anaphase promoter complex/ cyclosome (APC/C), an E3 ubiquitin ligase. Specifically APC/C and its activator Cdc20 are inhibited by the main effector of the SAC, called the mitotic checkpoint complex (MCC). The MCC consists of Cdc20, Mad2, Mad3 and Bub3 (except S. pombe) proteins, which are recruited to the unattached kinetochores to promote MCC assembly. Once the chromosomes stably attach to the spindle, the SAC is turned off, MCC disassembles, and APC/CCdc20 is released from the inhibition. Activated APC/CCdc20 then targets its two main substrates, securin and cyclin B, for proteasomal degradation, and thereby triggers anaphase onset and mitotic exit. SAC signalling involves many protein components, whose activities are essentially regulated by direct protein-protein interactions and/ or post-translational modifications. One of these major modifications is phosphorylation, which is mediated by the SAC kinases such as Aurora B, Mps1 and Bub1. A number of studies have characterised SAC related substrates of Aurora B and Mps1 kinases in several model organisms. On the other hand, Bub1 kinase activity has been thought to play a key role in chromosome bi-orientation and more of an auxiliary role in SAC activation. The aim of this study is to investigate the importance of Bub1 kinase activity for SAC response in fission yeast Schizosaccharomyces pombe (S. pombe). SAC activation assays, using various degrees of spindle perturbation, have demonstrated that Bub1 kinase activity plays an important role in SAC maintenance. In order to examine the pathways downstream of Bub1, we set out to indicate Bub1 substrates which may be involved in SAC signalling. According to studies in various species, Cdc20 appears to be a prominent candidate, whose phosphorylation by Cdk1 and Bub1 kinases has been reported to regulate its mitotic activity. To investigate whether Cdc20 is phosphorylated by Bub1 in vitro, we purified recombinant S. pombe proteins from insect cells. Subsequent kinase assays identified Cdc20 as an in vitro substrate of Bub1, and the phosphorylated sites in Cdc20 were mapped by mass spectrometry. To address if this phospho-modification is involved in SAC regulation, phosphorylation mutants of Cdc20 were analysed in terms of their abilities to activate and silence SAC in vivo. Results show that phosphorylation of Cdc20 C-terminus promotes SAC maintenance in response to spindle damage. Furthermore, the mutations mimicking Bub1-mediated phosphorylation of Cdc20 Cterminus restore the SAC defects in the absence of Bub1 kinase activity. In addition, we purified S. pombe mitotic checkpoint complex (MCC) from insect cells, and analysed the interactions between its components (Cdc20, Mad2 and Mad3) by cross-linking mass spectrometry. Crystal structure of S.pombe MCC has been determined recently, which lacks Mad3 C-terminus and flexible C-terminal tail of Cdc20. Using an MCC with full length Mad3, we identified novel interactions between the C-terminal tails Mad3 and Cdc20, which are in close proximity to the identified Cdc20 phosphorylation sites. Briefly, in this study we confirm the previously known roles of Bub1 kinase activity (chromosome bi-orientation). Moreover, we propose a new pathway (in addition to the well-established H2A pathway) mediated by Cdc20, that may be important to maintain the SAC response.

M.Tech. (Chemical Technology) / Please refer to full text to view abstract

The co-chaperone murine stress-inducible protein 1 (mSTI1), a heat shock protein 70 (Hsp70)/ heat shock protein 90 (Hsp90) organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. mSTI1 is phosphorylated in vitro by cell cycle kinases, proximal to a putative nuclear localization signal (NLS), substantiating a predicted CKII-cdc2-NLS (CcN) motif at position 189-239. Stable transfectants of NIH 3T3 fibroblasts that expressed mSTI1-EGFP, NLSmSTI1-EGFP and EGFP, were prepared. Fluorescence microscopy revealed mSTI1 was cytoplasmically localized, and that this localization was not affected by the fusion of mSTI1 with the EGFP moiety. NLSmSTI1-EGFP was targeted to the nucleus compared to EGFP, suggesting that the NLSmSTI1 was a functional NLS. The localization of mSTI1 was determined under normal and heat shock conditions, inhibition of nuclear export (leptomycin B), inhibition of CKII 5,6-dichlorobenzimidazole riboside, DRB), inhibition of cdc2 kinase (olomoucine), and G1/S phase arrest (hydroxyurea). mSTI1-EGFP and mSTI1 were excluded from the nucleus in the majority of resting cells, but accumulated in the nucleus following leptomycin B treatment, implying that mSTI1 possibly undergoes a functional import process, and export via the chromosomal region maintenance 1 (CRM-1)-mediated export pathway. Hydroxyurea and olomoucine (but not DRB or heat shock) treatment increased the proportion of cells in which mSTI1-EGFP exhibited cytoplasmic and nuclear localization. 2D gel electrophoresis detected three endogenous mSTI1 isoforms, which changed following hydroxyurea treatment. Furthermore, point inactivation and mimicking of phosphorylatable residues in mSTI1 altered the translocation of the protein and the isoform composition. Modification of mSTI1 at S189 and T198 decreased the number of isoforms of mSTI1-EGFP, suggesting that the protein is modified at these sites in vivo. The removal of the in vitro cdc2 kinase site at T198 promoted a nuclear localization during G1/S phase arrest. Therefore active cdc2 kinase, but not CKII, may be required for cytoplasmic localization of mSTI1. The CKII site appears to have no regulatory role under heat shock conditions or during the cell cycle. In vitro phosphorylation studies on untagged mSTI1 further supported the prediction that S189 is the only site recognised by CKII. The cdc2 kinase site at T198, however, although the major site, was not the only site phosphorylated in vitro. However, mSTI1 and cdc2 kinase did not interact in a detectable stable complex. Bioinformatic analysis of mSTI1 revealed NLS residues were conserved in STI1 proteins, and the NLS and TPR2A motifs were in close proximity. This may have mechanistic implications for the formation of the Hsp90-mSTI1 heterocomplex. The cytoplasmic or nuclear localization of mSTI1 is predicted to be the result of a dynamic equilibrium between nuclear import and nuclear export, the fulcrum of which may be shifted under different cell cycle conditions. These data provide the first evidence of regulated nuclear import/export of a major Hsp70/Hsp90 co-chaperone, and the regulation of this nuclear import by cell cycle status and cell cycle kinases.

Presently, progress in the field of phosphoproteomics has been accelerated by mass spectrometry. This is not a surprise owing to not only the accuracy, precision and high-throughput capabilities of MS but also due to the support it receives from informaticians whom allow the automated analysis; making the task of going from a complex sample to a statistically satisfactory set of phosphopeptides and corresponding site positions with relative ease. However, the process of identifying and subsequently pinpointing the phosphorylation moiety is not straightforward and remains a challenging task. Furthermore, it has been suggested that not all phosphorylation sites are of equal functional importance, to the extent that some may even lack function altogether. Clearly, such sites will confound the efforts towards functional characterisation. The work in this thesis is aimed at these two issues; accurate site localisation and functional annotation. To address the first issue, I adopt a multi-tool approach for identification and site localisation; utilising the different underlying algorithms of each tool and thereby allowing an orthogonal perspective on the same tandem mass spectra. Doing so enhanced accuracy over any single tool by itself. The power of this multi-tool approach stemmed from its ability to not predict more true positives but rather by removal of false positives. For the second issue, I first investigated the hypothesis that those of functional consequence exhibit stronger phosphorylation-characteristic features such as the degree of conservation and disorder. Indeed, it was found that some features were enriched for the functional group. More surprisingly, there were also some that were enriched for the less-functional; suggesting their incorporation into a prediction algorithm would hinder functional prediction. With this in mind, I train and optimise several machine-learning algorithms, using different combinations of features in an attempt to (separately) improve general phosphorylation and functional prediction.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Background - The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. Methods and Results - Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. Conclusions - Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Tyrosine phosphorylation is an important post-translational modification in cells that modulates key cellular pathways. Tyrosine phosphatases are the class of enzymes that remove this modification from proteins, yet we know relatively little about how they are regulated in various signaling contexts. Activity-based probes that successfully target active sites of tyrosine phosphatases and report on their activities can fill in this gap. We show the assessment of various thiol-reactive groups for their ability to target catalytic cysteine residues with specificity. Then we construct and screen a library of fragment-like compounds for their on-target and off-target reactivities. We also discuss theoretical considerations for screening covalent inhibitors for their kinetic parameters and show this using our experimental data. Lastly, we augment compounds selected from the library to enable click chemistry for reporter group attachment for use on the whole proteome, ultimately through mass spectrometry-based proteomics methods. We show enrichment of target proteins. These target-centric design efforts will yield new insights into the general development processes of activity-based probes or inhibitors.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 β chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function. Looking upstream the kinase cascade, the identity of the kinase (s) that brings about the phosphorylation of the tyrosine residues remains to be elucidated. It has been suggested that the non receptor tyrosine kinase Src family is involved in the capacitation associated phosphorylation cascade. Using an immunological approach we show that the only Src family member present in mouse sperm extract is Src. The capacitation associated tyrosine phosphorylation is greatly reduced in the presence of Src specific inhibitors (SU6656 and SKI606) in vivo. As a means of control for the activity of Src inhibitors in our system, parallel experiments assaying the activity of PKA both in vivo and in vitro were realized. Surprisingly, Src inhibitors down regulates the phosphorylation of serine/threonine residues that correlate on earlier events in the capacitation, as assayed by western blot with PKA substrates antibody. However, in vitro kinase activity of PKA showed no effect of Src inhibitors in the phosphorylation of the PKA specific substrate, kemptide.