A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this study are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.

A large number of evolutionarily conserved genes have been identified by comparative genomics approaches. However, a considerable fraction of these genes lack functional characterization despite the availability of several bioinformatics approaches for prediction of protein function. Moreover, with the advent of genome sequencing efforts, numerous disease associated genes have been identified. While high throughput approaches aid in identification of genes, studying individual genes is important to understand their cellular roles. During studies on cyclic AMP metabolism in mycobacteria conducted in the laboratory, a Class III cyclic nucleotide phosphodiesterase, Rv0805 was identified from Mycobacterium tuberculosis. Interestingly, additional bioinformatics analysis identified orthologs were in higher eukaryotes. These were members of the metallophosphoesterase-domain-containing protein 1 (MPPED1) and metallophosphoesterase-domain-containing protein 2 (MPPED2) family. Class III cyclic nucleotide phosphodiesterases were previously reported only in prokaryotes and are distinct from Class I cyclic nucleotide phosphodiesterases generally found in eukaryotes. Thus MPPED1 and MPPED2 proteins were the first identified eukaryotic Class III cyclic nucleotide phosphodiesterases. In humans, MPPED2 is located on chromosome 11 in the region p13-14 that has been associated with WAGR (Wilms’ tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome. Inspection of this region across sequenced mammalian genomes has revealed a shared synteny. Most interestingly, a stretch of 200 bp within the coding sequence of MPPED2 is identified to be one of 481 ultra conserved regions within the human genome. Furthermore, orthologs of MPPED2 can be traced all the way back to Drosophila melanogaster and Caenorhabditis elegans. All of these observations indicate that MPPED2 is highly conserved and hints at its likely importance in many organisms. MPPED1 and MPPED2 have been reported to be expressed in adult and fetal brain respectively and have been annotated as metallophosphoesterases. Metallophosphoesterases are a superfamily of proteins that show wide phyletic distribution and exhibit diversity in their substrate utilization and function. Previous studies from the laboratory have shown that MPPED1 and MPPED2 are indeed metallophosphoesterases and demonstrate cyclic nucleotide phosphodiesterase activity. The crystal structure of MPPED2 was obtained in collaboration with Dr. Marjetka Podobnik (National Institute of Chemistry, Slovenia). Interestingly, the crystal structure of MPPED2 revealed the presence of bound 5’GMP molecule at the active site, and this finding was investigated further in this thesis. MPPED2 bound 5’GMP and 5’AMP with high affinity (IC50 of ~70 nM) which inhibited the activity of MPPED2. Key residues involved in stabilising the 5’ nucleotide have been identified by structure guided mutational analysis. The MPPED2-G252H mutant, generated to mimic the active site of MPPED1, also bound 5’GMP or 5’AMP but with much lower affinity. Given the high affinity of MPPED2 towards 5’GMP/5’AMP, it can be speculated that MPPED2 may show poor phosphodiesterase activity in the cell, and could function in a catalytically-independent manner, perhaps as a scaffolding protein. MPPED1 on the other hand may have a catalytic role that could be regulated by intracellular levels of 5’AMP, 5’GMP and their respective cyclic nucleotides. In order to investigate the biological role of the MPPED1/MPPED2 family of proteins, Drosophila melanogaster was chosen as a model organism owing to the presence of a single ortholog, CG16717, in its genome. Biochemical characterization of CG16717 revealed that the protein was in fact a metallophosphodiesterase capable of hydrolysing cyclic AMP and cyclic GMP, albeit poorly. CG16717 could be inhibited by 5’ nucleotides at high concentrations that may seldom be achieved in-vivo, suggesting that CG16717 may have roles in the organism that depend on its catalytic activity. CG16717 has not been functionally characterized previously. In this thesis, a detailed analysis of CG16717 expression pattern has been performed. CG16717 was found to be expressed in all stages of the fly lifecycle. In adult female flies, levels of CG16717 increased across age. Moreover, CG16717 was not differentially regulated under conditions of starvation, paraquat-induced oxidative stress or in the presence of heavy metals. Spatial expression analysis revealed that CG16717 was expressed in all adult tissues tested, with maximal expression in the brain, suggesting that neuronal expression of CG16717 may be important for its function. Attempts to identify specific cells expressing CG16717 using an enhancer-promoter analysis were not successful. In order to elucidate the physiological role of CG16717, and after having ruled out options of using a P-element insertion mutant and RNA interference approaches, a targeted knock-out of CG16717 was generated using homologous recombination based genomic engineering. CG16717KO flies generated were homozygous viable suggesting that CG16717 was dispensable for fly survival at least under normal laboratory conditions. In line with high expression of CG16717 in the brain and in-vitro ability of CG16717 to hydrolyse cAMP and cGMP, CG16717KO flies showed two to three-fold higher levels of cyclic nucleotides in the head fraction than wild-type flies. C25E10.12, one of the three C. elegans orthologs of CG16717 has been identified to be a target of the transcription factor daf-16 (FOXO) that is inhibited by active insulin signalling. Moreover, knock-down of C25E10.12 reduced the lifespan of age-1 (PI3K) mutant worms. In contrast to this, CG16717 was not found to be differentially regulated in dFOXO null flies. CG16717KO flies however, showed median lifespan that was shorter than control wild-type flies even in the presence of functional PI3K. Various genetic approaches were employed to verify if reduced lifespan was indeed a consequence of loss of CG16717. In the first approach, a wild-type copy of CG16717 was re-introduced at the genomic locus of CG16717 in the CG16717KO flies using attP-attB recombination. However, this approach could not rescue the reduced lifespan of CG16717KO flies, probably due to very low expression of CG16717. In the second approach, CG16717 was reconstituted using genomic constructs containing a copy of CG16717. Finally, CG16717 was expressed ubiquitously using the bipartite Gal4/UAS system. Both the genomic construct and the expression of CG16717 using the Gal4/UAS approach were able to restore the lifespan of CG16717KO flies. More importantly, overexpression of CG16717 in an otherwise wild-type fly led to enhanced lifespan over and above that of control flies. All of these together suggested that CG16717 plays a critical role in regulating lifespan. Mutants of the insulin and target of rapamycin (TOR) signalling pathways have previously been reported to show lifespan extension. Moreover, these mutants have also been associated with reduced growth, increased stress resistance and reduced fecundity. Given the reduction in lifespan of CG16717KO flies, the other insulin/TOR signalling associated phenotypes were tested. While CG16717KO flies showed no difference in terms of developmental growth, and resistance to starvation or paraquat induced oxidative stress, CG16717KO flies were less fecund compared to wild-type controls. Multiple approaches were adopted even in the case of reduced fecundity to verify if the observed phenotype was a consequence of loss of CG16717. However, neither reconstitution of CG16717 using the genomic construct nor ubiquitous expression of CG16717 using the bipartite Gal4/UAS system were able to rescue the reduced fecundity phenotype of CG16717KO flies. This suggested that reduced fecundity in CG16717KO flies was probably not linked to CG16717 and was a consequence of a second mutation at a site distinct from CG16717. Two other approaches were employed to confirm these observations. When CG16717KO/Deficiency lines were tested, these showed fecundity comparable to wild-type control flies despite the lack of CG16717. CG16717KO flies were extensively out-crossed in an attempt to segregate the second site mutation from the CG16717 locus and their fecundity was tested. However, these flies which retained the deletion of CG16717, showed fecundity comparable to wild-type control flies, reiterating that reduced fecundity was not linked to loss of CG16717. In an attempt to find possible links between reduced longevity of CG16717KO flies and the well-established insulin/TOR pathways, transcript levels of key players of these pathways were measured by qRT-PCR. The translational repressor 4EBP was found to be upregulated in CG16717KO flies compared to wild-type control flies. Interestingly, increased 4EBP levels have been associated with enhanced lifespan but in this case despite higher levels of 4EBP, CG16717KO flies showed reduced lifespan. Phosphorylation status of 4EBP and other players involved in the insulin/TOR phosphokinase signalling cascade would shed light on the activity of these pathways. In summary, this thesis has attempted to understand the biochemistry and physiological functions of an evolutionarily conserved metallophosphoesterase. Its apparent role in regulating life span in the fly suggests that the functions of this protein are likely to impinge on a number of diverse and important pathways involved in basic physiological processes in the organism. Further investigation would shed light on the molecular basis by which CG16717 affects lifespan, and opens up new avenues to understanding the contributions of CG16717 in regulating lifespan and diverse neurological functions.

Les structures d'acto-myosine sont impliquées dans de nombreuses fonctions cellulaires. Comprendre leur organisation et leur comportement collectif est toujours difficile. Nous avons étudié l'anneau cytokinétique dans les cellules de mammifères et dans les levures de fission, en orientant les cellules dans les microcavités, ce qui permet de voir l'anneau dans un seul plan focal. Avec cette configuration, nous révélons de nouvelles structures et des dynamiques distinctes pour les deux systèmes cellulaires. Dans les cellules de mammifères, nous trouvons des motifs réguliers de la myosine et la formine. Les caractéristiques de ces motifs sont stables tout au long de sa fermeture et leur apparition coïncide avec la constriction. Nous proposons que ce phénomène est une propriété inhérente du réseau d'acto-myosine et que la formation de ces motifs entraîne une augmentation du stress. Ces hypothèses sont confirmées par notre modèle en champ moyen. Par contraste, l'anneau de levure de fission montre des inhomogénéités tournantes de l'actine, de la myosine, des protéines de la construction de la paroi (Bgs) et d'autres protéines. La dynamique des inhomogénéités de myosine est inchangée, si la croissance de la paroi est inhibée. Cependant, l'inhibition du mouvement des inhomogénéités conduit à l'arrêt de la fermeture. Nous proposons que la fermeture de l'anneau est entraînée par la rotation de l'actine et de la myosine qui tirent des protéines Bgs, lesquelles construisent ainsi le septum. Cette hypothèse est confirmée par nos calculs et par des simulations numériques. Nous suggérons que la transition entre les états de différents ordres et dynamiques pourrait être une façon de réguler in vivo les systèmes d'acto-myosine. / Actomyosin structures are involved in many cell functions. Understanding their organization and collective behavior is still challenging. We study the cytokinetic ring in mammalian cells and in fission yeasts, by orienting cells in microcavities. This allows seeing the ring in a single plane of focus. With this setup, we reveal new structures and distinct dynamics for both cellular systems. In mammalian cells we find a pattern of regular clusters of myosin and formin. The characteristics of this pattern are stable throughout closure and its formation coincides with the onset of constriction. We propose that its characteristic is an inherent property of the actomyosin network and that its formation leads to an increase in stress generation. These hypotheses are supported by our theoretical mean field model. In contrast, fission yeast rings show rotating inhomogeneities (speckles), i.e. rotations of actin, myosin, cell wall building proteins (Bgs) and other proteins. Myosin speckles dynamic is unchanged, if wall growth is inhibited. However, the inhibition of speckle motion leads to stalled closure. We propose that the ring closure is driven by the rotation of actin and myosin, which pull Bgs thereby building the septum. This model is supported by our calculations and by numerical simulations. We suggest that the transition between states of different orders and dynamics might be a way to regulate actomyosin systems in vivo.

In mammals, freshly ejaculated spermatozoa do not possess the ability to fertilize a mature oocyte. They acquire fertilization competence upon residing for a period of time in the female reproductive tract. The physiological changes that bring about these time-dependent changes in motility pattern and acquisition of fertilizing ability of spermatozoa are collectively referred to as capacitation, culminating in sperm hyperactivation. Capacitation-associated increase in sperm protein tyrosine phosphorylation (PYP), exhibited by mammalian sperm, is one of the major downstream events, regulating hyperactivated motility. However, it is still unclear which are the tyrosine kinases and phosphatases involved in modulating the capacitation-associated increase in global PYP. In order to determine this, our laboratory earlier showed the role of PYP in hamster sperm capacitation using a specific EGFR protein tyrosine kinase (PTK) inhibitor, tyrphostin A47 (TP-47). Interestingly, inhibition of capacitation by 0.5 mM TP-47 was associated with induction of a slow circular motility pattern, accompanied by inhibition of PYP of certain proteins (Mr. 45,000-52,000), localized to the principle piece of the sperm flagellum. Two such proteins, hypo-tyrosine phosphorylated, were found to be tektin-2 and ODF-2, using 2D-PAGE followed by MS/MS analysis. Interestingly, a global phosphoproteome analysis of human spermatozoa showed that PYP changes are associated with capacitation and asthenospermic condition in infertile men is attributed to the failure of capacitation-associated increase in PYP. Such individuals exhibited impaired sperm motility. There is a need to understand the exact mechanism of phosphorylation of sperm flagellar proteins, which is necessary to assess sperm’s ability to fertilize the mature oocyte. Therefore, the focus of the present work was to elucidate the role of receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) in mammalian (hamster) sperm capacitation. Recent studies have shown that apart from EGFR other RTKs like IGF1R, FGFR, VEGFR, MuSK, TrkA are expressed in mammalian spermatozoa and actively involved in sperm capacitation. However, there is very little information available in the context of sperm capacitation and associated PYP. Therefore, attempts were made to understand the role of various RTKs (IGF1R, FGFR and VEGFR) in hamster sperm capacitation and associated PYP. Initially, the role of IGF1R tyrosine kinase during sperm capacitation was studied. Immunolocalization of IGF1R in spermatozoa showed a strong signal in the sperm acrosome and the principal piece of the sperm flagellum. Inhibition of IGF1R kinase with an IGF1R-specific inhibitor TP-1-O-Me-AG538 (TP-538) showed inhibitory effect on sperm capacitation and the associated hyperactivation. But, inhibitors of FGFR and VEGFR tyrosine kinases did not show such an effect. Interestingly, inhibition of IGF1R by TP538 was associated with inhibition of PYP of certain proteins (Mr. 45,000-120,000), localized to head, mid piece and principle piece regions of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 17 differentially phosphorylated protein spots. Out of the 17 spots, 12 were identified by MALDI-MS/MS analysis. The proteins identified to be differentially phosphorylated, upon inhibition of IGF1R, were PDHE1, ODF-2, Tubulin β 2C chain, PDHE2 and ATP synthase β subunit. The RTKs being present in the membrane level may not be directly involved in the phopshorylation of downstream target proteins associated with the mitochondrial membrane, sperm axonemal structures and outer dense fibers. Therefore, the RTKs may interact directly or indirectly with the downstream NRTKs, which may be involved in the phosphorylation of target sperm proteins. Till date, six different families of NRTKs are shown to be expressed in mammalian spermatozoa. The major family of NRTKs involved in sperm function is the Src family of kinases. However, there is very little information available in the context of sperm capacitation and the associated PYP. Therefore, studies were carried out to understand the role of Src family of NRTKs in sperm capacitation and associated PYP. Presence of active Src signaling was observed by the immunolocalization of activated Src (pY416) in the acrosome, mid piece and the principal piece regions of the sperm flagellum. Inhibition of Src family of kinase with a specific Src family kinase inhibitor PP2, showed inhibition of sperm capacitation and the associated hyperactivation. Inhibition of Src family of kinases with PP2 was associated with decrease in PYP of several proteins (Mr. 45,000-120,000), localized mainly to the mid piece region, followed by the principle piece region of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 38 differentially phosphorylated protein spots. Out of the 38 spots, 16 were identified by MALDIMS/MS analysis and these corresponded to seven proteins which included PDHE1, ODF-2, Tubulin β 2C chain, Tektin-2, GAPDS, PDHE2 and ATP synthase β subunit. Additionally, the biochemical and molecular characteristics of the identified proteins were also studied. Bioinformatic analysis predicted the presence of phosphorylation motifs for several kinases and interestingly, all the proteins identified had a Src kinase motif. Comparing the current observations and the previous work in the laboratory, two proteins ODF-2 and Tektin-2 were found to be regulated by EGFR, IGF1R and Src family of kinases. Therefore, characterization of the capacitation-associated tyrosine phoposphorylated proteins ODF-2 and Tektin-2 was performed. By employing PCR and Northern blotting techniques, the presence of the transcripts of both the proteins was shown. Additionally, the ontogeny of expression of ODF2 and Tektin-2 in hamster testis development was studied and the results indicated that the expression of both the proteins started from week 3 onwards till week 8. To confirm the meiotic stage-associated expression of ODF-2 and Tektin-2, germ cells were sorted based on their DNA content. ODF-2 and Tektin-2 transcripts were first expressed in the meiotic germ cells (pachytene spermatocytes) and their expression was upregulated in the post-meiotic germ cells (round spermatids). Sequential extraction of sperm proteins showed that, Tektin-2 was majorly extracted out in the Triton X-100 and DTT fraction, whereas, ODF-2 was maximally extracted in the presence of urea and DTT. In conclusion, these observations indicate that IGF1R and Src family of tyrosine kinases are critical for mammalian sperm capacitation and associated global PYP. Inhibition of sperm capacitation was associated with hypo-tyrosine-phospohorylation of certain proteins associated with mitochondrial membrane, axonemal structures and outer dense fibers of the sperm flagellum. Future work can be directed towards understanding the role of other RTKs and NRTKs involved in sperm capacitation and the molecular characterization of hypophosphorylated proteins critical for sperm function and its fertilization competence.

The process of translation initiation is the most highly regulated step of protein synthesis. In bacteria, three initiation factors (IF1, IF2 and IF3) play crucial roles during initiation. IF3 acts as an anti-association factor for the two ribosomal subunits. Eubacterial IF3 also permits initiator tRNA (i-tRNA) selection at the P site of the ribosome. Two features of i-tRNA, i. e. the characteristic 3GC base pairs in the anticodon stem and the cognate interaction of the anticodon sequence with the initiation codon of the mRNA contribute to IF3 based selection and/or proofreading. However, the exact mechanism of this discrimination and the contribution of the individual domains towards this process of selection/ proofreading are unclear. Further, there are exceptional instances in the natural world where either the codon-anticodon interaction or the anticodon stem composition deviates from the norm. For instance, in mammalian mitochondria, non-AUG codons such as AUU and AUA are present in the genome although they are notoriously poor initiation codons. In addition, some species of Mycoplasma have i-tRNAs with variations in the typically conserved 3GC base pairs of the anticodon stem. In this study, we have investigated the mechanism of proofreading activity of IF3 of E. coli, mitochondrial and mycoplasmal origins. Part I: Proofreading function of IF3 in E. coli IF3 is composed of N and C terminal domains joined by a flexible linker region. By means of complete and partial IF3 knockouts, we show that the C-terminal domain (CTD) is essential for the survival of E. coli while the N-terminal (NTD) is required for cellular fitness. Using reporter assays, we have established the role of the NTD in proofreading, while polysome profile analyses reaffirm that the CTD alone can bind to the 30S and carry out ribosome anti-association. Therefore, we show that the CTD is the ribosome binding and anti-association domain, while the NTD is the major proofreading domain. Unpublished cryoEM structures from Prof. Ramakrishnan’s lab indicate that the NTD of IF3 pushes the i-tRNA at its elbow and helps in P site accommodation of the i-tRNA. We propose that when the codon-anticodon interaction is non-cognate or if the 3GC base pairs of the anticodon stem are not intact, then the dynamic action of the NTD destabilises the tRNA at the P site and leads to its rejection. Part II: Proofreading function of mitochondrial IF3 (IF3mt) Of the 13 protein-coding genes in mammalian mitochondria, 3 utilise the non-canonical AUA codon and one utilises the non-canonical start codon AUU. Since IF3mt does not possess many of the generally conserved residues implicated in proofreading, we decided to characterise the proofreading function of IF3mt and its role in initiation with non-canonical start codons. Structurally, IF3mt is similar to EcoIF3 with its N and C terminal domains joined by a linker region. However, IF3mt additionally possesses N- and C-terminal extensions which are generally disordered in structure. In vivo studies of mitochondrial translation factors have been mired by the lack of methodologies to manipulate mitochondria. We have developed an E. coli strain to study the proofreading functions of mitochondrial IF3 (IF3mt) with the help of reporter genes. Consistent with its function in mitochondria, IF3mt allowed promiscuous initiation from non-AUG codons. However, IF3mt avoided initiation with i-tRNAs lacking evolutionarily conserved 3GC pairs in anticodon stems. Interestingly, expression of IF3mt N-terminal domain or IF3mt devoid of its typical N-, and C-terminal extensions significantly improved its proofreading activity. Our immunoblot assays from polysome profile fractions indicate that the IF3mt derivative lacking extensions is capable of superior 30S ribosome binding. The two derivatives of IF3mt missing the Next (IF3mtΔNext) or both the Next and Cext (IF3mtΔNextCext) display an affinity for the 50S ribosome. We propose that the extensions of IF3mt may have evolved to reduce the affinity of IF3mt to the ribosome and thereby permit initiation with non-canonical start codons like AUU and AUA. Our studies suggest that E. coli provides an excellent heterologous model to study distinctive features of mitochondrial factors. Part III: Fidelity of translation initiation in mycoplasma One of the many singular features of mycoplasma is the presence of many anticodon stem variants of the i-tRNA across different species. In general, i-tRNAs are characterized by the presence of the typical feature of the conserved 3 consecutive GC base pairs (GC/GC/GC) in the anticodon stem. However, many mycoplasmal species have i-tRNAs with AU/GC/GC, GC/GC/GU or AU/GC/GU sequences. Interestingly, the mycoplasmal species which harbour the AU/GC/GU i-tRNA are also human pathogens. Therefore, we decided to investigate whether these organisms possess any unique features to accommodate the i-tRNA variants, by investigating the usage of Shine Dalgarno sequences and by carrying out multiple sequence alignments of genes encoding initiation factors, ribosomal proteins S9 and S13 and 16S rRNA. Since IF3 plays a crucial role in i-tRNA selection, we carried out computational analysis of mycoplasmal IF3 sequences, which revealed many interesting features. Most striking amongst them was the variation of the highly conserved R at position 131 in some species. Interestingly, these were the very mycoplasmal species which possessed the anticodon stem variant AU/GC/GU, suggesting a strong correlation between these two features. It is known that the R131P mutation of EcoIF3 is characterised by an enormous loss of proofreading activity. It seemed unusual that such compromised proofreading would be tolerated in the cell, so we decided to investigate other components of the translational machinery as well. The C-terminal SKR tail of the ribosomal protein S9, which contacts the P-site tRNA, is highly conserved across bacteria. Analysis of the C-terminal sequences of S9 proteins in various mycoplasmal species revealed a surprising variation- the presence of a TKR tail in strains with the AU/GC/GU tRNA. In this study we have investigated the co-occurrence of S9 and IF3 variations in i-tRNA selection in E. coli. We see that the R131P polymorphism of IF3 leads to a tremendous loss of proofreading, but this loss is significantly tempered by the presence of the S9 TKR variation. Our bioinformatics studies revealed that the mycoplasmal species which are sustained on AU/GC/GU i-tRNAs also tend to use a higher percentage of non-AUG codons. By means of our reporter assays in E. coli, we have shown once again that the R131P polymorphism of IF3 leads to a tremendous increase in initiation with the non-canonical start codon AUA, but this increase is significantly tempered by the presence of the S9 TKR variation.

O objetivo deste trabalho foi estudar o processo pelo qual hormônios e fatores de crescimento controlam a proliferação celular em mamíferos. O modelo experimental utilizado foi linhagens de células estabelecidas em cultura. Os estudos centraram-se em dois tipos básicos de células: fibroblastos e células adrenais e o ataque experimental foi feito sob dois pontos de vista: bioquímico e genético. O ataque bioquímico envolveu desenvolver estudos cinéticos da síntese de DNA não só durante o carenciamento de células para soro, como também durante a reestimulação de células carenciadas por:soro, hormônios e fatores de crescimento. Medidas do conteúdo intracelular de cAMP foram efetuadas com o intuito de adquirir informações à respeito do mecanismo de ação destes fatores. Um modelo de ciclo celular foi proposto no qual o controle do crescimento seria exercido através de reguladores positivos e negativos que agiriam estimulando ou inibindo a passagem de células do estado de repouso (Go) para a fase proliferativa. Entre os reguladores positivos (estimuladores) do sistema fibroblasto, encontra-se hormônios clássicos, como esteróides e insulina, e fatores de crescimento de natureza hormonal como EGF, PF (fator proteico extraído de glândulas pituitárias) e prostaglandina F2α. O esteróide hidrocortisona pode agir como regulador negativo, inibindo o crescimento de fibroblastos. Medidas do período de tempo transcorrido desde a estimulação de células carenciadas (Go), até o aparecimento da onda de síntese de DNA (período definido operacionalmente como Gl) foram feitas. Em fibroblastos 3T3 este período foi de 12 a 13 horas tanto para células estimuladas com soro como com hormônios clássicos (hidrocortisona, insulina) ou fatores de crescimento (EGF, PF) ou ainda com combinações deles (EGF + PF + insulina; PF + hidrocortisona; PF + hidrocortisona + insulina). No sistema células adrenais, adrenocorticotropina (ACTH) foi o único hormônio clássico que apresentou atividade sobre o crescimento destas células e também o único efetuador negativo encontrado. Neste sistema PF mostrou-se como o único fator com atividade estimulatória sobre o crescimento. Gl aqui foi de 11 horas tanto para células estimuladas com soro como com PF. Além disso os hormônios clássicos hidrocortisona e insulina não apresentaram atividade estimulatória por si só ou em combinação com PF. A análise da ação de hidrocortisona no sistema fibroblasto e de ACTH no sistema células adrenais estimuladas, forneceu evidências de que após deixar Go, em direção a S, numa certa altura de Gl as células tornam-se irreversivelmente comprometidas com o processo replicativo. Este comprometimento parece ocorrer 5 horas antes de S, sendo referido como Glc. Em face destes resultados foi proposto que os reguladores agem estimulando ou inibindo a transição Go → Glc. Na tentativa de obter maior definição do sistema de controle do crescimento, aproveitamo-nos das vantagens oferecidas pelo modelo experimental usado, para a busca de mutantes do tipo regulatório. Esta busca resultou no isolamento das linhagens ST1 e AR-1, derivadas, respectivamente, de fibroblastos 3T3 e células adrenais Y-l. Entre os vários aspectos interessantes da linhagem ST1 destaca-se: a) o dramático efeito de hidrocortisona causando mudança nas características das células as quais passam de um fenótipo tipicamente transformado para normal. Este fenômeno foi observado tanto \"in vitro\" (através de medidas de parâmetros de crescimento) como \"in vivo\" (através de ensaios de tumorogenicidade); b) as alterações morfológicas de caráter antagônico provocadas, por um lado, pela adição de hidrocortisona (causando achatamento) e, por outro, pela retirada do soro ou adição de cAMP ao meio de cultura (arredondamento). Através do estudo da ação de inibidores, obteve-se evidências do envolvimento de microtúbulos nestas alterações morfológicas. A análise do conteúdo intracelular de cAMP indicou que este nucleotídeo não atua como mediador da ação de hidrocortisona. Sua ação parece ser devida à indução de alterações no sistema superfície celular - membrana -citoesqueleto. Ao contrário de outros variantes de células Y-l resistentes à ACTH, células AR-1 mostraram-se também resistentes a cAMP. A utilidade destas células nos estudos da postulada mediação deste nucleotídeo na ação de ACTH, é óbvia. / The aim of this work was to study the process by which hormones and growth factors control proliferation of mammalian cells. Cell lines established in culture were used as the experimental model. The studies were centered on two basic types of cells: fibroblasts and adrenal cells and the experimental approach was made from two viewpoints: biochemical and genetic. The biochemical approach involved kinetic studies of the DNA synthesis process not only during serum starvation but also during restimulation of serum starved cells by serum, hormones and growth factors. Intracellular cyclic AMP determinations were made in order to gain informations on the mechanism of action of these factors. A cell cycle model was proposed in which cell growth control would be exerted by positive and negative regulators that would act by stimulating or inhibiting the flow of cells from a resting state (Go) to the proliferative phase. Among the positive regulators (stimulators) found for the fibroblast system are: classical hormones, like steroids and insulin, and growth factors of homonal nature, like EGF, PF (protein factor extracted from pituitary glands) and prostaglandin F2α. The steroid hydrocortisone can also act as a negative regulator, inhibiting fibroblast growth. Measurements of the time interval between stimulation of serum starved (Go) cells and the onset of DNA synthesis (period that is operationally defined as Gl) were made. In 3T3 fibroblasts this period was 12 to 13 hours for cells stimulated not only by serum but also by classical hormones (hydrocortisone, insulin) or growth factors (EGF, PF) or even by combinations of these factors (EGF + PF + insulin; PF + hydrocortisone; PF + hydrocortisone + insulin). In the adrenal system, adrenocorticotropin (ACTH) was the only classical hormone to present activity on the growth of these cells and also the only negative regulator found. In this system PF was shown to be the only factor with growth stimulatory activity. GL was estimated as 11 hours for cells stimulated with serum or PF. Moreover hydrocortisone and insulin had no stimulatory activity \"per si\" or in combination with PF. The analysis of hydrocortisone action on the fibroblast system on one hand and of that of ACTH on the adrenal system, on the other, indicated that upon leaving Go, towards S, at a certain point in Gl, cells become irreversibly committed to the replicative process. This commitment seems to occur 5 hours before S and is referred to as Glc. In view of these data we proposed that regulators act by stimulating or inhibiting the transition Go → Glc. In an attempt to obtain a better definition of the growth control system and taking advantage of the experimental model utilized, we searched for mutants of the regulatory type. This search resulted in the isolation of the lines ST1 and AR-l from 3T3 fibroblasts and Y-l adrenal cells, respectively. Among several interesting aspects of the STl cell line, we point out: a) the dramatic effect of hydrocortisone changing the characteristics of these cells from a typically transformed phenotype to a normal pattern. This phenomenon was observed both \"in vitro\" (by measuring a number of growth parameters) and \"in vivo\" (by tumorogenicity assays). b) morphological alterations of antagonistic nature caused by hydrocortisone (flattening) on one hand, and by the removal of serum or cAMP addition to the culture medium (rounding) on the other. Evidence for the involvement of microtubules in these alterations were obtained through studies on the action of several inhibitors. Quantitative analysis of intracellular cAMP indicated that this nucleotide does not act as a mediator of hydrocortisone action. Rather, this action seems to be due to the induction of alterations on the cell surface-membrane-cytoskeleton system. Contrary to other variants of the Y-1 line which are resistant to ACTH, AR-1 cells are also resistant to cAMP. The usefulness of these cells in studies of the postulated mediation by cAMP of the ACTH action, is obvious.

Atualmente, o Brasil encontra-se na privilegiada posição de maior produtor e exportador mundial de carne bovina, tornando a pecuária uma das atividades nacionais mais importantes e rentáveis. Este dado enfatiza a importância de pesquisa e desenvolvimento em reprodução bovina, especialmente em hormônios estimuladores da ovulação, tais como a gonadotrofina coriônica equina (eCG). Os produtos comerciais à base de eCG comercialmente disponíveis são purificados a partir do sangue de éguas gestantes, apresentando variabilidade de lote para lote e presença de contaminantes. Estes fatos, juntamente com a limitação do material de partida (sangue equino), enfatizam a necessidade de haver um sistema de expressão de eCG recombinante passível de ser explorado comercialmente. Neste quesito, as células de mamíferos se mostram um sistema robusto para tal finalidade, visto que são capazes de adicionar modificações pós-traducionais às cadeias polipeptídicas, tais como a glicosilação, o que é essencial para o correto dobramento, maturação e montagem das duas subunidades, além de interferir diretamente com a meia-vida, o reconhecimento do receptor, a solubilidade e a atividade biológica das proteínas. No entanto, mesmo entre os sistemas de expressão heteróloga em células de mamífero, encontra-se muita variabilidade nos padrões de glicosilação adicionado. No presente trabalho, foi realizado um estudo comparativo através da clonagem e expressão de uma forma fusionada de eCG (reCGβα) em duas linhagens celulares diferentes: (1) CHO-DG44, um dos sistemas de expressão mais utilizados pelas indústrias farmacêuticas, capaz de adicionar N-glicanos complexos; e (2) 293T, uma linhagem humana capaz de produzir glicoproteínas carreando oligossacarídeos complexos e sialilados. Os resultados de atividade biológica (in vitro e in vivo) apontam uma maior atividade de reCG produzido por células CHO-DG44. O perfil de N-glicosilação de reCG produzido pelas células CHOD-G44 assemelhou-se mais à eCG selvagem, quando comparado a reCG produzido por células 293T. Por fim, estudos clínicos foram realizados com reCG produzido em meio livre de soro fetal bovino e parcialmente purificado, onde atividade específica de reCG produzido por células CHO-DG44 mostrou-se similar ao produto comercial selvagem. / Brazil is currently the major beef producer and exporter, rendering to livestock one of the country´s most economically relevant activities. This emphasizes the importance of research and development in bovine reproduction, especially at ovulation-stimulatory hormones, such as equine gonadotropin (eCG). The commercially available eCG-based products are purified from blood of pregnant heifers, presenting batch-to-batch variability and the presence of contaminants. These facts, together with the limitation of the bulk material (equine blood), emphasize the need of an eCG expression system able to be commercially explored. In this aspect, mammalian cells are a robust system, capable of add post-translational modifications to polypeptide chains, such as glycosylation, which is essential for the correct folding, maturation and assembly of both eCG subunits. In addition, glycosylation directly interferes with the protein half-life, receptor recognition, solubility and biological activity. In the present work, a comparative study was carried out by cloning and expressing a fusion form of eCG (reCGβα) in two different mammalian cell lines: (1) CHO-DG44, one of the most used by pharmaceutical companies expression systems, capable of add complex-type N-glycans; and (2) 293T, a human cell line capable of produce glycoproteins carrying complex and sialylated oligosaccharides. The in vitro and in vivo biological activity results show a higher potency of reCG produced by CHO-DG44 cells. The N-glycosylation pattern produced by CHO-DG44 cells was more similar to native eCG in comparison to the N-glycosylation produced by 293T cells. Finally, clinical studies were performed with serum absent media produced and partially purified reCG, showing that the specific activity of reCG produced by CHO cells was similar to the commercial wild type product.

pH is a key parameter in the optimization of animal cell processes, and has be linked to specific patterns of consumption and production of extracellular metabolites. However, the effect of extracellular pH on intracellular metabolism has not been fully elucidated. Metabolic flux analysis is a mathematical method that can be used to generate the intracellular flux distributions in cells, e.g. as a function of some environmental parameter. In this work, the overall objective was to develop a culture system and experimental protocol for cultivation of CHO cells, which can be used to generate data for analysis of the relationship between extracellular pH and intracellular fluxes in CHO cells by metabolic flux analysis. First, shake-flask culture of an IgG-producing cell line was performed to select an academic and chemically-defined medium with known composition. This was followed by subsequent adaptation of the cells. It was found that the originally selected medium had to be supplemented with a commercial medium to produce acceptable growth and viability. Shake-flask culture was also performed to evaluate the effect of the biological buffer HEPES on cell growth and viability, and the pH-stability during culture. HEPES-concentrations in the investigated range (7.5-45 mM) did not show an apparent effect on cell growth or viability. The higher concentrations gave slightly better buffering capacity at inoculation, however were not sufficient to keep pH stable during culture. As a result, the idea of using shake flask culture and similar techniques for cultivation of cells at various pH set-points was dismissed. Instead, a culture system and protocol based on a 100 mL Spinner flask with pH-regulation was custom-designed for the project. Features of the final design included continuous monitoring of pH and DO, stable temperature at 37 °C, adjustable agitation rate, as well as the option to incorporate inflow of air, O2 and CO2. In addition, the possibility to disconnect the flask unit to perform medium exchange and sample collection away from the reactor site (i.e. in a laminar flow workbench) was integrated into the design and protocol. The system was demonstrated for pseudo-perfusion culture with the adapted IgG-producing cell line at pH 7.0 during 24 days. Optimized regulation settings were identified. It was shown that the system could support viable cell densities of up to 11 MVC/mL and high viability (> 90 %). During the final phase of culture, stable growth, at specific growth rates of approximately 0.7 Day-1, was achieved. The specific rates of consumption and production of the key metabolites glucose, glutamine, lactate and NH4+, as well as 20 amino acids were analyzed. A majority of the rates were in accordance with CHO cell metabolism. The expected consumption of a majority of the essential amino acids and main carbon sources glucose and glutamine were confirmed, as well as the associated production of by-products lactate and NH4+. The system and protocol developed in this work can be used in future experiments to generate data describing metabolic profiles as a function of various pH-set points. This data may then be used in metabolic flux analysis to further elucidate the metabolism behind pH effects in CHO cells. / pH är en viktig parameter i optimeringen av animalcellsprocesser och har sammankopplats med specifika konsumtions- och produktionsmönster rörande extracellulära metaboliter. Det extracellulära pH-värdets effekt på den intracellulära metabolismen är dock inte fullt klarlagd. Metabolisk flux analys är en matematisk metod som kan användas för att generera intracellulära fluxfördelningar i celler, exempelvis som en funktion av någon yttre parameter. Det övergripande målet i detta arbete var att utveckla ett odlingssystem och experimentellt protokoll för odling av CHO-celler som kan användas för att generera data för metabolisk flux analys där målet är att studera effekten av pH på den intracellulära cellmetabolismen. En IgG-producerande CHO-cellslinje odlades först i skakkolv för att välja ut ett akademiskt kemiskt definierat medium med känd sammansättning. Därefter följde försök att anpassa cellerna till det valda mediet. Det visade sig att ett kommersiellt medium behövde tillsättas för att ge godtagbar tillväxt och viabilitet. Effekten av den biologiska bufferten HEPES på cellernas tillväxt och viabilitet, samt pH-stabiliteten under odling, undersöktes också genom odling i skakkolv. HEPES-koncentrationer i det undersökta intervallet (7.5 – 45 mM) hade ingen större effekt på tillväxt och viabilitet. För de högre koncentrationerna var buffertkapaciteten något bättre precis vid inokulering. Dessa koncentrationer var dock ej tillräckliga för att ge stabilt pH under odlingen. Baserat på dessa resultat övergavs tanken på att använda skakkolvsodling för att odla celler vid olika pH-värden. Ett odlingssystem och ett protokoll baserat på en 100 mL Spinnerflaska med pH-reglering specialdesignades istället för projektet. I det färdiga systemet fanns lösningar för kontinuerlig övervakning av pH och DO, stabil temperatur vid 37 °C, justerbar omrörningshastighet, samt valmöjligheten att flöda in luft, O2 och CO2. Dessutom infördes möjligheten att koppla loss flaskenheten från reglersystemet för byte av medium och provtagning. För att demonstrera systemet genomfördes en odling med den anpassade IgG-producerande cellinjen enligt principen för pseudo-perfusion vid pH 7.0. Odlingen pågick under 24 dagar och optimerade reglerinställningar identifierades. Det visades att systemet kunde understödja cellkoncentrationer upp till 11 miljoner celler per milliliter, samt hög viabilitet (> 90 %). Under den senare delen av odlingen uppnåddes stabil tillväxt, vid specifika tillväxthastigheter omkring 0.7 per dygn. Den specifika konsumtions- och produktionshastigheten för metaboliterna glukos, glutamin, laktat och NH4+, samt 20 aminosyror analyserades. Majoriteten av hastigheterna stämde överens med typisk CHO-cellsmetabolism. Den förväntade konsumtionen av majoriteten av de essentiella aminosyrorna och huvudsakliga kolkällorna glukos och glutamin konfirmerades, såväl som den associerade produktionen av bi-produkterna laktat och NH4+. Odlingssystemet och det experimentella protokollet som utvecklades i detta arbete kan användas i framtida experiment för att generera data som beskriver metaboliska profiler som funktion av extracellulärt pH. Dessa data kan sedan användas i metabolisk flux analys för att dra slutsatser om pH-effekter i CHO-celler.

No description available.