Dissertations / Theses on the topic 'PHD finger'

Monocytic leukaemia zinc-finger protein (MOZ) is a histone acetyltransferase (HAT) implicated in haematopoiesis and acute myeloid leukaemia, as well as embryonic and postnatal development. MOZ contains multiple domains, including a MYST HAT domain and a double PHD finger domain (DPF) suggesting it interacts with histones. This work has established for the first time that the MOZ DPF exhibits dual functionality in establishing and sensing post-translational modifications (PTMs) of histones. Firstly, our data detected the direct interaction of MOZ with the N-terminal tails of histones H3 and H4 and shows that the MOZ DPF domain mediates such binding. Both PHD fingers are required and functionally cooperate to establish the DPF histone binding preference in terms of PTMs. We demonstrate that H3K4me3 prevents MOZ DPF association with H3, although H3K4me2 is tolerated. Similarly, H4Kac acts as a dominant exit signal that excludes MOZ from chromatin. This ability to sense H3K4 PTM status was confirmed in a collaborative effort establishing the crystal structure of MOZ DPF in complex with an unmodified H3 peptide. The H3 peptide adopted an α-helical conformation in the complex, which has not previously been observed. Secondly, we present novel data showing that the MOZ DPF domain exhibits a mild histone H3-specific acetyltransferase activity. This provides the first report of a possible enzymatic role in chromatin modification attributed to a PHD finger. Furthermore, the combined DPF and MYST domains were found to influence the reaction rate and substrate specificity of MOZ-induced histone acetylation. Our studies revealed that the MOZ DPF could associate with heterochromatic PTMs, namely H3K9me3. We report here that both the H3K9-specific methyltransferase SUV39H1 and heterochromatin protein 1 (HP1) form interactions with MOZ, implicating its function in both corepressor and coactivator complexes. Thus, our data suggest that like several other chromatin-associated proteins, MOZ is a multi-functional regulator of chromatin modification and gene expression.

Sp140 is an IFNg-inducible leukocyte-specific member of the Sp100 family proteins. Along with the PML tumor suppressor, Sp100 family proteins are major components of PML-nuclear bodies (PML-NBs), they interact with DNA and various regulatory factors and may be involved in chromatin-dependent transcriptional activation or repression. Sp140 is expressed in all human mature B cells and plasma cell lines, as well as some T cells, suggesting an important role in cellular functions that are peculiar to these cells. In mammalian cells Sp140 behaves as a transcription co-activator for a reporter gene (Bloch et al, 2000). Despite the involvement in B-cell Chronic Lymphocytic Leukaemia (CLL, OMIM151400) (Di Bernardo et al, 2008) and HIV-1 replication (Guldner et al, 1992), until now Sp140 physiological and pathological role is unknown and unexplored, both at cellular and molecular level. The predicted Sp140 amino acid sequence, 867 residues in length, indicates a modular structure similar to other Sp100 family members (Bloch et al, 1996). The N-terminus HSR domain (amino acids 36-157) is responsible both for PML-NBs targeting and homo/hetero-dimerization. The region between residues 306-404 is strongly negatively charged, while the central portion contains a putative bipartite nuclear localization signal, a SAND domain (residues 588-661), a PHD finger (residues 692-734) and a bromodomain (residues 756-859). In this thesis we investigated Sp140 PHD finger both at functional and structural level. Structural determination was performed in solution by means of NMR spectroscopy. Analysis of 1H-15N HSQC spectra of Sp140 15N PHD finger wild type and 15N PHD finger P45A mutant revealed peptidyl-prolyl cis trans isomerization of the T44-P45 peptide bond and a ratio between cis and trans conformers of 1:2. As the NetPhos 2.0 server predicts phosphorylation of the T44 residue by the p38 mitogen-activated protein kinase (p38 MAPK), we suppose that the phosphorylated T44-P45 bond might be a site of regulation of the domain structure through the activity of PIN-1, an enzyme that efficiently and specifically bind to and isomerizes the phosphorylated S/T-P motifs in proteins (Wulf et al, 2005). Using triple resonance NMR experiments we assigned 95% of backbone resonances, 96% of side chain 1H resonances and 73% of side chain non-1H resonances (side chain resonances of OH, SH, CO, NH and NH2 groups were not assigned) of the Sp140 PHD finger trans conformer. The tautomeric state of the two histidines was determined, analyzing a 1H-15N long range correlation HSQC spectrum. The 3J(HNHa) coupling constants and the corresponding phi dihedral angles were calculated by analysis of the 3D HNHA spectrum and were then compared to those predicted by TALOS+ software. We manually assigned more than 850 NOE cross-peaks of the 2D and 3D NOESY spectra. NOEs restraints and 12 dihedral angles were employed for structure calculation of the Sp140 trans conformer by means of ARIA 2.1.3 software. The best NMR ensemble achieved up to now showed a compact globular fold with two short a-helices (stretches 12-EVCR-15 and 50-FCRM-53) as the sole elements of secondary structure. Analysis of the electrostatic surface potential revealed the strong negative charged character of the Sp140 PHD finger trans conformer, with a positive patch only on one side of the domain. Heteronuclear NOE experiments revealed that loop 2, containing the T44-P45 bond which undergoes cis trans isomerization is flexible from E40 to N47. Also the N-terminal tail of the domain is flexible up to residue L8. Flexibility caused a limited number of available NOE restraints in these two regions, especially in loop 2, and consequently an overall high backbone RMSD of the NMR ensemble. In order to increase the number of NOE restraints to calculate the structure we have recently acquired NOESY spectra at higher magnetic fields (900 MHz 1H frequency) that will be soon analyzed. We next investigated the functional role of Sp140 PHD finger and verified its ability to work as histone binding modules. According to sequence alignments, Sp140 PHD finger belongs to the PHD finger subclass specifically recognizing the H3K4me0 epigenetic mark. Indeed, it has an N-terminal conserved aspartic acid which should be involved in electrostatic interactions with the unmodified histone K4 side-chain, as previously demonstrated by both AIRE PHD1 (Chignola et al, 2009) and BHC80 PHD finger (Lan et al, 2007) structures in complex with an unmodified H3 peptide. Unexpectedly tryptophan fluorescence and NMR titrations of Sp140 PHD finger with a 15mer H3K4me0 peptide showed no binding, indicating that the presence of the N-terminal acidic hallmark is not sufficient to predict binding to the H3K4me0 epigenetic mark. We next explored the possibility that SP140 PHD finger might recognize other epigenetic modifications, we therefore extended our binding assays to other synthetic peptides carrying different types of epigenetic marks (such as methylation or acetylation). We also applied a large screening approach using the MODifiedTM Histone Peptide Arrays, which allows the screening of 384 unique modification combinations on the N-terminal tails of histone H3 (up to residue 45), H4 (up to residue 30), H2A and H2B (up to residue 19). However, until now we identified no specific binding of Sp140 PHD finger to the unmodified histone tails and to any combination of histone epigenetic marks spotted on the array (acetylation, methylation, phosphorylation and citrullination). Taken together these data suggest that Sp140 PHD finger is not a classical epigenetic reader and other functions might be attributed to this domain, such as a SUMO E3 ligase activity towards the adjacent bromodomain. This hypothesis was supported by the presence of a typical KxE SUMOylation site at the N-terminus of the Sp140 bromodomain (761-LKCE-764) and by recent data on KAP1 PHD finger, which functions as SUMO E3 ligase for the adjacent bromodomain (Ivanov et al, 2007; Zeng et al, 2008). In support to this hypothesis NMR titration experiments revealed binding of recombinant SUMO E2 ligase Ubc9 to a small, well defined surface of Sp140 PHD finger. We next expressed and purified in E.coli Sp140 PHD finger – bromodomain (PB) tandem and through in vitro SUMOylation test and mass spectrometry analysis we found that the construct was SUMOylated on lysine K837. This residue constitutes an atypical SUMOylation site, located at the bromodomain BC loop. Importantly, sequence alignment reveals that this position corresponds to one of the four SUMOylation sites indentified in KAP1 bromodomain. The intramolecular SUMO E3 ligase activity of Sp140 PHD finger is also supported by the observation that Sp140 PHD finger and bromodomain interact with each other, as deduced by superposition of the 1H-15N HSQC spectra of Sp140 PHD finger alone and PB tandem. Interaction could occur through the hydrophobic core made by stretch 765-FLLLKV-770 in the bromodomain and V695, F712, F718, F732 residues in the PHD finger. Indeed, this is the same core that mediates KAP1 PHD finger and bromodomain interaction, leading to a structural and functional unit whose integrity is fundamental for the KAP1 PHD finger SUMO E3 liagse activity (Zeng et al, 2008). Through NMR titrations we also demonstrated binding of SUMO-1 to both Sp140 PHD finger alone and PB tandem. The binding surface mapped on the homology model of the PB tandem is in agreement with the covalent addition of one SUMO-1 moiety to both the bromodomain K873 and K762 (in the typical KxE SUMOylation site at the bromodomain N-terminus). We are currently performing mutagenesis experiments to confirm the two Sp140 PB tandem SUMOylation sites by means of in vitro SUMOylation tests and mass spectrometry analysis on the Sp140 PB tandem single mutants K762R and K837R. To further validate the intramolecular SUMO E3 ligase activity of the PHD finger we will perform in vitro SUMOylation tests on a PB tandem mutant, in which the function of the PHD finger is inhibited trough an unfolding mutation. In this thesis we have collected strong evidences that in vitro Sp140 PHD finger has SUMO E3 ligase activity for the adjacent bromodomain. This finding might have an important biological relevance in the context of the full length protein: first of all the SUMO E3 ligase activity correlates well with Sp140 localization in leukocytes PML-NBs. PML-NBs are nuclear sub-compartments in which 48% of their protein components show one or more SUMOylation sites which, once they are SUMOylated, work as PML-NBs recruitment signal for these proteins (Van Damme et al, 2010). A similar mechanism could be hypothesized for Sp140 protein, whereby the PHD finger mediates the SUMOylation of the adjacent bromodomain in order to enable the protein recruitment to PML-NBs. SUMOylation might also have a role in the Sp140 transcription co-activating function, other than localization. It is conceivable that Sp140 SUMOylation might recruit proteins able to interact non-covalently with the conjugated SUMO-1 through their SIMs (SUMO Interaction Motifs). Hereby SUMOylated Sp140 protein might serve as a platform for the assembly of a leukocyte-specific transcription complex, whose target genes are still to be identified. Importantly, a similar mechanism has been observed for the co-repressor KAP1; SUMOylation is required for KAP1-mediated gene silencing, because the SUMOylated bromodomain serves as a scaffold and recruits the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex through recognition of the SUMO moieties by the SIMs of these proteins (Ivanov et al, 2007). Through NMR and ITC titrations we found that Sp140 PB tandem is able to bind to a 15mer H3K9ac synthetic peptide. We excluded any PHD finger involvement in this interaction, because when we titrated the H3K9ac peptide to Sp140 PHD finger alone we did not see any binding. Therefore Sp140 bromodomain seems to retain the characteristic ability of bromodomains to bind to acetylated histone tails, despite it does not show the conserved Tyr, Tyr and Asn residues employed by bromodomains in order to bind to acetylated lysines. If confirmed, these results suggest that Sp140 bromodomain is characterized by a new, peculiar mode of recognition of acetylated histone tails. In conclusion, we are collecting important functional and structural data on two domains (PHD finger and bromodomain) of Sp140, a leukocyte-specific nuclear protein involved in B-cell Chronic Lymphocytic Leukaemia and HIV-1 replication, but unexplored up to now. Our functional data strongly indicate that the two domains interact with each other constituting a structural and functional unit in which the PHD finger mediates SUMOylation of the bromodomain. We are solving the NMR solution structure of the Sp140 PHD finger trans conformer. With the exception of the T44-P45 peptidyl-prolyl cis trans isomerization, we are not observing great differences with the solved structures of PHD fingers recognizing histone H3 epigenetic marks, on the contrary of Sp140 PHD finger. Therefore, our data further support the concept that PHD fingers are versatile domains able to perform different activities according subtle but significant structural differences.

Point Mutations or translocation in NSD1 cause the overgrowth disorder Sotos syndrome and acute myeloid leukemia (AML), respectively (Berdasco M. et al, 2009, Wang G et al, 2003). NSD1 contains several chromatin related domains including a SET domain responsible for histone methyltranferases activity (H3K36 and H4K20), two nuclear receptor-interaction (NID) motifs, five zinc finger domains (PHD1-5), a variant PHD finger (C5HCH), two proline-tryptophan-proline-tryptophan (PWWP1-2) domains (Lucio-Eterovic AK, et al , 2011), suggesting a role in chromatin regulation and gene expression. 20 pathological Sotos mutations have been detected on the PHD tandem domain composed by NSD1-PHD5 and NSD1-C5HCH (NSD1-P5C5). The tandem domain is essential for the pathogenesis of acute myeloid leukaemia (AML) caused by the chimeric protein NUP98/NSD1 that forces the abnormal activation of Hox-A and Meis1 genes (Wang at al 2007). The deletion of this tandem domain is sufficient to abolish NUP98/NSD1 interaction with chromatin, preventing both the transcription activation of HOX genes and the immortalization of myeloid progenitors. The biological role of NSD1-P5C5 is still unclear. It was proposed that this tandem domain is involved in the recognition of both H3K4me3 and H3K9me3 histone marks, (Pasillas M et al. 2011). However, biophysical experiments in our laboratory did not confirm these results challenging the idea that this tandem domain can really work as epigenetic reader. Previous biochemical studies suggested that NSD1-P5C5 can also work as protein-protein interaction motif, being able to bind to the co-repressor Nizp1 by its C2HR zinc fingers motif (Nizp1-C2HR) thus mediating gene repression (Nielsen AL et al, 2004). The structural determinants of this interaction are still unknown and have been object of this thesis. In order to get more insights into the physiological and pathological role of NSD1-P5C5, we have solved its (i) solution structure by NMR spectroscopy and (ii) characterized its interaction with Nizp1-C2HR. NSD1-P5C5 folds as unique functional unit adopting a “face to side orientation”. In particular NSD1-PHD5 (or NSD1-P5) presents the canonical PHD finger fold, whereas the NSD1-C5HCH (or NSD1-C5) domain displays an atypical topology characterized by the presence of an additional two stranded β-sheet. In order to investigate the impact of Sotos point mutation on NSD1-P5C5 we expressed and purified seven mutants and analyzed them by NMR. The majority of them destabilize the fold, with the exception of the solvent exposed mutation Arg2152Gln and His2205Arg suggesting a functional role for these residues. We next solved the solution structure of the zinc finger Nizp1-C2HR, an atypical Cys2His2-type zinc finger in which the fourth zinc chelating residue is substituted by an arginine residue. Its fold consists of a short α-helix and of a short two-stranded β-sheet hold together by one zinc ion. Importantly, we showed that three zinc ligands are sufficient to maintain the protein domain fold and functionality. NMR titrations of 15N labelled NSD1-P5C5 with Nizp1-C2HR and 15N labelled Nizp1-C2HR with NSD1-P5C5 clearly show that the two proteins directly interact. Analysis of the chemical shift displacements upon complex formation allowed to identify the residues of the two protein domains involved in protein-protein interaction. The interaction surface is located on the interface between NSD1-P5 and NSD1-C5 and on the α-helix of Nizp1-C2HR, respectively. Based on this information using the software HADDOCK we have computed a data driven docking model of the protein complex. In the model Nizp1-C2HR places its α-helix in the groove at the interface between NSD1-P5 and NSD1-C5, creating both hydrophobic and polar intermolecular contacts. The thermodynamic parameters that govern complex formation were studied by ITC titrations: the binding reaction is entropy-driven, with a stoichiometry of 1:1 and a Kd of 3,80±0,66 μM. In order to solve the structure of the protein complex we performed crystallographic screenings, and we have found preliminary conditions for obtaining single crystals. In conclusion, the presented results provide novel information on the interaction between a tandem PHD finger domain and zinc finger motif. The results represent, to the best of our knowledge, the first biophysical characterization between two zinc binding domains. Most importantly, these data give the first molecular details of the interaction between NSD1 and Nizp1 and may provide useful insights into the function of NSD1 and its role in pathological conditions both in Sotos Syndrome and AML. Future work will be dedicated to the full three-dimensional characterization of the complex and to the analysis of Sotos mutations on complex formation.

The use of chemical probes is a powerful tool that can help to address important biological questions. The study presented in this thesis aims to target reader domains of chromatin-associated proteins with small molecules, in order to provide information on their ligandability, useful to develop - potent chemical probes. This thesis work is divided in three parts. In the first and second part it is shown how structural information obtained by the use of synthetic peptides to study the binding mode of reader domains with their natural binding partner can be combined with fragment screening to gauge future optimization of small molecules. The first section described the disclosure of the binding mode of the H3 histone tail by the PHD zinc finger of BAZ2A. A crystal structure of the complex of BAZ2A with the H3 10-mer peptide identified a helical conformation of H3 upon binding with the PHD. This information coupled with further structural and biophysical analysis led to the identification of a subfamily of PHD characterized by an acidic patch on the helical turn, which is responsible of inducing helicity on H3 tail upon binding. The second part of the work investigated the ligandability of the PHD zinc finger domains of BAZ2A and BAZ2B. Using a combination of biophysical techniques and X-ray crystallography it was probed that it is possible to target these reader domains. Despite the similarities of the two PHDs, comparison of the fragment-bound crystal structures of the two proteins highlighted some differences in the binding mode. The last part of the project describes the several attempts performed in trying to elucidate the histone binding partner of the PHD-BrD tandem of the chromatin-related proteins BAZ1B and TRIM66, both involved in diseases.

The nucleosome remodeling factor (NURF) is a mutli-protein complex that plays a role in the regulation of gene expression through its ability to remodel nucleosomes. The largest subunit of this complex, Bptf (Bromodomain PHD Finger Transcription Factor) is important for many cellular processes as a transcriptional regulator and improper function results in disease or malignancy. To further understand the genome-wide recruitment of the NURF complex, the interaction partner for the N-terminal PHD finger domain of Bptf was investigated through pull down assays followed by mass spectrometry. It was determined that this domain does not recognize histones; instead it recognizes a nonhistone protein, Thoc4 or Hmgb1. The expression of a cDNA corresponding to Bptf was also tested for expression in mouse ES cells after the addition of two exons found to be missing in the original cDNA. Addition of this sequence did not allow for exogenous Bptf expression in ES cells.

This thesis summarise the results of an investigation of the tribological interactions of the human finger pad with different surfaces and tactile displays. In the wide range of analyses of the mechanical properties of the finger pad, an attempt has been made to explain the nature of the interactions based on critical material parameters and experimental data. The experimental data are presented together with detailed modelling of the contact mechanics of the finger pad compressed against a smooth flat surface. Based on the model and the experimental data, it was possible to account of the loading behaviour of a finger pad and derive the Young’s modulus of the fingerprint ridges. The frictional measurements of a finger pad against smooth flat surfaces are consistent with an occlusion mechanism that is governed by first order kinetics. In contrast, measurements against a rough surface demonstrated that the friction is unaffected by occlusion since Coulombic slip was exhibited. The thesis includes an investigation of critical parameters such as the contact area. It has been shown that four characteristic length scales, rather than just two as previously assumed, are required to describe the contact mechanics of the finger pad. In addition, there are two characteristic times respectively associated with the growth rates of junctions formed by the finger pad ridges and of the real area of contact. These length and time scales are important in understanding how the Archardian-Hertzian transition drives both the large increase of friction and the reduction of the areal load index during persisting finger contacts with impermeable surfaces. Established and novel models were evaluated with statistically meaningful experiments for phenomena such as lateral displacement, electrostatic forces and squeeze-film that have advanced applications.

The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.

The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.

This thesis presents the Intelligent Gripper, a low cost instrumented robot finger pad which is equipped with an 8 x 8 tactile sensing array and a 2 x 2 proximity sensing grid. The complete system is composed of three modules. The capacitive tactile sensing array utilizes a dual strip construction to improve its robustness and simplicity, a technology initially developed by Sarcos Research Inc. The proximity network is based on infrared range sensing technology. The microprocessor based interface module gives the system intelligent capability. In order to reduce the noise and to improve the system modularity, all electric circuitry is localized. The modular architecture gives the system excellent portability.<br>Following the comprehensive evaluation and characterization of the tactile sensing array and its associated electronic system, an experimental exploration to use the tactile sensor in transient contact force control is presented. It is found that using feedback from tactile sensor stabilizes the force control. However, the highest performance for transient force control is achieved from a combined feedback from tactile sensor and joint force sensor.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2003.<br>Includes bibliographical references (leaves 56-59).<br>by Louis H. Buell, Jr.<br>S.M.

Selenium is an important nutritional element that is required in a minute amount for maintenance of proper health in both humans and animals. Many biochemical processes in human and animal depends on Se and selenoprotein function, and various studies have suggested that Se supplementation can improve fundamental immune function in both humans and animals. Se status in the UK is very low; therefore, there is a need to enhance selenium intake through diet. One way of doing that is through the introduction of selenium to farmed animals. In this Ph.D. study, we fed rainbow trout with diet containing different spiked concentrations (0.8 - 8.9 μg g-1) of Se for 14 weeks. Fish were sampled every two weeks and liver, kidney, muscle, gills and whole blood were collected and processed. The first phase of this Ph.D. study focused on selenium distribution and biotransformation in tissues of rainbow trout. Three methods were developed and used to determine total selenium concentration; species' distributions in tissues and Se peptide sequence to determine the possible incorporation of Se into proteins respectively. In the first method total selenium concentration was determined using ICP-MS and the second and third methods using HPLC-ICP-MS/ESI-MS. Total selenium concentrations in trout tissues were determined, and the highest selenium concentrations were found in liver followed by kidney, gills, and muscle. SeMet and SeCys were found to be the major species in all tissues followed by inorganic and other unknown species. To determine the possibility of Se incorporation in to protein, peptide de novo sequencing was carried out. Few selenoprotein were identified using automated de novo sequence and database search. Se species distributions in tissues of beached Pilot whales were studied. Several low molecular weights Se species were identified with selenite pre-dominating other species in most of the adult whales tissues analysed.

This document provides an elaboration of the critical, contextual and methodological rationale for a practice‐based PhD research project undertaken at London Metropolitan University 2009-2013. This four‐year project was an exploration in identity, space and location. It looks at the transitions, journeys and stories of migrant women. Specifically this exploration has been developed through the language of the cultural practices of Iban women. The Iban are an indigenous group of people from Borneo, predominantly living within the Malaysian state of Sarawak. Significantly the Iban practices have migrated from the jungle, to urban areas, and globally, and inevitably the identity of these practices has developed as the locations have changed, much like the women performing them. My father is Iban and my mother white New Zealander, and I grew up in both Sarawak and New Zealand before coming to live in the UK in my 20s. My performance training has been within a Western context, both in New Zealand and the UK. This project has been a personal exploration, which has wider consequences in developing performance practice and understanding the discourses of home, belonging, migration and identity. This has led to questions around migrating Iban performance and cultural practices to a western contemporary context. These questions have been investigated through the cultural practices of the Iban pantun (chapter three), the Iban ngajat (chapter two), Iban weaving (chapter four) and the use of space in the Iban longhouse (chapter one). This project was an interdisciplinary investigation; in each chapter I pull together performance theory from western practitioners and post‐colonial feminist literature with the Iban performance practice. This project has asked the question: "Can Iban cultural and performance practices be ‘migrated’ to a contemporary western performance context in order to explore experiences of women’s migration?" My research question was central to the practice‐based research I conducted, the methodologies developed through practice as research, and are central to all the work covered in this thesis. Within this context the practice is submitted as an outcome alongside this written narrative. Additional details can be found on the website: www.fromthejungle.co.uk.

"Personal health record (PHR) service is an emerging model for health information exchange. It allows patients to create, manage, control and share their health information with other users as well as healthcare providers. In reality, a PHR service is likely to be hosted by third-party cloud service providers in order to enhance its interoperability. However, there have been serious privacy concerns about outsourcing PHR data to cloud servers, not only because cloud providers are generally not covered entities under HIPAA, but also due to an increasing number of cloud data breach incidents happened in recent years. In this thesis, we propose a privacy-preserving PHR system using attribute-based encryption (ABE). In this system, patients can encrypt their PHRs and store them on semi-trusted cloud servers such that servers do not have access to sensitive PHR contexts. Meanwhile patients maintain full control over access to their PHR files, by assigning fine-grained, attribute-based access privileges to selected data users, while different users can have access to different parts of their PHR. Our system also provides extra features such as populating PHR from professional electronic health record (EHR) using ABE. In order to evaluate our proposal, we create a Linux library that implement primitive of key-policy attribute-based encryption (KP-ABE) algorithms. We also build a PHR application based on Indivo PCHR system that allow doctors to encrypt and submit their prescription and diagnostic note to PHR servers using KP-ABE. We evaluate the performance efficiency of different ABE schemes as well as the data query time of Indivo PCHR system when PHR data are encrypted under ABE scheme."

This PhD provides the first organized view of art therapy education in Australia. It focuses on the theories that are used in this specialized teaching and learning process. It evolved from the authors’ immersion in the field as a migrant art therapy educator to Australia from the UK and a desire to be reflexive on this experience. The research questions aimed to discover the field of art therapy education in Australia: to find out what theories and practices were taught; and where the theoretical influences were coming from, in order to develop understanding of this emerging field. Positioned as a piece of qualitative research a bricolage of methods were used to gather and analyse information from several sources (literature, institutional sources, and key participants, including the author) on the theories and practices of art therapy training programs in Australia. This also included investigating other places in the world shown to be influential (USA and UK). The bricolage approach (McLeod, 2006) included: phenomenology; hermeneutics; semi-structured interviews; practical evaluation (Patton, 1982, 1990/2002); autoethnography (Ellis and Bochner, 2000); heuristic (Moustakas, 1990); and visual methodologies (Kapitan, 2010). These were used to develop a body of knowledge in the form of institution/program profiles, educator profiles, country profiles and an autoethnographic contribution using visual processes. Epistemologically, the project is located in a paradigm of personal knowledge and subjectivity which emphasizes the importance of personal experience and interpretation. The findings contribute knowledge to support the development of art therapy education and the profession in Australia, towards the benefit, health and wellbeing of people in society. The findings show a diverse and multi-layered field of hybrid views and innovative approaches held within seven programs in the public university and private sectors. It was found that theories and practices are closely linked and that theoretical views have evolved from the people who teach the programs, location, professional contexts (health, arts, education, social, community) and the prevailing views within these contexts, which are driven by greater economic, socio-political forces and neo-liberal agendas. The university programs generally teach a range of the major theories of psychotherapy underpinned with a psychodynamic or humanistic perspective. Movement towards a more integrative and eclectic approach was found. This was linked to being part of more general masters programs and economic forces. The private sector programs are more distinctly grounded in a particular theoretical perspective or philosophical view. Key words distilled from the profiles included: conflict, transpersonal, survival through art, pedagogy, epistemology, theory driven by context and mental health. Important issues for art therapy education were identified as: the position and emphasis on art; working with the therapy/education tension; the gender imbalance in the profession; Indigenous perspectives; intercultural issues and difference. The horizons of the field revealed the importance of developing the profile of the profession, reconciling differences towards a more inclusive view and the growth of research. A trend towards opportunities in the social, education and community areas was found, driven by the increasing presence of discourses on arts and wellness.

L'étude de problèmes de physique hadronique dans le cadre de la théorie des champs nécessite l'emploi de méthodes non-perturbatives, les approches perturbatives ne pouvant s'appliquer pour QCD à basse énergie. L'équivalence formelle existant entre la théorie des champs et le problème à N corps nous a conduit à adapter des techniques non-perturbatives usuelles de la théorie du problème à N corps, comme l'approximation de champ moyen (ou approximation gaussienne) et l'approximation de la phase aléatoire (RPA).<br> En se plaçant au-delà du champ moyen où seules sont prises en compte les corrélations entre une particule et un potentiel "moyen" à un corps, la RPA va permettre de rajouter dans le calcul de l'état fondamental des corrélations entre particules.<br> Afin de mettre en place le formalisme, on applique la RPA, sons différentes formes (standard, renormalisée, en termes de fonctions de Green), à l'une des plus simples théories des champs en interaction, la théorie scalaire lambda x phi^4. On montre qu'il se produit une transition de phase due à une brisure dynamique de symétrie dont le paramètre critique se rapproche des résultats obtenus sur réseaux et par la technique des "clusters". Les résultats sont aussi présentés à température finie pour le champ moyen.<br> On étudie également un modèle effectif réaliste de la transition de phase chirale, le modèle sigma-linéaire et on montre que le théorème de Goldstone est restauré, contrairement à l'approximation gaussienne.<br> Enfin pour éclaircir quelques points de la RPA et, aller au-delà des corrélations obtenues dans la forme renormalisée, on considère l'oscillateur anharmonique en mécanique quantique, en introduisant les corrélations minimales au-delà du champ moyen et on montre que les corrélations RPA améliorent grandement le résultat obtenu en champ moyen.

博士<br>國立臺灣大學<br>分子醫學研究所<br>87<br>Maintenance of chromosome integrity is a fundamental requirement in all-living organisms. Genes are expressed in a temporal or tissue-specific manner or activated in response to extracellular stimuli. Gene expression can be tightly controlled at several levels, including transcriptional, post-transcriptional (mRNA processing and transport), translational (protein synthesis) and post-translational levels (glycosylation or phosphorylation etc.). For most genes transcriptional controls are paramount. The initiation of transcription in eukaryotes is a highly and tightly controlled process. Genes exist at some time in an inert state that was tightly packaged with histones in to chromatin. Before transcription can occur, this inert structure must be decondensed so that transcriptional control sequences are made available to regulatory proteins. Once decondensation of chromatin has occurred, the scene is set for the second activation steps: the interaction of regulatory proteins, commonly referred to as transcription factors or trans-acting factors, with specific DNA sequences. Such interactions of these bound factors with general transcription factors mediate gene transcription. Protein-protein interaction and posttranslational modifications are important for regulating activities of these trans-acting factors. There are two models for the initiation of transcription by RNA pol II : the stepwise assembly model and the holoenzyme model. The conventional model (stepwise assembly model) for ordered transcriptional initiation by RNA pol II is characterized by a distinct series of events : (1) recognition of core promoter elements by TFIID, (2) recognition of the TFIID-promoter complex by TFIIB and TFIIA, (3) recruitment of the TFIIF/pol II complex, (4) binding of TFIIE and TFIIH to complete the preinitiation complex, (5) promoter melting and formation of an "open" initiation complex, (6) synthesis of the nascent mRNA transcript, (7) release of pol II contact with the promoter ("promoter clearance"), and (8) elongation of the RNA transcript. Recently, the discovery that a subset of the GTFs (General Transcription Factor) exists in a preassembled form in an RNA pol II holoenzyme, suggests that the majority of the initiation machinery can bind to a promoter in a single step (RNA pol II holoenzyme model). The assembly of the GTFs is subject to regulation by activator and repressor proteins. Activators can recruit GTFs to a promoter, thereby accelerating the assembly process, whereas repressor proteins can inhibit transcription by blocking the assembly of GTFs. The mechanism of activator can function during multiple stages of transcription. These include (1) removal of repressor molecules from promoter DNA, (2) recruitment of GTFs and pol II to a promoter, (3) induction of conformation changes in the preinitiation complex, (4) induction of covalent modification of proteins in the preinitiation complex, and (5) stimulation of promoter clearance and elongation. These steps involve direct interactions between activators and GTFs. The condensation of eukaryotic DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. PHD-finger domain, a zinc finger-like motif, has a unique Cys4-His-Cys3 pattern, spanning approximately 50-80 residues. Generally the PHD-fingers are protein-protein interaction domains or that they recognize a set of similar nuclear targets related to chromatin structure and chromatin regulation, such as the differentially modified tails of the nucleosomal histones. We used the alpha-1 acid glycoprotein (AGP) gene as a model for studying transcriptional regulation. AGP is an acute responsive, liver-specific gene. Its expression is regulated by both positive and negative trans-acting factors. At least five cis elements have been identified within the 180-bp region of AGP promoter. Four of these sites, A, C, D and E are recognized by AGP/EBP or AGB/EBP-like factors in liver nuclear extracts. A, C, D and E regions are positive regulatory elements enhancing the expression of AGP gene while B motif is negative regulatory element repressing the expression of AGP gene. There are two parts in this thesis : (1) Biochemical purification and characterization of the negative transcription factors for AGP gene expression. The purified factor has been identified as nucleolin by amino acid analysis. Furthermore, we show that nucleolin can function as a transcription repressor for the AGP gene. (2) Identification, molecular cloning and biological characterization of a novel PHD-finger protein DPKAP (DNA-PK Activating Protein). DPKAP interacts with DNA-PK physically and functionally. DPKAP stimulates the DNA-PK kinase activity in vitro. Genetic evidence strongly suggests for the crucial role of DNA-PK in DNA double-strand break (DSB) repair and V(D)J recombination. However, the precise regulatory mechanism of DNA-PK in these processes remains to be elucidated. The potential in vivo functions of DPKAP on DNA DSB repair and V(D)J recombination remain to be investigated.

碩士<br>國立臺灣大學<br>生化科學研究所<br>85<br>cDNA encoding a PHD Finger protein, Requiem 6, was cloned. The expression of Requiem 6 mRNA was found in many tissues and cells .Requiem 6 is associated with a complex containing DNA -dependent protein kinase catalytic subunit (DNA-PKcs). Recently, patients have been identified with antibodies to the DNA-PKcs that appears to be associated with anti-Ku antibody. Anti-Requiem 6 auto-antibody was found to be present in patients of systemic lupus erythematosus, Sj" gren's syndrome , rheumatoid arthritis, asthma, palindromic syndrome and multiple sclerosis. The appearance of anti-Requiem 6 and anti-DNA-PKcs auto-antibodies in patients with autoimmune conditions is strikingly similar. These results suggest that Requiem 6 and DNA-PKcs exist in the same complex .

CCR5 is the major co-receptor for HIV-1 entry and an important target for drug development. The recent finding that hematopoietic stem cell [1] transplantation from a CCR5-negative donor to an HIV-infected patient produces long-term virus control in the absence of antiretroviral drugs suggests the potential of stem cell and gene therapies targeting CCR5. To this end, we are developing a protocol to knock-out CCR5 in a patient's own HSC using CCR5-targeted zinc finger nucleases (ZFNs) ZFNs are sequence specific proteins that generate a double-stranded break in DNA, which is converted into a gene-disrupting lesion by host repair processes. We have optimized the delivery and function of ZFNs targeting CCR5 in human CD34-positive HSC, achieving up to 27% disruption of CCR5 alleles. We confirmed that ZFN-treated HSC remain fully functional by transplanting a mouse model of human hematopoiesis, the NOD/SCID/IL2gammacnull (NSG) mouse, where the modified HSC retained the ability to differentiate into multiple hematopoietic lineages Humanized NSG mice are additionally capable of supporting HIV-1 infection. Following challenge with an R5-tropic virus, control animals demonstrated altered CD4:CD8 ratios, profound loss of human cells in the thymus and GALT, and high viral loads in multiple tissues sampled. In contrast, ZFN-treated animals had significantly lower acute viral loads and very low levels of virus in tissues by 10-12 weeks post-infection. At this stage the numbers of human cells in tissues that are targets for HIV-1 infection had normalized, including the GALT and thymus. FACS and PCR analysis revealed a rapid and dramatic selection for CCR5-negative cells in these populations. These findings demonstrate that ZFN-treated HSC can generate HIV-resistant cells in vivo that rapidly replace cells depleted by HIV-1 infection, and importantly, preserve GALT populations. Transient ZFN treatment resulting in permanent disruption of CCR5 in autologous HSC could therefore represent a viable clinical approach for HIV-infected patients<br>acase@tulane.edu

Thesis (Ph.D.)--State University of New York at Buffalo, 2009.<br>Title from PDF title page (viewed on Jan. 14, 2009) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Jacobi, Robert D. Includes bibliographical references.

Current discourse on photography turns, invariably, on questions of the camera's social, political, and aesthetic influence---does this new medium serve or subvert the status quo, and what power does it exert over other representational practices? Despite the recent publication of several full-length studies exploring the influence of photography on fiction, this is the first critical examination of the relationship between modern photography and modern American poetry, whose similarities have been noted frequently by practitioners of both arts Part One looks at the historical and theoretical similarities between poetry and photography in America from 1900 to the late 1920's. It examines how the photographers and poets who crafted American Modernism (namely, Pound, Stieglitz, Weston, Williams, Stevens, and Moore) forged a radical and complementary vision which helped push America to the forefront of the modern linguistic and visual arts Part Two extends the analysis through the early years of the Great Depression. Here I use Susan Sontag's assertion that 'Photography [is] a social rite, a defense against anxiety, and a tool of power' as a framework to discuss the changing aesthetic and political landscape against the backdrop of the poems of Hart Crane and the final photographs of Alfred Stieglitz. Part Three continues with an examination of the 'witness culture' of the mid- to late-1930's and a critical analysis of Walker Evans's documentary photographs alongside the proletarian prose-poetry of James Agee in the defining 'document' of the Depression era, Let Us Now Praise Famous Men Part Four explores in detail the work of four successful women poets and photographers of the 1920's and '30's (Edna St. Vincent Millay, Margaret Bourke-White, Dorothea Lange, and Marianne Moore). Drawing on what feminist critics Sandra Gilbert and Susan Gubar have coined 'strategies of female-female impersonation' (the deliberate construction of stereotypical 'masks' behind which women artists of the period might work with relative autonomy), I examine how each was able, despite the limiting gaze of a patriarchal cultural and critical establishment, to inscribe her name on the 'blank page' of the New On the whole, this analysis explores the historical, theoretical, and aesthetic forces which helped to forge an alliance between photography and poetry during the high modernist period in America---an alliance which persists to this day. It examines the relation between still photographs and printed words in light of classical oppositions between image and word, between art and the body, articulated by contemporary cultural, semiotic, and feminist critics, and in the belief that understanding this relationship can help us perceive how art grows out of experience and how it transposes history's 'decisive moments' into forms which continue to be haunted, empowered, and sometimes subverted by them<br>acase@tulane.edu

My dissertation discusses the theoretical dispute over coherence and incoherence in The Cantos by analysing Pound's use of the ideogrammic method and the visual art allusions in the poem. I analyse the ideogrammic method and some modern developments, such as cubism, cinematic montage and linguistics. Even though Pound's intention was to enact the ideogrammic method in his Cantos, as he approaches the Pisan Cantos the poet seems unable to pursue his quest; after this sequence the poem seems to crumble. The foundation of the poem turns more undecidable, and closer to the concept of 'mise en abyme.' In the ideogram the relation of two things creates a new concept, a synthesis; in 'mise en abyme,' however, the juxtaposition of two fragments does not produce a new concept, but more disjunctions. In 'mise en abyme' an image is reflected in a mirror, and then another mirror, 'ad infinitum,' as in the painting Las Meninas, by Velazquez In Chapter One I discuss the concepts of closure and coherence and the ideogrammic method In Chapter Two I test my theory of coherence in the ideogrammic method and analyse some Pisan Cantos that I consider modal points in the poem In Chapter Three I compare the technique of some paintings alluded to in the poem with the technique of the poem itself. Though Pound's writings show his preference for the Vorticists, his technique seems to reflect the fragmentation of the Cubists, and the 'aporia' of Velazquez's 'mise en abyme' in Las Hilanderas and Las Meninas I conclude that Pound is a true representative of the modern temper, but that he becomes a weaver, a precursor of the Postmodern era, as his text is fragmentary, highly allusive, non-closural, palimpsestic and so unstable that the poet himself, like Arachne in the myth, is unable to stop weaving his web. Pound's search for order actually appears to be related to the etymology of this word, since 'ordiri' means to begin a web or to prepare to weave a continuous web<br>acase@tulane.edu

The purpose of this dissertation is the exploration of the relationship between painting, music, and literature in some selected novels of Vicente Blasco Ibanez. The fundamental similarity between the two arts and literature is established through the similar function of their basic units. Colors, notes, and words are considered the raw materials or signs by which the artists, the composers, and the writer create their works. It is by looking at the relationships of painting, music, and literature from this viewpoint of structure that the exploration of the ways in which the three disciplines interact as sign systems is made A conceptualization of an interdisciplinary sign which combines painting and literature is presented in four of Blasco Ibanez's novels: La barraca, Flor de mayo, Canas y barro, and La maja desnuda. The common ground of music and literature is established in three of his novels: Entre naranjos, Arroz y tartana and La catedral. In the novels, the manner in which a pictoric sign or a musical sign can operate within the literary text, is examined. In attempting to define how these signs can operate within the literary text, the distinction established by Roman Jakobson between two axis along which a linguistic sign may function is used. A vertical or metaphoric axis of selection and a horizontal or metonymic axis of combination are demonstrated in each novel. The metaphoric use of painting and music in the novels is studied as a substitution of one co-existent idea for another. The metonymic axis relies on the association and combination of painting and music and the narrative of the literary works The literary utilization of painting is presented through the works of Goya, Benlliure, Sorolla, and Picasso. The weaving of music into the texts is established through the music of Wagner, Berlioz and Beethoven Rather than relying on the separation of the arts, the main focus of the dissertation relies in their union. The novel becomes infused with, rather than being a referent for, painting or music<br>acase@tulane.edu

Excessive PM2.5 emissions in China threaten peoples’ health and cause massive economic burdens to society. Under pressure from the public, and the international community, China published PM2.5 standards for the first time in March 2012. Following the introduction of standards, several pilot cities began to build PM2.5 monitoring networks. This paper is designed to explore whether PM2.5 monitoring can be effectively undertaken and implemented in China and whether monitoring results can offer a technical basis to facilitate a significant reduction in actual PM2.5 emissions and protect public health. PM2.5 monitoring is essential in helping the government and public monitor pollution levels and supervise local compliance with PM2.5 standards. Key aspects to facilitate an effective monitoring process are discussed in the analysis. In addition, a case study – Lanzhou’s PM2.5 monitoring network – is provided to analyze and improve current PM2.5 monitoring practices at local levels, as well as suggest credible technical support to local authorities so as to cut PM2.5 emissions levels. Based on detailed analysis, the results suggest that PM2.5 monitoring can be successfully implemented in China by following several key principles – designing a representative PM2.5 monitoring network, applying QA/QC to ensure data quality, interpreting the data scientifically to understand real pollution levels, etc. In addition, this paper recommends three measures critical to realizing PM2.5 reduction goals: (1) emissions source control, (2) public participation to add input to the decision-making process and supervise local compliance with PM2.5 standards, and (3) non-governmental organization/international cooperation to improve local government and environmental agencies’ capacity with regards to environmental protection. Lessons derived from the case study can help improve PM2.5 monitoring performance not just in Lanzhou, but in cities that share similar monitoring issues across China. Scientific monitoring, together with the application of the above three measures, can more effectively curb PM2.5 emissions, improve air quality, and mitigate negative health effects associated with air pollution.<br>acase@tulane.edu

At the end of the Thirties and during the following years, we find in Cuba a socio-political and cultural atmosphere very different from that of the previous period. The revolution of the Thirties succeeded in overthrowing Machado, but at the same time the achievement of its deeper social goals was frustrated. A spirit of demoralization dominated the Cuban political scene and a group of writers and artists of the Third Generation of the Republic started to perceive politics as a meaningless comedy in which they did not want to participate. Simultaneously, an increase in the involvement of the United States in Cuban domestic affairs caused those creators to believe that they were experiencing a period of progressive disintegration of their national identity Disappointed with the political options available, they began to conceive of 'culture' as the only feasible way to save national dignity and awareness. For those creators, culture was the only real aspect of an otherwise disheartening socio-political farce. Culture was also a refuge in which the nation could survive until better political circumstances and new incorruptible leaders would emerge. Culture, for them, had an essentially ethical and holistic meaning. Thus, their cultural undertaking could be considered a chapter in the history of Cuban ethics, as Cintio Vitier pointed out in his Ese sol del mundo moral. This ethical perspective is the most appropriate for our understanding of this group, and constitutes the nucleus that gives meaning to the operational structure of the group's various artistic and literary reviews which reveal their conformity as a whole. Another factor in the group's uniformity was the presence of a leader, Jose Lezama Lima, who eventually became an ethical model The group is known as 'Origenes' and includes writers, musicians, painters, sculptors, and art critics. Its reviews are Verbum (1937), Espuela de Plata (1939-1941), Clavileno (1942-1944), Nadie Parecia (1942-1943), Poeta (1943), and Origenes (1944-1956) The object of this dissertation is to bring into the foreground the ethical holistic meaning of the 'Origenes' group, as reflected in its reviews, particularly in Origenes as a culmination of the process begun by Verbum<br>acase@tulane.edu