Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Effect of gamma-irradiation on total organic carbon and trihalomethane formation potential

This research was conducted to study the use of radiation in water treatment as an alternative to chlorination which has caused health concerns due to the formation of harmful disinfection by-products. Groundwater solutions from the Biscayne aquifer were radiated with Cobalt-60 gamma radiation and studied for changes in dissolved organic carbon (DOC), UV absorbance at 254 nm (UV254), fluorescence and trihalomethane formation potential (THMFP). Molecular fractionations were conducted by ultrafiltration. Effect of the combination of radiation/peroxide was studied for DOC and UV254. Radiation showed significant removal in DOC and THMFP. Similar results were seen in the fluorescence and UV absorbance experiments. Radiation/peroxide did not improve the DOC removal. Radiation of the groundwater samples broke the larger molecular weight fractions in to smaller fractions.

Instantaneous photoinduced patterning of an azopolymer colloidal nanosphere assembly

Colloidal azopolymer nanospheres assembled on a glass substrate were exposed to a single collimated laser beam. The combination of photo-fluidic elongation of the spherical colloids and light induced self-organization of the azopolymer film allows the quasiinstantaneous growth of a large amplitude surface relief grating. Pre-structuration of the sample with the nanosphere assembly supports faster creation of the spontaneous pattern. Confinement into the nanospheres provides exceptionally large modulation amplitude of the spontaneous relief. The method is amenable to any kind of photoactive azo-materials.

Synthesis of novel organic semiconductors for optoelectronic devices

This thesis describes the synthesis and characterisation of novel conjugated organic materials with optoelectronic application. The first chapter provides an introduction about organic semiconductors and in particular about their working principle from a physical and chemical point of view. An overview of the most common types of solar cells is provided, including examples of some of the best performing materials. The second chapter describes the synthesis of a new library of flavin derivatives as potential active materials for optoelectronic applications. Flavins are natural redox-active molecules, which show potential application in optoelectronics, thanks to their stability and versatility. FPF-Flavins, for instance, could be used either as acceptor units in push-pull polyconjugated systems or as acceptor unit in dyes for DSSCs. In the same chapter a first attempt of synthesising bis-flavins to be used as N-type semiconductors in BHJ devices is described. The third chapter describes the successful synthesis and characterization of a series of conjugated organic molecules based on the benzothiadiazole moiety. Among these, three molecules containing ferrocene as donor unit were tested as sensitizers for DSSCs, reporting a PCE of 0.3% as the best result. Further studies indicated a significant problem of charge recombination which limits the performance. A near-infrared absorbing push-pull polymer, based on BbT as acceptor unit, was also synthesised and tested in BHJ devices as P-type semiconductor in blend with PC71BM, showing a VOC of 0.71 V. Finally, the last chapter describes the synthesis of several tetrathiafulvalene derivatives in order to explore this moiety as donor unit in dyes for DSSCs and as HTM for perovskite-based solar cells. In particular, two very simple dyes were synthesised and implemented in DSSCs reporting a PCE 0.2% and 0.4%, respectively. The low efficiency was associated to the tendency to aggregate at the solid state, with the absorption shifting from the visible to the infrared range. A conjugated molecule, containing a DPP core, was also synthesised and tested as HTM for perovskite solar cells. The best reported PCE of 7.7% was obtained without any additives. A case study about dehalogenation and “halogen dance” in TTF iodide is also presented.

Peptide-based low molecular weight gelators for the preparation of self-assembled materials

Low molecular weight gelators (LMWGs) based on pseudo-peptides are here studied for the preparation of supramolecular materials. These compounds can self-assemble through non-covalent interactions such as hydrogen bonds and π-π stacking, forming fibres and gels. A wide variety of materials can be prepared starting from these building blocks, which can be tuned and functionalised depending on the application. In this work, derivatives of the three aromatic amino acids L-Phenylalanine, L-Tyrosine and L-DOPA (3,4-dihydroxiphenylalanine) were synthesised and tested as gelators for water or organic solvents. First, the optimal gelating conditions were studied for each compound, varying concentration, solvent and trigger. Then the materials were characterised in terms of mechanical properties and morphology. Water remediation from dye pollution was the first focus of this work. Organogels were studied as absorbent of dyes from contaminated water. Hydrogels functionalised with TiO2 nanoparticles and graphene platelets were proposed as efficient materials for the photo-degradation of dyes. An efficient method for the incorporation of graphene inside hydrogels using the gelator itself as dispersant was proposed. In these materials a high storage modulus coexists with good self-healing and biocompatibility. The incorporation of a mineral phase inside the gel matrix was then investigated, leading to the preparation of composite organic/inorganic materials. In a first study, the growth of calcium carbonate crystals was achieved inside the hydrogel, which preserved its structure after crystal formation. Then the self-assembled fibres made of LMWGs were used for the first time instead of the polymeric ones as reinforcement inside calcium phosphate cements (CPCs) for bone regeneration. Gel-to-crystal transitions occurring with time in a metastable gel were also examined. The formation of organic crystals in gels can be achieved in multicomponent systems, in which a second gelator constitutes the independent gel network. Finally, some compounds unable to gelate were tested as underwater adhesives.

I) Improving low photocurrent of pseudorotaxane based solar cell II) Synthesis and characterization of new Fe(0) cyclopentadienone complexes

Next to conventional solar panels that harvest direct sunlight, p-type dye-sensitized solar cells (DSSCs) have been developed, which are able to harvest diffuse sunlight. Due to unwanted charge recombination events p-type DSSCs exhibit low power conversion efficiencies (PCEs). Previous research has shown that dye-redox mediator (RM) interactions can prevent these recombination events, resulting in higher PCEs. It is unknown how the nature of dye-RM interactions affects the PCEs of pseudorotaxane-based solar cells. In this research this correlation is investigated by comparing one macrocycle, the 3-NDI, in combination with the three dyes that contains a recognition sites. 2D-DOSY-NMR experiments have been conducted to evaluate the diffusion constants (LogD) of the three couple. The research project has been stopped due to the coronavirus pandemic. The continuation of this thesis would have been to synthesize a dye on the basis of the data obtained from the diffusion tests and attempt the construction of a solar cell to then evaluate its effectiveness. During my training period I synthetized new Fe(0) cyclopentadienone compounds bearing a N-Heterocyclic Carbene ligand. The aim of the thesis was to achieve water solubility by modifications of the cyclopentadienone ligand. These new complexes have been modified using a sulfonation reaction, replacing an hydroxyl with a sulfate group, on the alkyl backbone of the cyclopentadienone ligand. All the complexes were characterized with IR, ESI-MS and NMR spectroscopy, and a new Fe(0) cyclopentadienone complex, involved as an intermediate, was obtained as a single crystal and was characterized also with X-Ray spectroscopy.

Design of luminescent metal complexes for polymer science

The program of my PhD studies has been dealing with the investigation of the research outcomes that may result from the use of luminescent Iridium(III) cyclometalated complexes in the field of polymer science. In particular, my activity has been focused on exploring two main applicative contexts, i.e. Ir(III) complexes for preparing polymers and in combination with polymers. In the first part, a new set of luminescent Ir(III) complexes was exploited as photocatalysts for light-assisted atom transfer radical polymerization of methyl methacrylate. The decoration of both cyclometalated and ancillary ligands with sp3 hybridized nitrogen substituents together with the use of specific counterions, imparted suitable photophysical and redox properties for an efficient photocatalyzed process. The second part has been focused on the employment of Ir(III) tetrazole complexes as phosphorescent dyes in polymeric materials. Colourless luminescent solar concentrators were prepared blending two Ir(III) cyclometalates with acrylate polymers. Their performances were investigated, leading to promising outcomes comparable, or superior, to those obtained from colourless LSCs based on organic fluorophores. As a complementary approach, Ir(III) complexes were covalently linked to polymers in the side chain, to obtain a new class of metallopolymers. To this extent, a bifunctional tetrazolate molecule, equipped with a coordination site and a polymerizable unit, was designed. The photophysical properties of the resultant luminescent polymeric films were discussed. In the end, an additional project involving both polymers and metal compounds was carried out during my experience as a visiting PhD student at Humboldt – University of Berlin. Polystyrene and polyethylene glycol -based ion-exchange resins were functionalized with peptides through a ligation pathway, for the selective chelation of Copper(II) in aqueous solutions. The coordinating capability of the materials towards Cu2+ ions was tested by ICP-MS analysis. The resins strategically modified with ion-selective peptides, may be exploited in the preparation of water-processing devices.

Nanocomposites based on poly(caprolactone)-chitosan electrospun nanofibers and metallic oxide nanoparticles. Their potential use to adsorption and photocatalytic degradation of organic contaminants

Pollution of water bodies is one of the most common environmental problems today. Organic pollutants are one of the main drawbacks in this natural resource, among which the following stand out long-lived dyes, pharmaceuticals, and pesticides. This research aims at obtaining nanocomposites based on polycaprolactone-chitosan (PCL-CS) electrospun nanofibers (NFs) containing TiO2 nanoparticles (NPs) for the adsorption and photocatalytic degradation of organic pollutants, using Rhodamine B as a model. The fabricated hybrid materials were characterized by FT-IR, TGA, DSC, SEM, TEM, tensile properties, and the contact angle of water drops. The photoactivity of the NFs was investigated using a batch-type system by following UV-Vis absorbance and fluorescence of rhodamine B (RhB). For this purpose, TiO2NPs were successfully ex-situ incorporated into the polymer matrix promoting good mechanical properties and higher hydrophilicity of the material. The results showed that CS in the NFs increased the absorption and degradation of RhB by the TiO2NPs. CS attracted the pollutant molecules to the active sites vicinity of TiO2NPs, favoring initial adsorption and degradation. In other words, a bait-hook-and-destroy effect was evidenced. It also was demonstrated that the sensitization of TiO2 by organic dyes (e.g., perylene derivative) considerably improves the photocatalytic activity under visible radiation, allowing the use of low amounts of TiO2. (≈0.05 g/1 g of fiber). Hence, the current study is expected to contribute with an environmentally friendly green alternative solution.

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

The Clinical Impact Of Cerebellar Grey Matter Pathology In Multiple Sclerosis.

The cerebellum is an important site for cortical demyelination in multiple sclerosis, but the functional significance of this finding is not fully understood. To evaluate the clinical and cognitive impact of cerebellar grey-matter pathology in multiple sclerosis patients. Forty-two relapsing-remitting multiple sclerosis patients and 30 controls underwent clinical assessment including the Multiple Sclerosis Functional Composite, Expanded Disability Status Scale (EDSS) and cerebellar functional system (FS) score, and cognitive evaluation, including the Paced Auditory Serial Addition Test (PASAT) and the Symbol-Digit Modalities Test (SDMT). Magnetic resonance imaging was performed with a 3T scanner and variables of interest were: brain white-matter and cortical lesion load, cerebellar intracortical and leukocortical lesion volumes, and brain cortical and cerebellar white-matter and grey-matter volumes. After multivariate analysis high burden of cerebellar intracortical lesions was the only predictor for the EDSS (p<0.001), cerebellar FS (p = 0.002), arm function (p = 0.049), and for leg function (p<0.001). Patients with high burden of cerebellar leukocortical lesions had lower PASAT scores (p = 0.013), while patients with greater volumes of cerebellar intracortical lesions had worse SDMT scores (p = 0.015). Cerebellar grey-matter pathology is widely present and contributes to clinical dysfunction in relapsing-remitting multiple sclerosis patients, independently of brain grey-matter damage.

Dna Extraction From Filter-paper Spots Of Vaginal Samples Collected After Sexual Violence.

To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human β-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. β-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

Analysis Of The Dna Fourier Transform-infrared Microspectroscopic Signature Using An All-reflecting Objective.

The Fourier transform-infrared (FT-IR) signature of dry samples of DNA and DNA-polypeptide complexes, as studied by IR microspectroscopy using a diamond attenuated total reflection (ATR) objective, has revealed important discriminatory characteristics relative to the PO2(-) vibrational stretchings. However, DNA IR marks that provide information on the sample's richness in hydrogen bonds have not been resolved in the spectral profiles obtained with this objective. Here we investigated the performance of an all reflecting objective (ARO) for analysis of the FT-IR signal of hydrogen bonds in DNA samples differing in base richness types (salmon testis vs calf thymus). The results obtained using the ARO indicate prominent band peaks at the spectral region representative of the vibration of nitrogenous base hydrogen bonds and of NH and NH2 groups. The band areas at this spectral region differ in agreement with the DNA base richness type when using the ARO. A peak assigned to adenine was more evident in the AT-rich salmon DNA using either the ARO or the ATR objective. It is concluded that, for the discrimination of DNA IR hydrogen bond vibrations associated with varying base type proportions, the use of an ARO is recommended.

Antiangiogenic And Finasteride Therapies: Responses Of The Prostate Microenvironment In Elderly Mice.

The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416+TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-β and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-β reactivity were verified in the finasteride, SU5416 groups and especially in SU5416+TNP-470 group. The TNP-470 group showed reduced AR and ER-β protein levels. The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416+TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-β.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Changes In The Bacterial Community Of Soil From A Neutral Mine Drainage Channel.

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.