Photophysics of Carbon Nanotubes: from Dispersion to Supramolecular Systems

The aim of this PhD thesis is the investigation of the photophysical properties of materials that can be exploited in solar energy conversion. In this context, my research was mainly focused on carbon nanotube-based materials and ruthenium complexes. The first part of the thesis is devoted to carbon nanotubes (CNT), which have unique physical and chemical properties, whose rational control is of substantial interest to widen their application perspectives in many fields. Our goals were (i) to develop novel procedures for supramolecular dispersion, using amphiphilic block copolymers, (ii) to investigate the photophysics of CNT-based multicomponent hybrids and understand the nature of photoinduced interactions between CNT and selected molecular systems such as porphyrins, fullerenes and oligo (p-phynylenevinylenes). We established a new protocol for the dispersion of SWCNTs in aqueous media via non-covalent interactions and demonstrated that some CNT-based hybrids are suitable for testing in PV devices. The second part of the work is focussed on the study of homoleptic and heteroleptic Ru(II) complexes with bipyridine and extended phenanthroline ligands. Our studies demonstrated that these compounds are potentially useful as light harvesting systems for solar energy conversion. Both CNT materials and Ru(II) complexes have turned out to be remarkable examples of photoactive systems. The morphological and photophysical characterization of CNT-based multicomponent systems allowed a satisfactory rationalization of the photoinduced interactions between the individual units, despite several hurdles related to the intrinsic properties of CNTs that prevent, for instance, the utilization of laser spectroscopic techniques. Overall, this work may prompt the design and development of new functional materials for photovoltaic devices.

Targeting the conversion of ceramide to sphingosine 1-phosphate as a novel strategy for cancer therapy

Sphingolipids not only function as structural components of cell membranes but also act as signaling molecules to regulate fundamental cellular responses, such as cell death and differentiation, proliferation and certain types of inflammation. Particularly the cellular balance between ceramide and sphingosine 1-phosphate seems to be crucial for a cell's decision to either undergo apoptosis or proliferate, two events which are implicated in tumor development and growth. Whereas ceramide possesses proapoptotic capacity in many cell types, sphingosine 1-phosphate acts as a counterplayer able to induce cell proliferation and protect cells from undergoing apoptosis. Therefore, tipping the balance in favour of ceramide production, i.e. by inhibiting ceramidase or sphingosine kinase activities has potential to support its proapoptotic action and hence represents a promising rational approach to effective cancer therapy. This review highlights most recent data on the regulation of cellular sphingolipid formation and their potential implication in tumor development, and provides perspectives for their use as targets in molecular intervention therapy.

Conversion of myoblasts to physiologically active neuronal phenotype.

Repressor element 1 (RE1)-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) can repress several terminal neuronal differentiation genes by binding to a specific DNA sequence (RE1/neuron-restrictive silencer element [NRSE]) present in their regulatory regions. REST-VP16 binds to the same RE1/NRSE, but activates these REST/NRSF target genes. However, it is unclear whether REST-VP16 expression is sufficient to cause formation of functional neurons either from neural stem cells or from heterologous stem cells. Here we show that the expression of REST-VP16 in myoblasts grown under muscle differentiation conditions blocked entry into the muscle differentiation pathway, countered endogenous REST/NRSF-dependent repression, activated the REST/NRSF target genes, and, surprisingly, activated other neuronal differentiation genes and converted the myoblasts to a physiologically active neuronal phenotype. Furthermore, in vitro differentiated neurons produced by REST-VP16-expressing myoblasts, when injected into mouse brain, survived, incorporated into the normal brain, and did not form tumors. This is the first instance in which myoblasts were converted to a neuronal phenotype. Our results suggest that direct activation of REST/NRSF target genes with a single transgene, REST-VP16, is sufficient to activate other terminal neuronal differentiation genes and to override the muscle differentiation pathways, and they suggest that this approach provides an efficient way of triggering neuronal differentiation in myoblasts and possibly other stem cells.

Radiocarbon Analysis of Elemental and Organic Carbon in Switzerland during Winter-Smog Episodes from 2008 to 2012 – Part I: Source Apportionment and Spatial Variability

While several studies have investigated winter-time air pollution with a wide range of concentration levels, hardly any results are available for longer time periods covering several winter-smog episodes at various locations; e.g., often only a few weeks from a single winter are investigated. Here, we present source apportionment results of winter-smog episodes from 16 air pollution monitoring stations across Switzerland from five consecutive winters. Radiocarbon (14C) analyses of the elemental (EC) and organic (OC) carbon fractions, as well as levoglucosan, major water-soluble ionic species and gas-phase pollutant measurements were used to characterize the different sources of PM10. The most important contributions to PM10 during winter-smog episodes in Switzerland were on average the secondary inorganic constituents (sum of nitrate, sulfate and ammonium = 41 ± 15%) followed by organic matter (OM) (34 ± 13%) and EC (5 ± 2%). The non-fossil fractions of OC (fNF,OC) ranged on average from 69 to 85 and 80 to 95% for stations north and south of the Alps, respectively, showing that traffic contributes on average only up to ~ 30% to OC. The non-fossil fraction of EC (fNF,EC), entirely attributable to primary wood burning, was on average 42 ± 13 and 49 ± 15% for north and south of the Alps, respectively. While a high correlation was observed between fossil EC and nitrogen oxides, both primarily emitted by traffic, these species did not significantly correlate with fossil OC (OCF), which seems to suggest that a considerable amount of OCF is secondary, from fossil precursors. Elevated fNF,EC and fNF,OC values and the high correlation of the latter with other wood burning markers, including levoglucosan and water soluble potassium (K+) indicate that residential wood burning is the major source of carbonaceous aerosols during winter-smog episodes in Switzerland. The inspection of the non-fossil OC and EC levels and the relation with levoglucosan and water-soluble K+ shows different ratios for stations north and south of the Alps (most likely because of differences in burning technologies) for these two regions in Switzerland.

Groundfish survey data from the German Bight from 1902-08, 1919-23, and 1930-1932 and ICES International Bottom Trawl Survey (IBTS) quarter 3 data from 1991 to 2009

Groundfish survey data from the German Bight from 1902-08, 1919-23, and 1930-1932 and ICES International Bottom Trawl Survey (IBTS) quarter 3 data from 1991 to 2009 were analysed with respect to species frequencies, maximum length, trends in catch-per-unit-effort, species richness parameters (SNR) and presence of large fish (Phi40), the latter defined as average presence of species per haul with specimens larger than 40 cm given. Four different periods are distinguished: (a) before 1914 with medium commercial CPUE and low landings, Phi40 approx. 2, high abundance in elasmobranchs and SNR conditions indicating highly diverse assemblages, (b) conditions immediately after 1918 with higher commercial CPUE, recovering landings, Phi40 at > 4 in 1919, and SNR conditions indicating highly diverse assemblages, (c) conditions from 1920 to the early 1930's with decreasing commercial CPUE, increased landings, decreasing Phi40, SNR conditions similar to later years indicating less diverse assemblages, and a decrease in elasmobranchs. In the IBTS series (d), Phi40 remains low indicating an increased rarity of large specimens, and SNR characteristics are similar to the third period. Dab, whiting and grey gurnard have increased considerably in the IBTS series as compared to the historic data. Phi40 is suggested an alternative indicator reflecting community functional diversity when weight based indicators cannot be applied.

Inluence of source and level of glycerin in the diet on growth performance, liver characteristics, and nturient digestibility in broilers from hatching to 21 days of age

The influence of source and level of inclusion of raw glycerin (GLYC) in the diet on growth performance, digestive traits, total tract apparent retention (TTAR), and apparent ileal digestibility of nutrients was studied in broilers from 1 to 21 d of age. There was a control diet based on corn and soybean meal and 8 additional diets that formed a 2 × 4 factorial with 2 sources of GLYC and 4 levels of inclusion (2.5, 5.0, 7.5, and 10%). The GLYC used were obtained from the same original batch of soy oil that was dried under different processing conditions and contained 87.5 or 81.6% glycerol, respectively. Type of processing of the GLYC did not affect any of the variables studied except DM and organic matter retention (P < 0.05) that was higher for the 87.5% glycerol diet. From d 1 to 21, feed conversion ratio (FCR) improved linearly (L, P ≤ 0.01) as the GLYC content of the diet increased, but ADG was not affected. On d 21, the relative weight (% BW) of the liver and the digestive tract increased (L, P < 0.01) as the level of GLYC in the diet increased, but lipid concentration in the liver was not affected. The TTAR of DM and organic matter increased quadratically (Q, P < 0.05) and the AMEn content of the diet increased linearly (L, P < 0.01) with increases in dietary GLYC. Also, the apparent ileal digestibility of DM (L, P < 0.05; Q, P = 0.07) and gross energy (L, P < 0.01) increased as the GLYC content of the diet increased. It is concluded that raw GLYC from the biodiesel industry can be used efficiently, up to 10% of the diet, as a source of energy for broilers from 1 to 21 d of age and that the energy content of well-processed raw GLYC depends primarily on its glycerol content.

Productive performance of brown-egg laying pullets from hatching to 5 weeks of age as affected by fiber inclusion, feed form, and energy concentration of the diet

The effects of fiber inclusion, feed form, and energy concentration of the diet on the growth performance of pullets from hatching to 5 wk age were studied in 2 experiments. In Experiment 1, there was a control diet based on cereals and soybean meal, and 6 extra diets that included 2 or 4% of cereal straw, sugar beet pulp (SBP), or sunflower hulls (SFHs) at the expense (wt/wt) of the whole control diet. From hatching to 5 wk age fiber inclusion increased (P < 0.05) ADG and ADFI, and improved (P < 0.05) energy efficiency (EnE; kcal AMEn/g ADG), but body weight (BW) uniformity was not affected. Pullets fed SFH tended to have higher ADG than pullets fed SBP (P = 0.072) with pullets fed straw being intermediate. The feed conversion ratio (FCR) was better (P < 0.05) with 2% than with 4% fiber inclusion. In Experiment 2, 10 diets were arranged as a 2×5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of AMEn (2,850, 2,900, 2,950, 3,000, and 3,050 kcal/kg). Pullets fed crumbles were heavier and had better FCR than pullets fed mash (P < 0.001). An increase in the energy content of the crumble diets reduced ADFI and improved FCR linearly, but no effects were detected with the mash diets (P < 0.01 and P < 0.05 for the interactions). Feeding crumbles tended to improve BW uniformity at 5 wk age (P = 0.077) but no effects were detected with increases in energy concentration of the diet. In summary, the inclusion of moderate amounts of fiber in the diet improves pullet performance from hatching to 5 wk age. The response of pullets to increases in energy content of the diet depends on feed form with a decrease in feed intake when fed crumbles but no changes when fed mash. Feeding crumbles might be preferred to feeding mash in pullets from hatching to 5 wk age.

Inclusion of fiber in diets for brown-egg laying pullets: Effects on growth performance and digestive tract traits from hatching to 17 weeks of age

We investigated the effects of fiber inclusion in the diet on growth performance and digestive traits in pullets from hatching to 17 wk of age. The control diets of the 3 feeding periods (0 to 5 wk, 5 to 10 wk, and 10 to 17 wk) were based on corn and soybean meal and did not include any additional fiber source. The experimental diets included 2 or 4% of cereal straw or sugar beet pulp (SBP) at the expense (wt:wt) of the control diet. From 0 to 5 wk of age, fiber inclusion did not affect pullet performance. From hatch to 17 wk of age, the inclusion of straw had little effect on pullet performance but the inclusion of 4% SBP reduced (ADG) (P < 0.05) and reduced feed conversion ratio (FCR; P < 0.001). Pullets fed straw had greater ADG (P < 0.05) and better energy conversion ratio (P < 0.01) than pullets fed SBP. An increase in fiber from 2 to 4% reduced FCR (P < 0.05). Body weight uniformity was not affected by diet. Fiber inclusion increased the relative weight (% BW) of the gizzard at 5 wk (P = 0.056) and 10 wk (P < 0.01) of age, but no differences were detected between fiber sources. At same ages, the relative length (cm/kg BW) of the pullets (P = 0.058 and P < 0.01, respectively) and tarsus (P = 0.079 and P < 0.05, respectively) was higher in pullets fed SBP than in pullets fed straw. Fiber inclusion, however, did not affect any of these traits at 17 wk of age. In summary, the inclusion of 2% straw at the expense (wt:wt) of the whole diet did not affect pullet performance at 17 wk of age. An increase in the level of straw from 2 to 4% reduced FCR but did not affect ADG. The inclusion of SBP, however, reduced pullet growth, with effects being more pronounced at the higher level.

Feed form and energy concentration of the diet affect growth performance and digestive tract traits of brown-egg laying pullets from hatching to 17 weeks of age

The influence of feed form and energy concentration of the diet on growth performance and the development of the gastrointestinal tract (GIT) was studied in brown-egg laying pullets. Diets formed a 2 x 5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of energy differing in 50 kcal AMEn/kg. For the entire study (0 to 17 wk of age) feeding crumbles increased ADFI (52.9 vs. 49.7 g; P < 0.001) and ADG (12.7 vs. 11.6 g; P < 0.001) and improved feed conversion ratio (FCR; 4.18 vs. 4.27; P < 0.001). An increase in the energy content of the diet decreased ADFI linearly (P < 0.001) and improved FCR quadratically (P < 0.01) but energy intake (kcal AMEn/d) was not affected. BW uniformity was higher (P < 0.05) in pullets fed crumbles than in those fed mash but was not affected (P > 0.05) by energy content of the diet. At 5, 10, and 17 wk of age, the relative weight (RW, % BW) of the GIT and the gizzard, and gizzard digesta content were lower (P < 0.05 to P < 0.001) and gizzard pH was higher (P < 0.05 to P < 0.001) in pullets fed crumbles than in pullets fed mash. Energy concentration of the diet did not affect any of the GIT variables studied. In summary, feeding crumbles improved pullet performance and reduced the RW of the GIT and gizzard, and increased gizzard pH at all ages. An increase in the energy content of the diet improved FCR from 0 to 17 wk of age. The use of crumbles and the increase in the AMEn content of the diet might be used adventageously when the objetive is to increase the BW of the pullets. However, crumbles affected the development and weight of the organs of the GIT, which might have negative effects on feed intake and egg production at the beginning of the egg laying cycle.

Del plan a la realidad: gobernanza y toma de decisiones en los procesos de regeneración urbana. El caso de Boston = From plan to reality: governance and decision-making in urban regeneration processes in Boston

En las últimas tres décadas, las dinámicas de restructuración económica a nivel global han redefinido radicalmente el papel de las ciudades. La transición del keynesianismo al neoliberalismo ha provocado un cambio en las políticas urbanas de los gobiernos municipales, que han abandonado progresivamente las tareas de regulación y redistribución para centrarse en la promoción del crecimiento económico y la competitividad. En este contexto, muchas voces críticas han señalado que la regeneración urbana se ha convertido en un vehículo de extracción de valor de la ciudad y está provocando la expulsión de los ciudadanos más vulnerables. Sin embargo, la regeneración de áreas consolidadas supone también una oportunidad de mejora de las condiciones de vida de la población residente, y es una política necesaria para controlar la expansión de la ciudad y reducir las necesidades de desplazamiento, promoviendo así ciudades más sostenibles. Partiendo de la hipótesis de que la gobernanza de los procesos de regeneración urbana es clave en el resultado final de las operaciones y determina el modelo de ciudad resultante, el objetivo de esta investigación es verificar si la regeneración urbana es necesariamente un mecanismo de extracción de valor o si puede mejorar la calidad de vida en las ciudades a través de la participación de los ciudadanos. Para ello, propone un marco de análisis del proceso de toma de decisiones en los planes de regeneración urbana y su impacto en los resultados de los planes, tomando como caso de estudio la ciudad de Boston, que desde los años 1990 trata de convertirse en una “ciudad de los barrios”, fomentando la participación ciudadana al tiempo que se posiciona en la escena económica global. El análisis se centra en dos operaciones de regeneración iniciadas a finales de los años 1990. Por un lado, el caso de Jackson Square nos permite comprender el papel de la sociedad civil y el tercer sector en la regeneración de los barrios más desfavorecidos, en un claro ejemplo de urbanismo “desde abajo” (bottom-up planning). Por otro, la reconversión del frente marítimo de South Boston para la construcción del Distrito de Innovación nos acerca a las grandes operaciones de regeneración urbana con fines de estímulo económico, tradicionalmente vinculadas a los centros financieros (downtown) y dirigidas por las élites gubernamentales y económicas (la growth machine) a través de procesos más tecnocráticos (top-down planning). La metodología utilizada consiste en el análisis cualitativo de los procesos de toma de decisiones y la relación entre los agentes implicados, así como de la evaluación de la implementación de dichas decisiones y su influencia en el modelo urbano resultante. El análisis de los casos permite afirmar que la gobernanza de los procesos de regeneración urbana influye decisivamente en el resultado final de las intervenciones; sin embargo, la participación de la comunidad local en la toma de decisiones no es suficiente para que el resultado de la regeneración urbana contrarreste los efectos de la neoliberalización, especialmente si se limita a la fase de planeamiento y no se extiende a la fase de ejecución, y si no está apoyada por una movilización política de mayor alcance que asegure una acción pública redistributiva. Asimismo, puede afirmarse que los procesos de regeneración urbana suponen una redefinición del modelo de ciudad, dado que la elección de los espacios de intervención tiene consecuencias sobre el equilibrio territorial de la ciudad. Los resultados de esta investigación tienen implicaciones para la disciplina del planeamiento urbano. Por una parte, se confirma la vigencia del paradigma del “urbanismo negociado”, si bien bajo discursos de liderazgo público y sin apelación al protagonismo del sector privado. Por otra parte, la planificación colaborativa en un contexto de “responsabilización” de las organizaciones comunitarias puede desactivar la potencia política de la participación ciudadana y servir como “amortiguador” hacia el gobierno local. Asimismo, la sustitución del planeamiento general como instrumento de definición de la ciudad futura por una planificación oportunista basada en la actuación en áreas estratégicas que tiren del resto de la ciudad, no permite definir un modelo coherente y consensuado de la ciudad que se desea colectivamente, ni permite utilizar el planeamiento como mecanismo de redistribución. ABSTRACT In the past three decades, the dynamics of global economic restructuring have radically redefined the role of cities. The transition from keynesianism to neoliberalism has caused a shift in local governments’ urban policies, which have progressively abandoned the tasks of regulation and redistribution to focus on promoting economic growth and competitiveness. In this context, many critics have pointed out that urban regeneration has become a vehicle for extracting value from the city and is causing the expulsion of the most vulnerable citizens. However, regeneration of consolidated areas is also an opportunity to improve the living conditions of the resident population, and is a necessary policy to control the expansion of the city and reduce the need for transportation, thus promoting more sustainable cities. Assuming that the governance of urban regeneration processes is key to the final outcome of the plans and determines the resulting city model, the goal of this research is to verify whether urban regeneration is necessarily a value extraction mechanism or if it can improve the quality of life in cities through citizens’ participation. It proposes a framework for analysis of decision-making in urban regeneration processes and their impact on the results of the plans, taking as a case study the city of Boston, which since the 1990s is trying to become a "city of neighborhoods", encouraging citizen participation, while seeking to position itself in the global economic scene. The analysis focuses on two redevelopment plans initiated in the late 1990s. The Jackson Square case allows us to understand the role of civil society and the third sector in the regeneration of disadvantaged neighborhoods, in a clear example of bottom-up planning. On the contrary, the conversion of the South Boston waterfront to build the Innovation District takes us to the big redevelopment efforts with economic stimulus’ goals, traditionally linked to downtowns and led by government and economic elites (the local “growth machine”) through more technocratic processes (top-down planning). The research is based on a qualitative analysis of the processes of decision making and the relationship between those involved, as well as the evaluation of the implementation of those decisions and their influence on the resulting urban model. The analysis suggests that the governance of urban regeneration processes decisively influences the outcome of interventions; however, community engagement in the decision-making process is not enough for the result of the urban regeneration to counteract the effects of neoliberalization, especially if it is limited to the planning phase and does not extend to the implementation of the projects, and if it is not supported by a broader political mobilization to ensure a redistributive public action. Moreover, urban regeneration processes redefine the urban model, since the choice of intervention areas has important consequences for the territorial balance of the city. The results of this study have implications for the discipline of urban planning. On the one hand, it confirms the validity of the "negotiated planning" paradigm, albeit under public leadership discourse and without a direct appeal to the leadership role of the private sector. On the other hand, collaborative planning in a context of "responsibilization" of community based organizations can deactivate the political power of citizen participation and serve as a "buffer" towards the local government. Furthermore, the replacement of comprehensive planning, as a tool for defining the city's future, by an opportunistic planning based on intervention in strategic areas that are supposed to induce change in the rest of the city, does not allow a coherent and consensual urban model that is collectively desired, nor it allows to use planning as a redistribution mechanism.

Productive performance of brown-egg laying pullets from hatching to 5 weeks of age as affected by fiber inclusion, feed form, and energy concentration of the diet1

The effects of fiber inclusion, feed form, and energy concentration of the diet on the growth performance of pullets from hatching to 5 wk age were studied in 2 experiments. In Experiment 1, there was a control diet based on cereals and soybean meal, and 6 extra diets that included 2 or 4% of cereal straw, sugar beet pulp (SBP), or sunflower hulls (SFHs) at the expense (wt/wt) of the whole control diet. From hatching to 5 wk age fiber inclusion increased (P < 0.05) ADG and ADFI, and improved (P < 0.05) energy efficiency (EnE; kcal AMEn/g ADG), but body weight (BW) uniformity was not affected. Pullets fed SFH tended to have higher ADG than pullets fed SBP (P = 0.072) with pullets fed straw being intermediate. The feed conversion ratio (FCR) was better (P < 0.05) with 2% than with 4% fiber inclusion. In Experiment 2, 10 diets were arranged as a 2×5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of AMEn (2,850, 2,900, 2,950, 3,000, and 3,050 kcal/kg). Pullets fed crumbles were heavier and had better FCR than pullets fed mash (P < 0.001). An increase in the energy content of the crumble diets reduced ADFI and improved FCR linearly, but no effects were detected with the mash diets (P < 0.01 and P < 0.05 for the interactions). Feeding crumbles tended to improve BW uniformity at 5 wk age (P = 0.077) but no effects were detected with increases in energy concentration of the diet. In summary, the inclusion of moderate amounts of fiber in the diet improves pullet performance from hatching to 5 wk age. The response of pullets to increases in energy content of the diet depends on feed form with a decrease in feed intake when fed crumbles but no changes when fed mash. Feeding crumbles might be preferred to feeding mash in pullets from hatching to 5 wk age.

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-l-cysteine, d-penicillamine, captopril, l-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of α-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH3- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.

Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis

Squalene epoxidase, a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis. In 1957, Bloch and colleagues identified a factor from rat liver cytosol termed “supernatant protein factor (SPF),” which promotes the squalene epoxidation catalyzed by rat liver microsomes with oxygen, NADPH, FAD, and phospholipid [Tchen, T. T. & Bloch, K. (1957) J. Biol. Chem. 226, 921–930]. Although purification of SPF by 11,000-fold was reported, no information is so far available on the primary structure or biological function of SPF. Here we report the cDNA cloning and expression of SPF from rat and human. The encoded protein of 403 amino acids belongs to a family of cytosolic lipid-binding/transfer proteins such as α-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Sec14p), and squid retinal binding protein. Recombinant SPF produced in Escherichia coli enhances microsomal squalene epoxidase activity and promotes intermembrane transfer of squalene in vitro. SPF mRNA is expressed abundantly in the liver and small intestine, both of which are important sites of cholesterol biosynthesis. SPF is expressed significantly in isolated hepatocytes, but the expression level was markedly decreased after 48 h of in vitro culture. Moreover, SPF was not detectable in most of the cell lines tested, including HepG2 and McARH7777 hepatomas. Transfection of SPF cDNA in McARH7777 significantly stimulated de novo cholesterol biosynthesis. These data suggest that SPF is a cytosolic squalene transfer protein capable of regulating cholesterol biosynthesis.

The life of Lady Guion / written in French, by herself, now abridged, and translated into English. Exhibiting her eminent piety, charity, meekness, resignation, fortitude and stability; her labours, travels, sufferings and services, for the conversion of souls to God; and her great success, in some places, in that best of all employments on the earth. To which are added, accounts of the lives of worthy persons, whose memories were dear to Lady Guion.

"From an English edition, published at Bristol: with a few abridgements, etc."