Mechanistic Importance of Redox Potentials and Conformational Flexibility in Electron-Transferring Flavoproteins

The mitochondrial matrix flavoproteins electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) are responsible for linking fatty acid β-oxidation with the main mitochondrial respiratory chain. Electrons derived from flavoprotein dehydrogenases are transferred sequentially through ETF and ETF-QO to ubiquinone and then into the respiratory chain via complex III. In this study, the effects of changes in ETF-QO redox potentials on its activity and the conformational flexibility of ETF were investigated. ETF-QO contains one [4Fe-4S]2+,1+ and one flavin adenine dinucleotide (FAD). In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. FAD redox potentials were measured by potentiometric titration probed by electron paramagnetic resonance (EPR) spectroscopy. The N338T and N338A mutations lowered the midpoint potentials, which resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e- catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore it is proposed that the iron-sulfur cluster is the immediate acceptor from ETF. It has been proposed that the αII domain of ETF is mobile, allowing promiscuous interactions with structurally different partners. Double electron-electron resonance (DEER) was used to measure the distance between spin labels at various sites and an enzymatically reduced FAD cofactor in Paracoccus denitrificans ETF. Two or three interspin distance distributions were observed for spin-labels in the αI (A43C) and βIII (A111C) domains, but only one is observed for a label in the βII (A210C) domain. This suggests that the αII domain adopts several stable conformations which may correspond to a closed/inactive conformation and an open/active conformation. An additional mutation, E162A, was introduced to increase the mobility of the αII domain. The E162A mutation doubled the activity compared to wild-type and caused the distance distributions to become wider. The DEER method has the potential to characterize conformational changes in ETF that occur when it interacts with various redox partners.

Applications of EPR with an Emphasis on Tau Fibril Structure

Substances containing unpaired electrons have been studied by electron paramagnetic resonance (EPR) for nearly 70 years. With continual development and enhancement of EPR techniques, questions have arisen regarding optimum method selection for a given sample based on its properties. In this work, radiation defects, natural lattice defects, solid organic radicals, radicals in solution, and spin-labeled proteins were analyzed using CW, pulse, and rapid scan EPR to compare methods. Studies of solid BDPA, EOe in quartz, Ns0 in diamond, and a-Si:H, showed that rapid scan could overcome many obstacles presented by other techniques, cementing rapid scan as an effective alternative to CW and pulse methods. Relaxation times of six nitroxide radicals were characterized from 0.25-34 GHz, guiding synthesis of improved nitroxides for in vivo imaging experiments. Processes contributing to T1 of DPPH in polystyrene were found through variable temperature measurements at X- and Q-band, resolving previously-reported discrepancies in relaxation properties and providing new insight into this commonly-used standard. In the history of EPR, the study of proteins is relatively new. Double electron-electron resonance (DEER) has emerged as a powerful technique for the study of amyloid fibrils, a class of protein aggregates implicated in a number of neurodegenerative disorders. Microtubule-associated protein tau forms fibrils linked to Alzheimerfs disease through seeded conversion of monomer. Self-assembly is mediated by the microtubule binding repeats in tau, and there are either three or four repeats present depending on the isoform. DEER was used to show that filaments of 3R and 4R tau are conformationally distinct and that 4R fibrils adopt a heterogeneous mixture of conformations. Populations of 4R fibril conformations, which were independently validated using a model system, can be modulated by introduction of mutations to the primary sequence or by varying fibril growth conditions. These findings provided unprecedented insights into the seed selection of tau monomers and established conformational compatibility as an important driving force in tau fibril propagation. Lastly, DEER acquisition was improved through addition of paramagnetic metal to spin-labeled protein, decreasing collection time, and through use of a novel spin label with increased T2, thereby lengthening the available acquisition window.

Sea lions, sacrifice, and ritual : The symbolism of Moche Island imagery

The Mochica, or Moche (c. 100-800 AD) culture, flourished along the northern Peruvian coast. The Moche did not have a formal written language; as such, contemporary scholars base their analysis on Moche iconography and archaeological burial remains. Especially renown for their ceramic artistry, Moche vessels exhibit a wide range of subject matter, including animal and enigmatic figural representations that evoke terrestrial, marine and possibly, spiritual realms. While research has focused on political organization and the interrelationship between sacrifice and warfare, many marine themes have not been fully explored in the discourse. An exploration of sea lion imagery and sacrifice themes suggests that the marine mammals were ritually hunted. A careful iconographic analysis of island scenes demonstrates ritual and gender affiliations held by the Moche about sea lions. In a multi-disciplinary approach, scientific, archaeological and ethnographic resources substantiate this claim.

Prevalence of chronic wasting disease in post burn areas

Chronic wasting disease is a fatal neurological disease found in deer and elk in 14 western and mid-western states and two Canadian provinces. It is believed to have been first observed in Colorado and Wyoming in 1967. It is a disease caused by prions by an unknown transmission vector and impossible to cure at this time. Most of the management options currently available are labor-intensive and costly. The potential use of controlled burns to reduce or eliminate the prions that cause the disease was shown to have no effect on the prevalence of the disease in either study area. The temperatures needed to destroy prions were not reached by either surface or crown fires.

Notebook of John Allen, 1772

Paper-covered notebook containing handwritten poems and verse by Harvard graduate John Allen. Some of the poems refer to Allen’s illnesses in October 1772. The notebook also contains a short list titled “The Gentleman that I wrote diplomas for," with a list of sixteen individuals who received degrees from Harvard. The inside cover includes the inscription: “John Allen – November 4, 1772. Poetic Composition.” “Dr. T. C. Gilman” is stamped on cover.

Student essay composed by Thomas Mason, 1796

Three-page folio-sized handwritten student essay composed by Thomas Mason as a Harvard undergraduate. The verso of the last page is inscribed "Mason February 1796." A quotation from Edward Young appears at the top of the first page: "Heaven gives us friends to bless the present science; / Resumes them, to prepare us for the rest." The essay discusses friendship and the death of friends, and begins, "The author of our nature has so constituted it, that pleasure is unknown without the intervention of pain."

Upper Penobscot Bay, Maine, Nautical Chart, 1776 (Image 2 of 2) (Raster Image)

This layer is a georeferenced raster image of the untitled, historic nautical chart: [A chart of Great Bluehill Bay, Penobscot River &c.] (sheet originally published in 1776). The map is [sheet 41] from the Atlantic Neptune atlas Vol. 3 : Charts of the coast and harbors of New England, from surveys taken by Samuel Holland and published by J.F.W. Des Barres, 1781. Scale [ca. 1:50,000]. This layer is image 2 of 2 total images of the two sheet source map, representing the eastern portion of the map. Covers a portion of Penobscot Bay, including Blue Hill Bay, Deer Island, and Eggemoggin Reach, Maine. The image is georeferenced to the surface of the earth and fit to the 'World Mercator' (WGS 84) projected coordinate system. All map collar information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This map shows coastal features such as harbors, inlets, rocks, channels, points, coves, shoals, islands, and more. Includes also selected land features such as cities and towns, and buildings. Relief is shown by hachures; depths by soundings. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection. The entire Atlantic Neptune atlas Vol. 3 : Charts of the coast and harbors of New England has been scanned and georeferenced as part of this selection.

Milwaukee County, Wisconsin, 1858 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of the county of Milwaukee, Wisconsin, from surveys under the direction of H.F. Walling ; engraved, printed, colored & mounted at H.F. Wallings Map Establishment. It was published by M.H. Tyler in 1858. Scale [1:31,680]. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Wisconsin South State Plane NAD 1983 coordinate system (in Feet) (Fipszone 4803). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This cadastral map shows features such as roads, railroads and stations, drainage, city wards, selected buildings, property lots, and names of landowners, and more. Includes insets: City of Milwaukee -- Humboldt (Milwaukee) -- Franklin -- Oak Creek -- Wauwatosa. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

Milwaukee and vicinity, Wisconsin, 1962 (Image 1 of 4) (Raster Image)

This layer is a georeferenced raster image of the historic, topographic paper map entitled: Milwaukee and vicinity, Wisconsin, 1959, mapped, edited and published by the Geological Survey. It was published by U.S. Dept. of the Interior, Geological Survey in 1962. Scale 1:24,000. Compiled from 1:24,000-scale maps of Thiensville, Menomonee Falls, Wauwatosa, Milwaukee, South Milwaukee, Greendale 1958, and Hales Corners 1959 7.5 minute quadrangles. Selected hydrographic data compiled from U.S. Lake Survey Charts 74 and 743 (1957). This layer is image 1 of 4 total images of the four sheet source map representing the northwest portion of the map. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Wisconsin South State Plane NAD 1927 coordinate system (Fipszone 4803). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This is a typical topographic map portraying both natural and manmade features. It shows and names works of nature, such as mountains, valleys, lakes, rivers, vegetation, etc. It also identify the principal works of humans, such as roads, railroads, boundaries, transmission lines, major buildings, etc. Relief shown by contours (interval 10 feet) and spot heights. Depths shown by contours and soundings. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

Milwaukee and vicinity, Wisconsin, 1962 (Image 2 of 4) (Raster Image)

This layer is a georeferenced raster image of the historic, topographic paper map entitled: Milwaukee and vicinity, Wisconsin, 1959, mapped, edited and published by the Geological Survey. It was published by U.S. Dept. of the Interior, Geological Survey in 1962. Scale 1:24,000. Compiled from 1:24,000-scale maps of Thiensville, Menomonee Falls, Wauwatosa, Milwaukee, South Milwaukee, Greendale 1958, and Hales Corners 1959 7.5 minute quadrangles. Selected hydrographic data compiled from U.S. Lake Survey Charts 74 and 743 (1957). This layer is image 2 of 4 total images of the four sheet source map representing the northeast portion of the map. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Wisconsin South State Plane NAD 1927 coordinate system (Fipszone 4803). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This is a typical topographic map portraying both natural and manmade features. It shows and names works of nature, such as mountains, valleys, lakes, rivers, vegetation, etc. It also identify the principal works of humans, such as roads, railroads, boundaries, transmission lines, major buildings, etc. Relief shown by contours (interval 10 feet) and spot heights. Depths shown by contours and soundings. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

Mécanismes moléculaires de l’hypertrophie vasculaire dans le modèle animal d’hypertension essentielle (SHR)

La voie de signalisation des phosphoinositides joue un rôle clé dans la régulation du tonus vasculaire. Plusieurs études rapportent une production endogène de l’angiotensin II (Ang II) et de l’endothéline-1 (ET-1) par les cellules musculaires lisses vasculaires (CMLVs) de rats spontanément hypertendus (spontaneously hypertensive rats : SHR). De plus, l’Ang II exogène induit son effet prohypertrophique sur les CMLVs selon un mécanisme dépendant de la protéine Gqα et de la PKCẟ. Cependant, le rôle de l’axe Gqα/PLCβ/PKCẟ dans l’hypertrophie des CMLVs provenant d’un modèle animal de l’hypertension artérielle n’est pas encore étudié. L’objectif principal de cette thèse est d’examiner le rôle de l’axe Gqα/PLCβ1 dans les mécanismes moléculaires de l’hypertrophie des CMLVs provenant d’un modèle animal d’hypertension artérielle essentielle (spontaneously hypertensive rats : SHR). Nos premiers résultats indiquent que contrairement aux CMLVs de SHR âgés de 12 semaines (absence d’hypertrophie cardiaque), les CMLVs de SHR âgés de 16 semaines (présence d’hypertrophie cardiaque) présentent une surexpression protéique endogène de Gqα et de PLCβ1 par rapport aux CMLVs de rats WKY appariés pour l’âge. L’inhibition du taux d’expression protéique de Gqα et de PLCβ1 par des siRNAs spécifiques diminue significativement le taux de synthèse protéique élevé dans les CMLVs de SHR. De plus, la surexpression endogène des Gqα et PLCβ1, l’hyperphosphorylation de la molécule ERK1/2 et le taux de synthèse protéique élevé dans les CMLVs de SHR de 16 semaines ont été atténués significativement par des antagonistes des récepteurs AT1 (losartan) et ETA (BQ123), mais pas par l’antagoniste du récepteur ETB (BQ788). L’inhibition pharmacologique des MAPKs par PD98059 diminue significativement la surexpression endogène de Gqα/PLCβ1 et le taux de synthèse protéique élevé dans les CMLVs de SHR. D’un côté, l’inhibition du stress oxydatif (par DPI, inhibiteur de la NAD(P)H oxidase, et NAC , molécule anti-oxydante), de la molécule c-Src (PP2) et des récepteurs de facteurs de croissance (AG1024 (inhibiteur de l’IGF1-R), AG1478 (inhibiteur de l’EGFR) et AG1295 (inhibiteur du PDGFR)) a permis d’atténuer significativement la surexpression endogène élevée de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. D’un autre côté, DPI, NAC et PP2 atténuent significativement l’hyperphosphorylation de la molécule c-Src, des RTKs (récepteurs à activité tyrosine kinase) et de la molécule ERK1/2. Dans une autre étude, nous avons aussi démontré que la PKCẟ montre une hyperphosphorylation en Tyr311 dans les CMLVs de SHR comparées aux CMLVs de WKY. La rottlerin, utilisée comme inhibiteur spécifique de la PKCẟ, inhibe significativement cette hyperphosphorylation en Tyr311 dépendamment de la concentration. L’inhibition de l’activité de la PKCẟ par la rottlerin a été aussi associée à une atténuation significative de la surexpression protéique endogène de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. De plus, l’inhibition pharmacologique de l’activité de la PKCẟ, en amont du stress oxydatif, a permis d’inhiber significativement l’activité de la NADPH, le taux de production élevée de l’ion superoxyde ainsi que l’hyperphosphorylation de la molécule ERK1/2, de la molécule c-Src et des RTKs. À notre surprise, nous avons aussi remarqué une surexpression protéique de l’EGFR et de l’IGF-1R dans les CMLVs de SHR à l’âge de 16 semaines. L’inhibition pharmacologique de l’activité de la PKCẟ, de la molécule c-Src et du stress oxydatif a permis d’inhiber significativement la surexpression protéique endogène de ces RTKs. De plus, l’inhibition de l’expression protéique de l’EGFR et de la molécule c-Src par des siRNA spécifiques atténue significativement le taux d’expression protéique élevé de Gqα et de PLCβ1 ainsi que le taux de synthèse protéique élevé dans les CMLVs de SHR. Des siRNAs spécifiques à la PKCẟ ont permis d’atténuer significativement le taux de synthèse protéique élevé dans les CMLVs de SHR et confirment le rôle important de la PKCẟ dans les mécanismes moléculaires de l’hypertrophie des CMLVs selon une voie dépendante du stress oxydatif. En conclusion, ces résultats suggèrent un rôle important de l’activation endogène de l’axe Gqα-PLCβ-PKCẟ dans le processus d’hypertrophie vasculaire selon un mécanisme impliquant une activation endogène des récepteurs AT1/ETa, de la molécule c-Src, du stress oxidatif, des RTKs et des MAPKs.

Mécanismes de transfert de proton d’une réaction acido-basique en phase aqueuse : une étude ab-initio

Les réactions de transfert de proton se retrouvent abondamment dans la nature et sont des processus cruciaux dans plusieurs réactions chimiques et biologiques, qui se produisent souvent en milieu aqueux. Les mécanismes régissant ces échanges de protons sont complexes et encore mal compris, suscitant un intérêt des chercheurs en vue d’une meilleure compréhension fondamentale du processus de transfert. Le présent manuscrit présente une étude mécanistique portant sur une réaction de transfert de proton entre un acide (phénol fonctionnalisé) et une base (ion carboxylate) en phase aqueuse. Les résultats obtenus sont basés sur un grand nombre de simulations de dynamique moléculaire ab-initio réalisées pour des systèmes de type « donneur-pont-accepteur », où le pont se trouve à être une unique molécule d’eau, permettant ainsi l’élaboration d’un modèle cinétique détaillé pour le système étudié. La voie de transfert principalement observée est un processus ultra-rapide (moins d’une picoseconde) passant par la formation d’une structure de type « Eigen » (H9O4+) pour la molécule d’eau pontante, menant directement à la formation des produits. Une seconde structure de la molécule d’eau pontante est également observée, soit une configuration de type « Zündel » (H5O2+) impliquant l’accepteur de proton (l’ion carboxylate) qui semble agir comme un cul-de-sac pour la réaction de transfert de proton.

Mécanismes de transfert de proton d’une réaction acido-basique en phase aqueuse : une étude ab-initio

Les réactions de transfert de proton se retrouvent abondamment dans la nature et sont des processus cruciaux dans plusieurs réactions chimiques et biologiques, qui se produisent souvent en milieu aqueux. Les mécanismes régissant ces échanges de protons sont complexes et encore mal compris, suscitant un intérêt des chercheurs en vue d’une meilleure compréhension fondamentale du processus de transfert. Le présent manuscrit présente une étude mécanistique portant sur une réaction de transfert de proton entre un acide (phénol fonctionnalisé) et une base (ion carboxylate) en phase aqueuse. Les résultats obtenus sont basés sur un grand nombre de simulations de dynamique moléculaire ab-initio réalisées pour des systèmes de type « donneur-pont-accepteur », où le pont se trouve à être une unique molécule d’eau, permettant ainsi l’élaboration d’un modèle cinétique détaillé pour le système étudié. La voie de transfert principalement observée est un processus ultra-rapide (moins d’une picoseconde) passant par la formation d’une structure de type « Eigen » (H9O4+) pour la molécule d’eau pontante, menant directement à la formation des produits. Une seconde structure de la molécule d’eau pontante est également observée, soit une configuration de type « Zündel » (H5O2+) impliquant l’accepteur de proton (l’ion carboxylate) qui semble agir comme un cul-de-sac pour la réaction de transfert de proton.

Pollen profiles from a Late Palaeolithic site in the community of Bad Buchau-Kappel, Baden-Wuerttemberg, Germany

Excavations were carried out in a Late Palaeolithic site in the community of Bad Buchau-Kappel between 2003 and 2007. Archaeological investigations covered a total of more than 200 m**2. This site is the product of what likely were multiple occupations that occurred during the Late Glacial on the Federsee shore in this location. The site is situated on a mineral ridge that projected into the former Late Glacial lake Federsee. This beach ridge consists of deposits of fine to coarse gravel and sand and was surrounded by open water, except for a connection to the solid shore on the south. A lagoon lay between the hook-shaped ridge and the shore of the Federsee. This exposed location provided optimal access to the water of the lake. In addition, the small lagoon may have served as a natural harbor for landing boats or canoes. Sedimentological and palynological investigations document the dynamic history of the location between 14,500 and 11,600 years before present (cal BP). Evidence of the deposition of sands, gravels and muds since the Bølling Interstadial is provided by stratigraphic and palynological analyses. The major occupation occurred in the second half of the Younger Dryas period. Most of the finds were located on or in the sediments of the ridge; fewer finds occurred in the surrounding mud, which was also deposited during the Younger Dryas. Direct dates on some bone fragments, however, demonstrate that intermittent sporadic occupations also took place during the two millennia of the Meiendorf, Bølling, and Allerød Interstadials. These bones were reworked during the Younger Dryas and redeposited in the mud. A 14C date from one bone of 11,600 years ago (cal BP) places the Late Palaeolithic occupation of the ridge at the very end of the Younger Dryas, which is in agreement with stratigraphic observations. Stone artifacts, numbering 3,281, comprise the majority of finds from the site. These include typical artifacts of the Late Palaeolithic, such as backed points, short scrapers, and small burins. There are no bipointes or Malaurie-Points, which is in accord with the absolute date of the occupation. A majority of the artifacts are made from a brown chert that is obtainable a few kilometers north of the site in sediments of the Graupensandrinne. Other raw materials include red and green radiolarite that occur in the fluvioglacial gravels of Oberschwaben, as well as quartzite and lydite. The only non-local material present is a few artifacts of tabular chert from the region near Kelheim in Bavaria. A unique find consists of two fragments of a double-barbed harpoon made of red deer antler, which was found in the Younger Dryas mud. It is likely, but not certain, that this find belongs to the same assemblage as the numerous stone artifacts. Although not numerous, animal bones were also found in the excavations. Most of them lay in sediments of the Younger Dryas, but several 14C dates place some of these bones in earlier periods, including the Meiendorf, Bølling, and Allerød Interstadials. These bones were reworked by water and redeposited in mud sediments during the Younger Dryas. As a result, it is difficult to attribute individual bones to particular chronological positions without exact dates. Species that could be identified include wild horse (Equus spec.), moose or elk (Alces alces), red deer (Cervus elaphus), roe deer (Capreolus capreolus), aurochs or bison (Bos spec.), wild boar (Sus scrofa), as well as birds and fish, including pike (Esox Lucius).