Abstract 3200 years ago a massive battle took place at the river Tollense in MecklenburgVorpommern, Germany, possibly involving thousands of people. 130 victims have been found so far. Several show signs of former violence. Axes, wooden clubs, knives and a vast number of arrowheads have been found among the human skeletal remains. According to isotope analyses on tooth enamel from human remains at the site, a considerable amount of the victims did not originate from the local area. The isotope analyses divided the victims into two major groups, one local and the other probably originating from Central Europe. In other words, the battlefield of the Tollense were not fought by local farmers and craftsmen. This, together with the extent of the battle, the weapon finds and traumata on the human remains, has led archaeologists to assume that the battle was fought by professional warriors. If this is true, the battlefield of the Tollense would be the first of its kind in the Northern European Bronze Age. Sammanfattning Uppsatsen diskuterar omfattningen och karaktären av slaget vid floden Tollense som utspelade sig för 3200 år sedan i vad som idag är Nordtyskland. Slagfältet har uppmärksammats för att vara den första konflikten med professionella krigare i bronsålderns Nordeuropa. Tidigare forskning om ämnet saknar en konsekvent definition av begreppet professionell krigare. För att avgöra om det var professionella krigare som stred vid Tollense definierar uppsatsen begreppet utifrån teoretiska perspektiv såsom antropologiska klassifikationer av hövdingadömen, krigare och soldater, men också genom att belysa kontaktnätverken och den långväga handel som växte fram under bronsåldern. Omfattningen och karaktären av slaget vid Tollense beskrivs utifrån hur många människor som deltog på slagfältet samt vilka arkeologiska fynd som talar för huruvida de var professionella krigare och inte lokala bönder och hantverkare som drabbade samman. Uppsatsen är en klassisk litteraturstudie. Utgrävningarna vid Tollense pågår i skrivande stund, vilket innebär att det inte finns någon slutpublikation med färdigställda resultat. För att besvara frågeställningarna om antalet deltagare i konflikten samt huruvida de var professionella krigare eller inte, redogör uppsatsen för analyser av de mänskliga kvarlevorna som återfunnits vid Tollense och vapnen som användes på slagfältet. Avslutningsvis ger uppsatsen en personlig tolkning av vad fyndmaterialet säger om hur många människor som var involverade i konflikten och vad som talar för att de var professionella krigare. Diskussionen lyfter fram hövdingadömen som centraliserade och hierarkiska samhällsstrukturer och hur detta möjliggör organiserad krigföring.

Os reservatórios, embora importantes para diversas atividades humanas, podem trazer modificações negativas ao ambiente. Tais sistemas aquáticos propiciam condições favoráveis ao maior desenvolvimento da comunidade fitoplanctônica, associado ao aporte de nutrientes e ao fenômeno da eutrofização artificial. O estudo do processo de assimilação de macronutrientes por esta comunidade pode orientar planos para mitigação dos efeitos da eutrofização e contribuir para o entendimento dos fatores limitantes ao fitoplâncton. A presente pesquisa teve como objetivo principal quantificar a assimilação de três formas de nitrogênio pelo fitoplâncton com uso do traçador 15N, de modo a contribuir para o entendimento da influência da temperatura e da disponibilidade de luz sobre esse processo em amostras ambientais e em culturas laboratoriais. Os objetivos específicos visaram: 1) estimar a variação temporal da assimilação de nitrato, amônio e ureia em amostras ambientais do reservatório do Lobo (SP); 2) quantificar e comparar a assimilação das mesmas três formas nitrogenadas por duas espécies fitoplanctônicas isoladas em laboratório (a cianobactéria Microcystis aeruginosa e a clorofícea Scenedesmus sp.), para verificar a influência da luz e da temperatura sobre o processo de assimilação. Para o primeiro objetivo específico, amostras foram coletadas em quatro períodos do ano no reservatório do Lobo (out /14; jan, abr e jul /15), próximo a São Carlos (SP). Foram reproduzidas, em incubadoras, as condições de Radiação Solar Fotossinteticamente Ativa (RSFA) e temperatura observadas em campo e relativas às profundidades em que estavam disponíveis 50% e 10% da RSFA. Em laboratório, os traçadores nitrato (15NO3-), amônio (15NH4+) e ureia [(15NH2)2CO] foram adicionados em frascos de 250 mL, incubados por uma hora. Após etapa de filtração, os filtros seguiram para análise elementar de quantificação do nitrogênio particulado e espectrometria de massa para a determinação da composição isotópica (15N/14N). Para o segundo objetivo específico, após a obtenção das culturas isoladas de uma cianobactéria e uma clorofícea, foi realizada a quantificação da assimilação das formas de nitrogênio. Esta análise foi realizada por experimento fatorial, com dois fatores e três níveis (temperatura: 20, 24 e 28°C e luz: 50, 250 e 450 µE.m-2.s-1). Os resultados apresentados para as amostras ambientais sugeriram maior preferência da comunidade fitoplanctônica em assimilar amônio em janeiro (23,7 µgN.L-1.h-1) e julho (54,4 µgN.L-1.h-1) de 2015. Em abril de 2015, a assimilação foi maior para nitrato (38,5 µgN.L-1.h-1) e ureia (43,1 µgN.L-1.h-1). Foi observado, ao longo dos meses de coleta, aumento na assimilação total do nitrogênio, da mesma forma como a densidade total de fitoplâncton, o biovolume e a clorofila-a, sugerindo que a assimilação deve acompanhar o aumento do grau de trofia do reservatório. Os resultados para as culturas laboratoriais indicaram uma assimilação quase que total do amônio pelas duas espécies estudadas. Scenedesmus sp. mostrou-se mais adaptada a assimilar amônio (517,1 µgN.L-1.h-1) em condições de alta intensidade luminosa (450 µE.m-2.s-1), enquanto Microcystis aeruginosa assimilou melhor o amônio (160,0 µgN.L-1.h-1) em condições de 50 µE.m-2.s-1. Para as duas espécies, a temperatura de 20°C resultou em maiores taxas de assimilação. Com base nos resultados obtidos, busca-se oferecer subsídios para o controle do estado trófico e manejo de reservatórios subtropicais por meio da identificação da forma de nitrogênio preferencialmente assimilada pelo fitoplâncton e os respectivos fatores intervenientes. / The reservoirs, despite their importance for different human activities, can bring negative changes to the environment. Such aquatic systems hold favorable conditions to the development of the phytoplankton community, which is associated with nutrient inputs and to the artificial eutrophication. The study of macronutrients uptake by this community can aid in plans for mitigating the effects of eutrophication and contribute to the understanding of the limiting factors to phytoplankton. This research aimed at quantifying the uptake of three forms of nitrogen by the phytoplankton with of use the 15N isotope, in order to contribute to the understanding of the influence of temperature and light availability on this process in environmental samples and in laboratory cultures. The specific objectives were: 1) to estimate the temporal variation of the uptake of nitrate, ammonium and urea in environmental samples from the Lobo Reservoir (SP, Brazil); 2) to quantify and compare the assimilation of the same three nitrogen forms for phytoplankton species isolated in laboratory (the Cyanobacteria Microcystis aeruginosa and the Chlorophyceae Scenedesmus sp.), to verify the influence of light and temperature in the uptake process. For the first specific objective, samples were collected in four periods of the year in the Lobo Reservoir (Oct/14; Jan, Apr and Jul/15), close to São Carlos (SP). Under controlled lab conditions, Photosynthetic Active Radiation (PAR) and temperature conditions observed in the field (at depths corresponding to 50% and 10% of the PAR) were reproduced. The tracers nitrate (15NO3-), ammonium (15NH4+) and urea [(15NH2)2CO] were added in bottles of 250 mL and incubated for one hour. After filtration, the filters were sent to elemental analysis for quantification of particulate nitrogen and mass spectrophotometry for the determination of isotopic composition (15N/14N). For the second of specific objective, after obtaining isolated cultures of the Cyanobacteria and the Chlorophyceae, the quantification of nitrogen uptake was performed. This analysis was carried out by factorial experiment with two factors and three levels (temperature: 20, 24 and 28°C and light: 50, 250 and 450 µE.m-2.s-1). The results obtained for the environmental samples suggested greater preference of the phytoplankton community for ammonium uptake in January (23.7 µgN.L-1.h-1) and July (54.4 µgN.L-1.h-1) from 2015. In April 2015, the uptake rates were greater for nitrate (38.5 µgN.L-1.h-1) and urea (43.1 µgN.L-1.h-1). It was observed temporal increase in total nitrogen uptake, following the patterns for total phytoplankton density, biovolume and chlorophyll-a, suggesting that uptake can be related with the increasing trophic state of the reservoir. The results from the laboratory cultures showed almost total ammonium uptake by both studied species. Scenedesmus sp. seemed to be more adapted to uptake ammonium (517.1 µgN.L-1.h-1) in conditions of high light intensity (450 µE.m-2.s-1), while the respective condition for Microcystis aeruginosa uptaking ammonium (160.0 µgN.L-1.h-1) was 50 µE.m-2.s-1. For the two species, the temperature of 20ºC resulted in higher uptake rates. We expect our results will aid in trophic state control and management of subtropical reservoirs through the identification of the nitrogen forms preferentially assimilated by phytoplankton and the factors involved.

Stable carbon isotope analyses of the humin fraction of the soil organic matter were conducted on more than 160 soil profiles from Tikal, Guatemala. The profiles were collected from near areas associated with the earthworks of Tikal; an ancient ditch and parapet construction hypothesized to have formed ancient boundaries of the polity. In addition to the isotope analyses, the physical and chemical characteristics of the horizons were determined. Maize, a C4 plant, formed an integral part of the ancient Maya diet and is the only known C4 plant cultivated by the Maya. Prior to and subsequent to the ancient Maya occupation of Tikal, the landscape was dominated by C3 forest vegetation. Over the centuries C4 plant biomass including rhizodeposition decomposed to form soil organic matter that contains a distinct C4 signature reflecting the vegetation history of the area. Forested areas anciently cleared for agriculture were identified through interpretation of significant isotopic shifts that signaled past vegetation changes. Buried horizons were encountered in the upland depressions and bajo wetlands. The aggraded soil deposits were likely the result of increased human activity related to settlement and agriculture. The buried horizons and the overlying sediments exhibited stable carbon isotope shifts associated with forest clearance and maize agriculture. Geospatial analysis of the stable carbon isotope ratios indicated that ancient Maya agriculture was focused on deeper footslope and toeslope soils in both bajos and upland depressions. Some evidence of infield agriculture or food processing was also encountered in connection with ancient settlement at upland locations. The soil data provide insight into ancient land use and sustainability that could potentially contribute to subsistence and population reconstruction models.

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

The relationship between protein structure, motions, and catalytic activity is an evolving perspective in enzymology. An interactive approach, where experimental and theoretical studies examine the same catalytic mechanism, is instrumental in addressing this issue. We combine various techniques, including steady state and pre-steady state kinetics, temperature dependence of kinetic isotope effects (KIEs), site-directed mutagenesis, X-ray crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations, to study the catalytic mechanisms of thymidylate synthase (TSase). Since TSase catalyzes the last step of the sole intracellular de novo synthesis of thymidylate (i.e. the DNA base T), it is a common target for antibiotic and anticancer drugs. The proposed catalytic mechanism for TSase comprises a series of bond cleavages and formations including activation of two C-H bonds: a rate-limiting C-H→C hydride transfer and a faster C-H→O proton transfer. This provides an excellent model system to examine the structural and dynamic effects of the enzyme on different C-H cleavage steps in the same catalyzed reaction. Our experiments found that the KIE on the hydride transfer is temperature independent while the KIE on the proton transfer is temperature dependent, implying the protein environment is better organized for H-tunneling in the former. Our QM/MM calculations revealed that the hydride transfer has a transition state (TS) that is invariable with temperature while the proton transfer has multiple subsets of TS structures, which corroborates with our experimental results. The calculations also suggest that collective protein motions rearrange the network of H-bonds to accompany structural changes in the ligands during and between chemical transformations. These computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide guidance for future experiments to verify the proposed mechanisms. In addition, we conducted experiments to examine the importance of long-range interactions in TSase-catalyzed reaction, using both kinetic and structural analysis. Those experiments found that a remote mutation affects the hydride transfer by disrupting concerted protein motions, and Mg2+ binds to the surface of TSase and affects the hydride transfer at the interior active site. Both our experiments and computations have exposed interesting features of ecTSase that can potentially provide new targets for antibiotic drugs targeting DNA biosynthesis. The relationship between protein structure, motions, and catalytic activity learned from this project may have general implications to the question of how enzymes work.

Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR. Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites. As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.

O objetivo da dissertação é provar que toda álgebra, sobre o corpo dos números reais, algébrica e absolutamente valuada é de dimensão nita, e portanto isótopa a D . Observamos que H é a álgebra real dos Quatérnios e D R , C , H ou a álgebra real dos Octônios. A demonstração do resultado é feita gradualmente, considerando inicialmente álgebras reais absolutamente valuadas algébrica com unidade, a seguir com unidade e nalmente, algébrica. Na demonstração do teorema será necessário combinar resultados não triviais de álgebras não associativas, análise funcional, álgebras de Banach e técnicas de ultraprodutos de espaços normados. As álgebra absolutamente valuadas não são necessariamente associativas. Abraham Adrian 1947 mostrou que R , C , H e D são as únicas álgebras reais absolutamente valuadas dimensão nita e com unidade; o mesmo Albert dois anos depois, em 1949 , caracterizou Albert em de essas mesmas álgebras como as únicas que são absolutamente valuadas algébricas e com unidade sobre os reais. Em 1960 Fred B. Wright e Kazimierz Urbanik provaram que R , C , D são as únicas álgebra reais absolutamente valuadas e com unidade. Recentemente, em 1997 , Kaidi El-Amin, Maria Isabel Ramírez e Ángel Rodríguez Palacios mostraram que H e toda álgebra real absolutamente valuadas e algébrica é isótopa a uma de estas quatro. Nosso objetivo é desenvolver e unicar os resultados obtidos nestes 4 trabalhos. / Our goal here is to study the absolute valued algebraic real algebras. In order to reach our intention, we regard an absolute valued real algebra and on which one we impose: First, such one is nite-dimensional algebra; second; such one is algebraic algebra; third, such one is with unity; and in the end such one is algebraic algebra. In the latter case, our aim, it needs of certain classic results of functional analysis and others one of Banach algebras; then, we reach that such one real algebra is isotope to one of the classical absolute valued real algebras algebra and D R , C , H or D . Where H is the Quaternions real is the Octonions real algebra. The absolute valued algebras are not necessarily associative. Abraham Adrian Albert was the rst mathematician considering absolute valued algebras in a context not necessarily associative. In 1947 , he proved that any nite-dimensional absolute valued real algebra with unit element is isomorphic to either real eld H or the Octonions algebra D . Two years R , the complex eld C , the Quaternions algebra later, he demonstrated that R , C , H and D are the unique absolute valued algebraic real algebras with unit element. Recently, in 1997 , Kaidi El-Amin, Maria Isabel Ramírez and Ángel Rodríguez Palacios proved that any absolute valued algebraic real algebra is nite-dimensional.

This dissertation research is a bioarchaeological investigation of Late Neolithic through Early Bronze Age (3600-1800 BC) burial populations from the Portuguese Estremadura. In this project macroscopic and isotopic analyses of skeletal and dental materials are used to gather information pertaining to diet, health status, and inter-lifetime mobility patterns for individuals interred at different burials within a small geographic area with the goal of evaluating the level of social differentiation in the region. The archaeological record for the transition between the Late Neolithic and the Early Bronze Age in southwestern Portugal demonstrates clear evidence of the rise of a socially-complex, non-state society. During the Early Bronze Age, however, this region underwent a period of social `devolution' which cumulated in widespread settlement abandonment. To date, it is unclear to what extent sociopolitical or environmental factors contributed to this social collapse. This study seeks to expand our knowledge of social differentiation in the Late Neolithic to Early Bronze Age of the Estremadura region of Portugal and provide insight into social structure during the emergence and collapse of early complex societies in Iberia. The results of this study found that there were statistically significant differences in dietary, mobility and demographic patterns between burials that suggest socially distinct populations were interred at different sites. In particular, one burial site, Cova da Moura, diverged significantly from the other sampled burial populations. However, based upon the data presented here, it was not possible to tie these biological markers of differentiation to particular aspects of Late Neolithic to Early Bronze Age social organization. Therefore, while this study successfully identified differences between burial populations, at this time, it is not possibly to relate these to particular hierarchical structures. It is suggested that aspects of burial practices in the region confound biologically-based investigations of social organization in a similar way that they have impeded researchers' abilities to identify elite versus non-elite individuals through grave goods alone. Nonetheless, despite these obstacles, this work provides strong evidence of population heterogeneity in the region, and has implications for our understanding of the evolution of complex societies in the Iberian Peninsula and elsewhere.

An important cause of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To gain a better understanding of the disease process, the changes in biochemical pathways and metabolism of pulmonary arterial smooth muscle cells (PASMCs) were studied using a unique surgical ovine model of increased pulmonary blood flow. PASMCs isolated from 4-week-old lambs with increased PBF (shunt) showed lower oxygen consumption rates and lower extracellular acidification rates linked to glutamine metabolism when compared to controls. Shunt and control PASMCs both exhibited a switch into the reverse tricarboxylic acid (TCA) cycle, while only shunt cells showed a decrease of glucose being transformed into Acetyl CoA to enter the forward TCA cycle. Shunt PASMCs also demonstrated increased levels of yes-associated protein 1 (YAP1) expression in the nucleus. These results indicate changes in glutamine metabolism, glucose metabolism, and protein signaling cascades associated with increased mechanical forces in the setting of increased PBF, as seen in PAH in children with CHD.

Understanding how karst aquifers store and transmit water and contaminants is an ongoing problem in hydrogeology. Flowpath and recharge heterogeneity contribute to the complexity of these systems. This thesis explores karst-conduit connectivity and water chemistry variability in the Houzhai catchment in Guizhou province, China. Artificial tracer tests were conducted during both the monsoon and dry seasons to understand temporal variability in connectivity and water velocity between karst features. Multiple flowpaths through the catchment are activated during the monsoon season and partially abandoned during the dry season. Additionally, gradient reversals during monsoonal high-flow events and as a result of pumping can be significant. Synoptic water samples from several karst features taken during both monsoon and dry seasons elucidate spatial and temporal variability within the catchment. In general, water residence time is longer during the dry season and flow within the Houzhai catchment is temporally dependent. Time-series sampling at the outlet spring during a monsoonal storm event captured chemical variability and identified multiple flowpaths. Overall, this study refines widely applicable methods for studying karst systems to this catchment and provides a foundation for future studies in similar settings.