Biophysics of V(D)J recombination and Genome Packaging: In singulo studies on RAG, HMGB1, and TFAM

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.

La mujer investigadora en el País Vasco: análisis de la presencia de la mujer en actividades de I+D y el gasto en I+D

[ES]La apuesta por la igualdad en cualquier ámbito de nuestro entorno es un aspecto clave para la mejora de él. La igualdad en la investigación científica y desarrollo tecnológico es todavía un reto entre las instituciones que componen el sistema. En el País Vasco, existe un potente sistema y estructura de apoyo institucional a la ciencia, la tecnología y la innovación, y es imprescindible su correcto funcionamiento para un adecuado desarrollo de la I+D en todas sus vertientes. Para conocer la situación actual del País Vasco, este trabajo de investigación tiene como objetivo estudiar la relación existente entre la evolución del gasto en I+D y la evolución de la presencia de la mujer investigadora en actividades de I+D en el País Vasco. En general, el estudio nos permite concluir que la presencia de la mujer investigadora y el gasto en I+D han evolución de manera similar en los últimos veinte años.

Theory of s-d exchange scattering in dilute magnetic alloys

The problem of s-d exchange scattering of conduction electrons off localized magnetic moments in dilute magnetic alloys is considered employing formal methods of quantum field theoretical scattering. It is shown that such a treatment not only allows for the first time, the inclusion of multiparticle intermediate states in single particle scattering equations but also results in extremely simple and straight forward mathematical analysis. These equations are proved to be exact in the thermodynamic limit. A self-consistent integral equation for electron self energy is derived and approximately solved. The ground state and physical parameters of dilute magnetic alloys are discussed in terms of the theoretical results. Within the approximation of single particle intermediate states our results reduce to earlier versions. The following additional features are found as a consequence of the inclusion of multiparticle intermediate states;

(i) A non analytic binding energy is pre sent for both, antiferromagnetic (J < o) and ferromagnetic (J > o) couplings of the electron plus impurity system.

(ii) The correct behavior of the energy difference of the conduction electron plus impurity system and the free electron system is found which is free of unphysical singularities present in earlier versions of the theories.

(iii) The ground state of the conduction electron plus impurity system is shown to be a many-body condensate state for J < o and J > o, both. However, a distinction is made between the usual terminology of "Singlet" and "Triplet" ground states and nature of our ground state.

(iv) It is shown that a long range ordering, leading to an ordering of the magnetic moments can result from a contact interaction such as the s-d exchange interaction.

(v) The explicit dependence of the excess specific heat of the Kondo systems is obtained and found to be linear in temperatures as T→ o and T ℓnT for 0.3 T_K ≤ T ≤ 0.6 T_K. A rise in (ΔC/T) for temperatures in the region 0 < T ≤ 0.1 T_K is predicted. These results are found to be in excellent agreement with experiments.

(vi) The existence of a critical temperature for Ferromagnetic coupling (J > o) is shown. On the basis of this the apparent contradiction of the simultaneous existence of giant moments and Kondo effect is resolved.

The variations and climatic significance of D/H ratios in the carbon-bound hydrogen of cellulose in trees

The σD values of nitrated cellulose from a variety of trees covering a wide geographic range have been measured. These measurements have been used to ascertain which factors are likely to cause σD variations in cellulose C-H hydrogen.

It is found that a primary source of tree σD variation is the σD variation of the environmental precipitation. Superimposed on this are isotopic variations caused by the transpiration of the leaf water incorporated by the tree. The magnitude of this transpiration effect appears to be related to relative humidity.

Within a single tree, it is found that the hydrogen isotope variations which occur for a ring sequence in one radial direction may not be exactly the same as those which occur in a different direction. Such heterogeneities appear most likely to occur in trees with asymmetric ring patterns that contain reaction wood. In the absence of reaction wood such heterogeneities do not seem to occur. Thus, hydrogen isotope analyses of tree ring sequences should be performed on trees which do not contain reaction wood.

Comparisons of tree σD variations with variations in local climate are performed on two levels: spatial and temporal. It is found that the σD values of 20 North American trees from a wide geographic range are reasonably well-correlated with the corresponding average annual temperature. The correlation is similar to that observed for a comparison of the σD values of annual precipitation of 11 North American sites with annual temperature. However, it appears that this correlation is significantly disrupted by trees which grew on poorly drained sites such as those in stagnant marshes. Therefore, site selection may be important in choosing trees for climatic interpretation of σD values, although proper sites do not seem to be uncommon.

The measurement of σD values in 5-year samples from the tree ring sequences of 13 trees from 11 North American sites reveals a variety of relationships with local climate. As it was for the spatial σD vs climate comparison, site selection is also apparently important for temporal tree σD vs climate comparisons. Again, it seems that poorly-drained sites are to be avoided. For nine trees from different "well-behaved" sites, it was found that the local climatic variable best related to the σD variations was not the same for all sites.

Two of these trees showed a strong negative correlation with the amount of local summer precipitation. Consideration of factors likely to influence the isotopic composition of summer rain suggests that rainfall intensity may be important. The higher the intensity, the lower the σD value. Such an effect might explain the negative correlation of σD vs summer precipitation amount for these two trees. A third tree also exhibited a strong correlation with summer climate, but in this instance it was a positive correlation of σD with summer temperature.

The remaining six trees exhibited the best correlation between σD values and local annual climate. However, in none of these six cases was it annual temperature that was the most important variable. In fact annual temperature commonly showed no relationship at all with tree σD values. Instead, it was found that a simple mass balance model incorporating two basic assumptions yielded parameters which produced the best relationships with tree σD values. First, it was assumed that the σD values of these six trees reflected the σD values of annual precipitation incorporated by these trees. Second, it was assumed that the σD value of the annual precipitation was a weighted average of two seasonal isotopic components: summer and winter. Mass balance equations derived from these assumptions yielded combinations of variables that commonly showed a relationship with tree σD values where none had previously been discerned.

It was found for these "well-behaved" trees that not all sample intervals in a σD vs local climate plot fell along a well-defined trend. These departures from the local σD VS climate norm were defined as "anomalous". Some of these anomalous intervals were common to trees from different locales. When such widespread commonalty of an anomalous interval occurred, it was observed that the interval corresponded to an interval in which drought had existed in the North American Great Plains.

Consequently, there appears to be a combination of both local and large scale climatic information in the σD variations of tree cellulose C-H hydrogen.

Analyses of planetary atmospheres across the spectrum : from Titan to exoplanets

Planetary atmospheres exist in a seemingly endless variety of physical and chemical environments. There are an equally diverse number of methods by which we can study and characterize atmospheric composition. In order to better understand the fundamental chemistry and physical processes underlying all planetary atmospheres, my research of the past four years has focused on two distinct topics. First, I focused on the data analysis and spectral retrieval of observations obtained by the Ultraviolet Imaging Spectrograph (UVIS) instrument onboard the Cassini spacecraft while in orbit around Saturn. These observations consisted of stellar occultation measurements of Titan's upper atmosphere, probing the chemical composition in the region 300 to 1500 km above Titan's surface. I examined the relative abundances of Titan's two most prevalent chemical species, nitrogen and methane. I also focused on the aerosols that are formed through chemistry involving these two major species, and determined the vertical profiles of aerosol particles as a function of time and latitude. Moving beyond our own solar system, my second topic of investigation involved analysis of infra-red light curves from the Spitzer space telescope, obtained as it measured the light from stars hosting planets of their own. I focused on both transit and eclipse modeling during Spitzer data reduction and analysis. In my initial work, I utilized the data to search for transits of planets a few Earth masses in size. In more recent research, I analyzed secondary eclipses of three exoplanets and constrained the range of possible temperatures and compositions of their atmospheres.

Direct simulation of proton-coupled electron transfer reaction dynamics and mechanisms

Proton-coupled electron transfer (PCET) reactions are ubiquitous throughout chemistry and biology. However, challenges arise in both the the experimental and theoretical investigation of PCET reactions; the rare-event nature of the reactions and the coupling between quantum mechanical electron- and proton-transfer with the slower classical dynamics of the surrounding environment necessitates the development of robust simulation methodology. In the following dissertation, novel path-integral based methods are developed and employed for the direct simulation of the reaction dynamics and mechanisms of condensed-phase PCET.

Sacode a poeira e dá a volta por cima: resiliência em mulheres que vivenciaram violência sexual

O objeto deste estudo foi o processo de construção da resiliência em mulheres que vivenciaram violência sexual. A violência sexual contra a mulher é um problema antigo no mundo, onde o Brasil dispõe de elevadas estatísticas. As justificativas para a violência contra a mulher constroem-se sob normas e preceitos sociais de gênero, os quais definem as diferenças nos papéis e responsabilidades dos homens e das mulheres na sociedade e na família. As consequências físicas e psicológicas para a mulher em situação de violência sexual são alarmantes, podendo ocasionar traumas por longo prazo ou até mesmo para a vida inteira, impedindo-a de retomar seus direitos humanos e de se reinserirem em suas famílias e na sociedade. Entretanto, após a vivência de uma violência algumas mulheres têm seus comportamentos transformados a fim de retomarem o curso de suas vidas. Tais comportamentos dizem respeito à postura resiliente diante à violência sexual vivida e à sua superação. Reconhecendo este comportamento como uma nova possibilidade de promoção da saúde dessas mulheres, traçou-se como objetivo geral do estudo compreender o processo de construção da resiliência em mulheres que vivenciaram violência sexual. Desenvolveu-se uma pesquisa exploratória com abordagem qualitativa, realizada através da coleta da história de vida com seis mulheres que vivenciaram violência sexual atendidas em um hospital municipal do Rio de Janeiro (Brasil), referência no atendimento dessas mulheres. Os dados produzidos foram interpretados à luz da modalidade temática da análise de conteúdo de Bardin. Deste processo emergiram duas categorias: A violência sexual vivida expressa nas atitudes do cotidiano: sentimentos e emoções e A resiliência de mulheres em situação de violência sexual. Na primeira categoria identificaram-se as atitudes, sentimentos e emoções decorrentes da adversidade. Destacaram-se os sentimentos de medo, tristeza, culpa e perda como sendo as principais mudanças ocorridas com a violência. Na segunda categoria emergiram elementos existentes na vida das mulheres que vivenciaram violência sexual e que favoreceram no processo de construção da resiliência, sendo os aspectos individuais, familiares e sociais. A pesquisa considerou que a resiliência é elemento fundamental na promoção da saúde das mulheres que vivenciaram violência sexual assim como uma oportunidade de melhoria de sua qualidade de vida, uma vez que reduz os agravos decorrentes dessa violência e incorpora sentido de vida, serenidade, autoconfiança, autossuficiência e perseverança na vida da mulher. Contudo, a resiliência para ser desenvolvida precisa além dos aspectos individuais da mulher, uma rede de apoio familiar e social significativa e eficaz. A consulta de Enfermagem estabelecida nos princípios da humanização, integralidade e dialogicidade entre profissional e a mulher, seja nas Estratégias de Saúde da Família ou nos ambientes ambulatoriais e hospitalares, caracteriza-se como campo fértil na promoção e apoio a essa rede familiar e social. A enfermeira torna-se facilitadora na construção da resiliência em mulheres em situação de violência sexual, onde é preciso oferecer escuta sensível e sem preconceitos, incentivar a construção de sentido de vida, a recuperação da autoestima e autoconfiança e de sua reinserção social.

Neutron-neutron scattering parameters from the ^3He(d,t)2p and ^3H(d,^3He)2n reactions

The energy spectra of tritons and Helium-3 nuclei from the reactions ³He(d,t)2p, ³H(d,³He)2n, ³He(d,³He)pn, and ³H(d,t)pn were measured between 6° and 20° at a bombarding energy of 10.9 MeV. An upper limit of 5 μb/sr. was obtained for producing a bound di-neutron at 6° and 7.5°. The ³He(d,t)2p and ³H(d,³He)2n data, together with previous measurements at higher energies, have been used to investigate whether one can unambiguously extract information on the two-nucleon system from these three-body final state reactions. As an aid to these theoretical investigations, Born approximation calculations were made employing realistic nucleon-nucleon potentials and an antisymmetrized final state wave function for the five-particle system. These calculations reproduce many of the features observed in the experimental data and indicate that the role of exchange processes cannot be ignored. The results show that previous attempts to obtain information on the neutron-neutron scattering length from the ³H(d,³He)2n reaction may have seriously overestimated the precision that could be attained.

Ratio of natural isotopes of nitrogen. I. Primary results: soils of Dombes [Translation from: Revue d'Ecologie et de Biologie du Sol 14, 279-287, 1977]

Utilization of the heavy isotope of nitrogen as a tracer has found numerous applications in soil biology. It allows better definition of different stages of the nitrogen cycle, in particular the immobilization-mineralization cycle. In this work, the authors report the results of calculations of natural isotope ratios of nitrogen in samples of water, soil and vegetation prevailing in Dombes and discuss the possibilities of errors and coefficients of fractionation.