Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Reactivity of titanocene methylidene with metal halides, alkene sulfides, and alkene oxides

Titanocene metallacyclobutanes show a wide variety of reactivites with organic and inorganic reagents. Their reactions include methylene transfer to organic carbonyls, formation of enolates, electron transfer from activated alkyl chlorides, olefin metathesis, ring opening polymerization. Recently, preparations of heterobinuclear µ-methylene complexes were reported. In this thesis, mechanistic, synthetic, and structural studies of the heterobinuclear µ-methylene complexes will be described. Also, the reaction of titanocene methylidene trimethylphosphine complex with alkene sulfide and styrene sulfide will be presented.

Heterobinuclear µ-methylene-µ-methyl complexes C_(p2)Ti(µ-CH_2)( µ-CH_3)M(1,5-COD) have been prepared (M = Rh, Ir). X-ray crystallography showed that the methyl group of the complex was bonded to the rhodium and bridges to the titanium through an agostic bond. The ^(1)H,^(13)CNMR, IR spectra along with partial deuteration studies supported the structure in both solution and solid state. Activation of the agostic bond is demonstrated by the equilibration of the µ-CH_3 and µ-CH_2 groups. A nonlinear Arrhenius plot, an unusually large kinetic isotope effect (24(5)), and a large negative activation entropy (-64(3)eu) can be explained by the quantum-mechanical tunneling. Calculated rate constants with Bell-type barrier fitted well with the observed one. This equilibration was best explained by a 4e-4c mechanism (or σ bond metathesis) with the character of quantum-mechanical tunneling.

Heterobinuclear µ-methylene-µ-phenyl complexes were synthesized. Structural study of C_(p2)Ti(µ-CH_(2))(µ-p-Me_(2)NC_(6)H_(4))Rh(l,5-COD) showed that the two metal atoms are bridged by the methylene carbon and the ipso carbon of the p-N,N-dimethylarninophenyl group. The analogous structure of C_(p2))Ti(µ-CH_(2))(µ-o-MeOC_(6)H_(4))Rh(1,5-COD) has been verified by the differential NOE. The aromaticity of the phenyl group observed by ^(1)H NMR, was confirmed by the comparison of the C-C bond lengths in the crystallographic structure. The unusual downfield shifts of the ipso carbon in the ^(13)C NMR are assumed to be an indication of the interaction between the ipso carbon and electron-deficient titanium.

Titanium-platinum heterobinuclear µ-methylene complexes C_(p2)Ti(µ-CH_(2))(µ -X)Pt(Me)(PM_(2)Ph) have been prepared (X= Cl, Me). Structural studies indicate the following:(1) the Ti-CH2 bond possesses residual double bond character, (2) there is a dative Pt→Ti interaction which may be regarded as a π back donation from the platinum atom to the 'Ti=CH_(2)'' group, and (3) the µ-CH_3 group is bound to the titanium atom through a three-center, two-electron agostic bond.

Titanocene (η^(2)-thioformaldehyde)•PMe_3 was prepared from C_(p2)Ti=CH_(2)•PMe_3 and sulfur-containing organic compounds (e.g. alkene sulfide, triphenylphosphine sulfide) including elemental sulfur. Mechanistic studies utilizing trans-styrene sulfide-d_1 suggested the stepwise reaction to explain equimolar mixture of trans- and cis-styrene-d_1 as by-products. The product reacted with methyl iodide to produce cationic titanocene (η_(2)-thiomethoxymethyl) complex. Complexes having less coordinating anion like BF_4 or BPh_4 could be obtained through metathesis. Together with structural analyses, the further reactivities of the complexes have been explored.

The complex C_(p2)TiOCH_(2)CH(Ph)CH_2 was prepared from the compound C_(p2)Ti=CH_(2)-PMe_3 and styrene oxide. The product was characterized with ^(1)H-^(1)H correlated 2-dimensional NMR, selective decoupling of ^(1)H NMR, and differential NOE. Stereospecificity of deuterium in the product was lost when trans-styrene oxide-d_1 was allowed to react. Relative rates of the reaction were measured with varying substituents on the phenyl ring. Better linearity (r = -0.98, p^(+) = -0.79) was observed with σ_(p)^(+)than σ(r = -0.87, p = -1.26). The small magnitude of p^+ value and stereospecificity loss during the formation of product were best explained by the generation of biradicals, but partial generation of charge cannot be excluded. Carbonylation of the product followed by exposure to iodine yields the corresponding β-phenyl γ-lactone.

Electron energy-loss spectroscopy study of polyatomic molecular systems under pyrolytic conditions

The technique of variable-angle, electron energy-loss spectroscopy has been used to study the electronic spectroscopy of the diketene molecule. The experiment was performed using incident electron beam energies of 25 eV and 50 eV, and at scattering angles between 10° and 90°. The energy-loss region from 2 eV to 11 eV was examined. One spin-forbidden transition has been observed at 4.36 eV and three others that are spin-allowed have been located at 5.89 eV, 6.88 eV and 7.84 eV. Based on the intensity variation of these transitions with impact energy and scattering angle, and through analogy with simpler molecules, the first three transitions are tentatively assigned to an n → π* transition, a π - σ* (3s) Rydberg transition and a π → π* transition.

Thermal decomposition of chlorodifluoromethane, chloroform, dichloromethane and chloromethane under flash-vacuum pyrolysis conditions (900-1100°C) was investigated by the technique of electron energy-loss spectroscopy, using the impact energy of 50 eV and a scattering angle of 10°. The pyrolytic reaction follows a hydrogen-chloride α-elimination pathway. The difluoromethylene radical was produced from chlorodifluoromethane pyrolysis at 900°C and identified by its X^1 A_1 → A^1B_1 band at 5.04 eV.

Finally, a number of exploratory studies have been performed. The thermal decomposition of diketene was studied under flash vacuum pressures (1-10 mTorr) and temperatures ranging from 500°C to 1000°C. The complete decomposition of the diketene molecule into two ketene molecules was achieved at 900°C. The pyrolysis of trifluoromethyl iodide molecule at 1000°C produced an electron energy-loss spectrum with several iodine-atom, sharp peaks and only a small shoulder at 8.37 eV as a possible trifluoromethyl radical feature. The electron energy-loss spectrum of trichlorobromomethane at 900°C mainly showed features from bromine atom, chlorine molecule and tetrachloroethylene. Hexachloroacetone decomposed partially at 900°C, but showed well-defined features from chlorine, carbon monoxide and tetrachloroethylene molecules. Bromodichloromethane molecule was investigated at 1000°C and produced a congested, electron energy-loss spectrum with bromine-atom, hydrogen-bromide, hydrogen-chloride and tetrachloroethylene features.

Síntese, caracterização e avaliação da capacidade bactericida de copolímeros à base de 2-vinilpiridina funcionalizados

Neste trabalho, dois copolímeros à base de 2-vinilpiridina (2Vpy), estireno (Sty) e divinilbenzeno (DVB) foram sintetizados empregando a técnica de polimerização em suspensão aquosa via radical livre. Os copolímeros com diferentes características morfológicas foram preparados variando a composição da mistura diluente, constituída por tolueno e n-heptano, solventes solvatantes e não solvatantes para as cadeias poliméricas. A caracterização estrutural desses materiais foi feita através de medidas de área específica, volume de poros, diâmetro médio de poros e densidade aparente. Além disso, esses materiais foram avaliados por microscopia ótica e eletrônica de varredura, por espectroscopia na região do infravermelho (FTIR), análise termogravimétrica (TGA) e análise elementar. Os copolímeros Sty-DVB-2Vpy foram modificados quimicamente através de reação de quaternização das unidades de 2Vpy usando dois reagentes: iodeto de metila e acrilonitrila. A quaternização dos copolímeros Sty-DVB-2Vpy foi confirmada através de espectroscopia de infravermelho, pelo aparecimento das bandas de absorção características do íon peridíneo e do grupo nitrila e também pela análise do comportamento térmico. Os produtos dessas reações foram submetidos à avaliação da capacidade bactericida através do método de contagem em placas contra suspensão de Escherichia coli. O copolímero do tipo gel quaternizado com iodeto de metila apresentou a maior ação bactericida registrada, com eficiência até a concentração de 104 células/mL. Contudo, a maioria dos materiais quaternizados não apresentou ação biocida significativa. Com o objetivo de maximizar a atividade bactericida dos materiais preparados, os copolímeros quaternizados e não quaternizados foram impregnados com iodo por meio de duas metodologias: em solução e em fase vapor. O teor de iodo incorporado foi quantificado por análise gravimétrica. Foi possível observar que os copolímeros quaternizados e impregnados com iodo se mostraram mais eficientes como agentes bactericidas que os copolímeros não funcionalizados impregnados com iodo. De uma forma geral, foi possível perceber que a ação bactericida dos polímeros é fruto da associação entre as suas características de porosidade, o grau de quaternização alcançado e o teor de iodo incorporado. Para efeito de comparação foram feitos também ensaios bactericidas com uma resina comercial à base de Sty-DVB com grupo amônio quaternário, VP OC 1950. Os testes mostraram que a resina comercial não possui atividade bactericida. A impregnação de iodo a essa resina comercial forneceu um material com ação biocida semelhante à do copolímero do tipo gel, quaternizado com iodeto de metila e impregnado com iodo

Silicide formation and the interaction of metals with polycrystalline Si

The main factors affecting solid-phase Si-metal interactions are reported in this work. The influence of the orientation of the Si substrates and the presence of impurities in metal films and at the Si-metal interface on the formation of nickel and chromium silicides have been demonstrated. We have observed that the formation and kinetic rate of growth of nickel silicides is strongly dependent on the orientation and crystallinity of the Si substrates; a fact which, up to date, has never been seriously investigated in silicide formation. Impurity contaminations in the Cr film and at the Si-Cr interface are the most dominant influencing factors in the formation and kinetic rate of growth of CrSi₂. The potentiality and use of silicides as a diffusion barrier in metallization on silicon devices were also investigated.

Two phases, Ni₂Si and NiSi, form simultaneously in two distinct sublayers in the reaction of Ni with amorphous Si, while only the former phase was observed on other substrates. On (111) oriented Si substrates the growth rate is about 2 to 3 times less than that on <100> or polycrystalline Si. Transmission electron micrographs establish-·that silicide layers grown on different substrates have different microcrystalline structures. The concept of grain-boundary diffusion is speculated to be an important factor in silicide formation.

The composition and kinetic rate of CrSi₂ formation are not influenced by the underlying Si substrate. While the orientation of the Si substrate does not affect the formation of CrSi₂ , the purity of the Cr film and the state of Si-Cr interface become the predominant factors in the reaction process. With an interposed layer of Pd₂Si between the Cr film and the Si substrate, CrSi₂ starts to form at a much lower temperature (400°C) relative to the Si-Cr system. However, the growth rate of CrSi₂ is observed to be independent of the thickness of the Pd2Si layer. For both Si-Cr and Si-Pd₂Si-Cr samples, the growth rate is linear with time with an activation energy of 1.7 ± 0.1 ev.

A tracer technique using radioactive ³¹Si (T_1/2 = 2.26 h) was used to study the formation of CrSi₂ on Pd₂Si. It is established from this experiment that the growth of CrSi₂ takes place partly by transport of Si directly from the Si substrate and partly by breaking Pd₂Si bonds, making free Si atoms available for the growth process.

The role of CrSi₂ in Pd-Al metallization on Si was studied. It is established that a thin CrSi₂ layer can be used as a diffusion barrier to prevent Al from interacting with Pd₂Si in the Pd-Al metallization on Si.

As a generalization of what has been observed for polycrystalline-Si-Al interaction, the reactions between polycrystalline Si (poly Si) and other metals were studied. The metals investigated include Ni, Cr, Pd, Ag and Au. For Ni, Cr and Pd, annealing results in silicide formation, at temperatures similar to those observed on single crystal Si substrates. For Al, Ag and Au, which form simple eutectics with Si annealing results in erosion of the poly Si layer and growth of Si crystallites in the metal films.

Backscattering spectrometry with 2.0 and 2.3 MeV ⁴He ions was the main analytical tool used in all our investigations. Other experimental techniques include the Read camera glancing angle x-ray diffraction, scanning electron, optical and transmission electron microscopy. Details of these analytical techniques are given in Chapter II.

Estudo da correlação entre as características da matéria-prima graxa e as propriedades físico-químicas do biodiesel

A possibilidade de utilização de várias matérias graxas na produção do biodiesel nacional é um fator diferencial em relação aos outros países e requer um conhecimento mais profundo das particularidades do biodiesel obtido a partir de matérias-primas tão distintas. Neste contexto, se insere a presente pesquisa, em que se buscou estabelecer correlações entre algumas propriedades do biodiesel e a fonte oleaginosa que lhe deu origem. O biodiesel foi obtido a partir da reação de transesterificação etílica de dez diferentes matérias-primas graxas (óleos refinados de soja, arroz, canola, girassol, milho, oliva, cyclus (mistura contendo óleos de canola, milho e girassol); óleos brutos de mamona e murumuru, além de resíduo de fritura), utilizando etanolato de potássio como agente catalítico, em um reator com sistema de refluxo a 70C durante uma hora. Após purificação, foram determinadas algumas propriedades do biodiesel (massa específica, viscosidade, índice de acidez e índice de iodo) e da matéria-prima (massa específica, viscosidade, índice de acidez, índice de iodo e composição) que lhe deu origem. Os resultados obtidos nas caracterizações geraram gráficos de correlações entre os diversos parâmetros e os dados foram analisados estatisticamente pelo método de correlações canônicas. Para todas as amostras de biodiesel, os valores obtidos nas caracterizações foram compatíveis com as especificações estabelecidas pela ANP para o produto, com exceção do biodiesel de murumuru (índice de acidez elevado e viscosidade baixa) e do biodiesel de mamona (viscosidade e massa específica elevadas). Os dados estatísticos demonstram altas correlações entre o biodiesel (massa específica (87,4%), viscosidade (98,5%) e índice de acidez (82,8%)) e as matérias-primas (massa específica (92,1%) e viscosidade (99,3%)). Além disso, existe uma grande correlação entre o índice de iodo (84,5%) do biodiesel e o índice de iodo (77,2%) e a massa molar dos ésteres na faixa de C16-C18 (MMTG) presentes na matéria-prima (89,1%). Estes resultados estatísticos ratificam as observações analíticas

Mechanism of mass transfer in the formation of hydrothermal deposits of sulphides [Translation from: Trudy Inst.Geol.rudn.Mesterozh. 16, 80 [pages 44-46 ”Solubility of sulphides of iron” only], 1958]

This partial translation of the original paper provides the summary of this study of the mechanism of mass transfer in the formation of hydrothermal deposits of sulphides. For determining the solubility of sulphides of iron, the radioactive isotope Fe59 was used. The solubility of two sulphides was determined.

Biossorção de tório com emprego de Sargassum filipendula

Com o aumento do rigor para descarte de efluentes contaminado com metais pesados, as pesquisas têm se intensificado na busca de métodos de remoção, que tragam bons resultados de captação dos metais, aliado a um baixo custo. O uso de biomassas como bactérias, fungos e algas marinhas como material adsorvente, tem se apresentado como uma alternativa, principalmente para soluções com baixo teor de metais. Neste trabalho, a alga marinha Sargassum filipendula foi avaliada na sua capacidade de remoção do metal tório de uma solução sintética e do efluente dos laboratórios de análises ambientais do IRD. A cinética desta biossorção foi estudada em regime de batelada e o equilíbrio foi alcançado com 180 min de reação. Dois modelos cinéticos foram utilizados nesta avaliação, um de primeira ordem e um modelo de pseudo segunda ordem, tendo o modelo de segunda ordem apresentado um melhor ajuste dos dados. Na avaliação da capacidade máxima de captação do tório pela biomassa marinha em regime de batelada, foi construída a isoterma que apresentou um perfil crescente na captação alcançando um máximo de 2,59 mol/g. Os modelos de Langmuir e Freundlich foram utilizados para ajustar os dados da isoterma, tendo apresentado maior correlação com os dados o modelo de Langmuir, resultando num valor de captação máxima calculado pelo modelo de 2,92 mol/g. A capacidade de remoção do metal tório da alga Sargassum filipendula também foi avaliada em regime contínuo. Um estudo de altura crítica de leito foi realizado preenchendo-se uma coluna com diferentes massas de alga correspondendo a diferentes alturas de leito. A concentração de tório residual foi quantificada na solução de saída e a menor concentração na saída foi alcançada com 40 cm de leito ou 96 g de biomassa. Após este estudo um sistema contínuo com duas colunas, com 96 g de biomassa cada, e uma bomba peristáltica foi utilizado para o tratamento do efluente real do IRD, contendo não apenas o metal tório, mas outros metais como urânio, cálcio, cromo, ferro, chumbo, etc. Cento e cinco litros de efluente foram tratados numa concentração de 6 mg/L, a concentração do efluente de saída foi de 3,75 mg/L. A caracterização do efluente quanto aos metais presentes foi realizada em ICP-MS, os resultados demonstraram que não houve competição pelos sítios ligantes da biomassa entre o tório e os demais metais. Além disso, alguns metais como cálcio, ferro e magnésio, tiveram um aumento na concentração de saída indicando a presença do mecanismo de troca iônica na biossorção do tório por Sargassum filipendula

Growth rates of bacteria in rivers

Three different methods for determining bacterial growth rate in rivers are described. Two of the methods are for bacteria in suspension: a recirculating experimental channel method and a radioactive tracer technique using super(35)SO sub(4). The third method is for bacteria attached to surfaces and specifically considers the surface of the common duckweed Lemna minor).

Long-term natural acidification of upland sites in Cumbria: Evidence from post-glacial lake sediments

Studies by the Freshwater Biological Association over the last 25 years have supplied data relevant to the levels of acidity in local soils and water before the onset of industrial pollution and current interest in acid rain. This article reviews published analysis from cores of lake sediments, in or near the catchment of the River Duddon. Electron spin resonance spectra of humic acids and iodine values confirm evidence from pollen analysis for a history of progressive acidification of the source material of lake sediments since before 5000 radiocarbon years, in upland catchments of the Lake District. Processes involved included: removal of basic ions from soils by rainfall, the effects of which were intensified by removal by man of deciduous forest; acidification of soils and waters by decomposition products of Calluna and further acidification of waters by Sphagnum species which colonized habitats where drainage became impeded by paludification processes.

Beta and gamma vibrational bands in deformed nuclei

Energies and relative intensities of gamma transitions in ¹⁵²Sm, ¹⁵²Gd, ¹⁵⁴Gd, ¹⁶⁶Er, and ²³²U following radioactive decay have been measured with a Ge(Li) spectrometer. A peak fitting program has been developed to determine gamma ray energies and relative intensities with precision sufficient to give a meaningful test of nuclear models. Several previously unobserved gamma rays were placed in the nuclear level schemes. Particular attention has been paid to transitions from the beta and gamma vibrational bands, since the gamma ray branching ratios are sensitive tests of configuration mixing in the nuclear levels. As the reduced branching ratios depend on the multipolarity of the gamma transitions, experiments were performed to measure multipole mixing ratios for transitions from the gamma vibrational band. In ¹⁵⁴Gd, angular correlation experiments showed that transitions from the gamma band to the ground state band were predominantly electric quadrupole, in agreement with the rotational model. In ²³²U, the internal conversion spectrum has been studied with a Si(Li) spectrometer constructed for electron spectroscopy. The strength of electric monopole transitions and the multipolarity of some gamma transitions have been determined from the measured relative electron intensities.

The results of the experiments have been compared with the rotational model and several microscopic models. Relative B(E2) strengths for transitions from the gamma band in ²³²U and ¹⁶⁶Er are in good agreement with a single parameter band mixing model, with values of z₂= 0.025(10) and 0.046(2), respectively. Neither the beta nor the gamma band transition strengths in ¹⁵²Sm and ¹⁵⁴Gd can be accounted for by a single parameter theory, nor can agreement be found by considering the large mixing found between the beta and gamma bands. The relative B(E2) strength for transitions from the gamma band to the beta band in ²³²U is found to be five times greater than the strength to the ground state band, indicating collective transitions with strength approximately 15 single particle units.

Construção e automação de um mapa de vulnerabilidade de solos ao 137Cs.

O comportamento dos radionuclídeos no solo pode variar de acordo com sua interação com os elementos que compões este solo. O fator de transferência (FT) é o parâmetro que descreve a interação que ocorre entre o solo e as plantas para um determinado radionuclídeo, observando que este solo tem propriedades químicas e físicas que favorecem seu crescimento. Através de ferramentas computacionais e baseado em solos extremos, com o FT conhecidos na literatura e nos parâmetros de solo que interferem no comportamento de 137Cs (como K trocável, capacidade de troca catiônica e pH), este trabalho visa aplicar técnicas de geoprocessamento para a criação de um mapa de vulnerabilidade de solos ao 137Cs e sua automação. Este estudo mostra que o uso de técnicas de geoprocessamento visando o mapeamento da vulnerabilidade ao 137Cs pode ser uma ferramenta importante para o planejamento de ações de emergência em áreas rurais, a identificação de áreas risco à contaminação radioativa, na escolha de ações corretivas adequadas, bem como no suporte a criação de políticas públicas.

Evaluación del impacto radiológico ambiental de una industria NORM

[ES]El proceso de soldadura más común es la soldadura por arco metálico con electrodos revestidos. A veces ese revestimiento contiene materiales radiactivos de origen natural (NORMs). En España los electrodos más utilizados son los recubiertos de rutilo mezclado con otros materiales. El rutilo contiene algunos radionúclidos naturales detectables, por lo que puede ser considerado como un NORM. Este trabajo principalmente se centra en la aplicación de la metodología expuesta en la Guía de Seguridad 11.3 del Consejo de Seguridad Nuclear (Metodología para la evaluación del impacto radiológico en las industrias NORM), como una herramienta para obtener las dosis en una fábrica que produce este tipo de electrodo y evaluar el impacto radiológico en una instalación específica. Para ello, se aplicaron en dicha instalación los pasos requeridos por la metodología para su cumplimiento. Se analizaron los beneficios inherentes al estudio y se identificaron las zonas de radiación más altas así como la posición de los trabajadores. Habiendo hecho uso de evaluaciones de dosis llevadas a cabo con anterioridad por otros procedimientos, métodos de simulación, se establecieron las pautas de protección radiológica a seguir para el cumplimiento de dicha guía, mediante la colocación de dosímetros personales y monitores de radiación.