Conditional expression of Sry, and Wnt4 by gene-targeting: Studies in sexual and skeletal development

Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) locus by gene targeting in embryonic stem (ES) cells to create a conditional gene expression system in mice. In the targeted alleles, expression of these cDNAs should be blocked by a neomycin resistance selection cassette that is flanked by loxP sites. Transgene expression should be activated after the blocking cassette is deleted by Cre recombinase. ^ To test this conditional expression system, I have bred R26-stop- Sry and R26-stop-Wnt4 heterozygotes with a MisRII-Cre mouse line that expresses Cre in the gonads of both sexes. Analysis of these two types of bigenic heterozygotes indicated that their gonads developed normally like those of wild types. However, one XX R26-Sry/R26-Sry; MisR2-Cre/+ showed epididymis-like structures resembling those of males. In contrast, only normal phenotypes were observed in XY R26-Wnt4/R26-Wnt4; MisR2-Cre /+ mice. To interpret these results, I have tested for Cre recombinase activity by Southern blot and transcription of the Sry and Wnt4 transgenes by RT-PCR. Results showed that bigenic mutants had insufficient activation of the transgenes in their gonads at E12.5 and E13.5. Therefore, the failure to observe mutant phenotypes may have resulted from low activity of MisR2-Cre recombination at the appropriate time. ^ Col2a1-Cre transgenic mice express Cre in differentiating chondrocytes. R26-Wnt4; Col2a1-Cre bigenic heterozygous mice were found to exhibit a dramatic alteration in growth presumably caused by Wnt4 overexpression during chondrogenesis. R26-Wnt4; Col2a1-Cre mice exhibited dwarfism beginning approximately 10 days after birth. In addition, they also had craniofacial abnormalities, and had delayed ossification of the lumbar vertebrate and pelvic bones. Histological analysis of the growth plates of R26-Wnt4; Col2a1-Cre mice revealed less structural organization and a delay in onset of the primary and secondary ossification centers. Molecular studies confirmed that overexpression of Wnt4 causes decreased proliferation and early maturation of chondrocytes. In addition, R26-Wnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF), suggesting that defects in vascularization may contribute to the dwarf phenotype. Finally, 9-month-old R26-Wnt4; Col2a1-Cre mice had significantly more fat cells in the marrow cavities of their metaphysis long bones, implying that long-term overexpression of Wnt4may cause bone marrow pathologies. In conclusion, Wnt4 was activated by Col2a1-Cre recombinase and was overexpressed in the growth plate, resulting in aberrant proliferation and differentiation of chondrocytes, and ultimately leads to dwarfism in mice. ^

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.

Seawater carbonate chemistry and larval development and settlement of the sea urchin Arbacia lixula in a laboratory experiment

We studied the effects of temperature and pH on larval development, settlement and juvenile survival of a Mediterranean population of the sea urchin Arbacia lixula. Three temperatures (16, 17.5 and 19 °C) were tested at present pH conditions (pHT 8.1). At 19 °C, two pH levels were compared to reflect present average (pHT 8.1) and near-future average conditions (pHT 7.7, expected by 2100). Larvae were reared for 52-days to achieve the full larval development and complete the metamorphosis to the settler stage. We analyzed larval survival, growth, morphology and settlement success. We also tested the carry-over effect of acidification on juvenile survival after 3 days. Our results showed that larval survival and size significantly increased with temperature. Acidification resulted in higher survival rates and developmental delay. Larval morphology was significantly altered by low temperatures, which led to narrower larvae with relatively shorter skeletal rods, but larval morphology was only marginally affected by acidification. No carry-over effects between larvae and juveniles were detected in early settler survival, though settlers from larvae reared at pH 7.7 were significantly smaller than their counterparts developed at pH 8.1. These results suggest an overall positive effect of environmental parameters related to global change on the reproduction of A. lixula, and reinforce the concerns about the increasing negative impact on shallow Mediterranean ecosystems of this post-glacial colonizer.

Elevated pCO2 causes developmental delay in early larval Pacific oysters, Crassostrea gigas

Increasing atmospheric CO2 equilibrates with surface seawater, elevating the concentration of aqueous hydrogen ions. This process, ocean acidification, is a future and contemporary concern for aquatic organisms, causing failures in Pacific oyster (Crassostrea gigas) aquaculture. This experiment determines the effect of elevated pCO2 on the early development of C. gigas larvae from a wild Pacific Northwest population. Adults were collected from Friday Harbor, Washington, USA (48°31.7' N, 12°1.1' W) and spawned in July 2011. Larvae were exposed to Ambient (400 µatm CO2), MidCO2 (700 µatm), or HighCO2 (1,000 µatm). After 24 h, a greater proportion of larvae in the HighCO2 treatment were calcified as compared to Ambient. This unexpected observation is attributed to increased metabolic rate coupled with sufficient energy resources. Oyster larvae raised at HighCO2 showed evidence of a developmental delay by 3 days post-fertilization, which resulted in smaller larvae that were less calcified.

Effects of pre-hatching pCO2 on development and survival of zoea stages of Arctic spider crab Hyas araneus

Sensitivity of marine crustaceans to anthropogenic CO2 emissions and the associated acidification of the oceans may be less than that of other, especially lower, invertebrates. However, effects on critical transition phases or carry-over effects between life stages have not comprehensively been explored. Here we report the impact of elevated seawater PCO2 values (3100 µatm) on Hyas araneus during the last 2 weeks of their embryonic development (pre-hatching phase) and during development while in the consecutive zoea I and zoea II larval stages (post-hatching phase). We measured oxygen consumption, dry weight, developmental time and mortality in zoea I to assess changes in performance. Feeding rates and survival under starvation were investigated at different temperatures to detect differences in thermal sensitivities of zoea I and zoea II larvae depending on pre-hatch history. When embryos were pre-exposed to elevated PCO2 during maternal care, mortality increased about 60% under continued CO2 exposure during the zoea I phase. The larvae that moulted into zoea II, displayed a developmental delay by about 20 days compared to larvae exposed to control PCO2 during embryonic and zoeal phases. Elevated PCO2 caused a reduction in zoea I dry weight and feeding rates, while survival of the starved larvae was not affected by the seawater CO2 concentration. In conclusion, CO2 effects on egg masses under maternal care carried over to the first larval stages of crustaceans and reduced their survival and development to levels below those previously reported in studies exclusively focussing on acute PCO2 effects on the larval stages.

Methods and Tools for Objective Assessment of Psychomotor Skills in Laparoscopic Surgery

Training and assessment paradigms for laparoscopic surgical skills are evolving from traditional mentor–trainee tutorship towards structured, more objective and safer programs. Accreditation of surgeons requires reaching a consensus on metrics and tasks used to assess surgeons’ psychomotor skills. Ongoing development of tracking systems and software solutions has allowed for the expansion of novel training and assessment means in laparoscopy. The current challenge is to adapt and include these systems within training programs, and to exploit their possibilities for evaluation purposes. This paper describes the state of the art in research on measuring and assessing psychomotor laparoscopic skills. It gives an overview on tracking systems as well as on metrics and advanced statistical and machine learning techniques employed for evaluation purposes. The later ones have a potential to be used as an aid in deciding on the surgical competence level, which is an important aspect when accreditation of the surgeons in particular, and patient safety in general, are considered. The prospective of these methods and tools make them complementary means for surgical assessment of motor skills, especially in the early stages of training. Successful examples such as the Fundamentals of Laparoscopic Surgery should help drive a paradigm change to structured curricula based on objective parameters. These may improve the accreditation of new surgeons, as well as optimize their already overloaded training schedules.

Development of mathematical models to elaborate strategies, select alternatives and development of plans for adaptation of communities to climate change in different geographical areas including costs to implement it.

There is evidence that the climate changes and that now, the change is influenced and accelerated by the CO2 augmentation in atmosphere due to combustion by humans. Such ?Climate change? is on the policy agenda at the global level, with the aim of understanding and reducing its causes and to mitigate its consequences. In most countries and international organisms UNO (e.g. Rio de Janeiro 1992), OECD, EC, etc . . . the efforts and debates have been directed to know the possible causes, to predict the future evolution of some variable conditioners, and trying to make studies to fight against the effects or to delay the negative evolution of such. The Protocol of Kyoto 1997 set international efforts about CO2 emissions, but it was partial and not followed e.g. by USA and China . . . , and in Durban 2011 the ineffectiveness of humanity on such global real challenges was set as evident. Among all that, the elaboration of a global model was not boarded that can help to choose the best alternative between the feasible ones, to elaborate the strategies and to evaluate the costs, and the authors propose to enter in that frame for study. As in all natural, technological and social changes, the best-prepared countries will have the best bear and the more rapid recover. In all the geographic areas the alternative will not be the same one, but the model must help us to make the appropriated decision. It is essential to know those areas that are more sensitive to the negative effects of climate change, the parameters to take into account for its evaluation, and comprehensive plans to deal with it. The objective of this paper is to elaborate a mathematical model support of decisions, which will allow to develop and to evaluate alternatives of adaptation to the climatic change of different communities in Europe and Latin-America, mainly in especially vulnerable areas to the climatic change, considering in them all the intervening factors. The models will consider criteria of physical type (meteorological, edaphic, water resources), of use of the ground (agriculturist, forest, mining, industrial, urban, tourist, cattle dealer), economic (income, costs, benefits, infrastructures), social (population), politician (implementation, legislation), educative (Educational programs, diffusion) and environmental, at the present moment and the future. The intention is to obtain tools for aiding to get a realistic position for these challenges, which are an important part of the future problems of humanity in next decades.

Development of mathematical models to elaborate strategies, to select alternatives and the development of plans for the adaptation of communities to climate change in different geographical areas including costs to implement it

Climate change is on the policy agenda at the global level, with the aim of understanding and reducing its causes and to mitigate its consequences. In most of the countries and international organisms UNO, OECD, EC, etc … the efforts and debates have been directed to know the possible causes, to predict the future evolution of some variable conditioners, and trying to make studies to fight against the effects or to delay the negative evolution of such. Nevertheless, the elaboration of a global model was not boarded that can help to choose the best alternative between the feasible ones, to elaborate the strategies and to evaluate the costs. As in all natural, technological and social changes, the best-prepared countries will have the best bear and the more rapid recover. In all the geographic areas the alternative will not be the same one, but the model should help us to make the appropriated decision. It is essential to know those areas that are more sensitive to the negative effects of climate change, the parameters to take into account for its evaluation, and comprehensive plans to deal with it. The objective of this paper is to elaborate a mathematical model support of decisions, that will allow to develop and to evaluate alternatives of adaptation to the climatic change of different communities in Europe and Latin-America, mainly, in vulnerable areas to the climatic change, considering in them all the intervening factors. The models will take into consideration criteria of physical type (meteorological, edaphic, water resources), of use of the ground (agriculturist, forest, mining, industrial, urban, tourist, cattle dealer), economic (income, costs, benefits, infrastructures), social (population), politician (implementation, legislation), educative (Educational programs, diffusion), sanitary and environmental, at the present moment and the future.

Large aperture antenna arrays with subband delay compensation for reception of wideband satellite signals

Con esta disertación se pretenden resolver algunos de los problemas encontrados actualmente en la recepción de señales de satélites bajo dos escenarios particularmente exigentes: comunicaciones de Espacio Profundo y en banda Ka. Las comunicaciones con sondas de Espacio Profundo necesitan grandes aperturas en tierra para poder incrementar la velocidad de datos. La opción de usar antennas con diámetro mayor de 35 metros tiene serios problemas, pues antenas tan grandes son caras de mantener, difíciles de apuntar, pueden tener largos tiempo de reparación y además tienen una efeciencia decreciente a medida que se utilizan bandas más altas. Soluciones basadas en agrupaciones de antenas de menor tamaño (12 ó 35 metros) son mas ecónomicas y factibles técnicamente. Las comunicaciones en banda Ka tambien pueden beneficiarse de la combinación de múltiples antennas. Las antenas de menor tamaño son más fáciles de apuntar y además tienen un campo de visión mayor. Además, las técnicas de diversidad espacial pueden ser reemplazadas por una combinación de antenas para así incrementar el margen del enlace. La combinación de antenas muy alejadas sobre grandes anchos de banda, bien por recibir una señal de banda ancha o múltiples de banda estrecha, es complicada técnicamente. En esta disertación se demostrará que el uso de conformador de haz en el dominio de la frecuencia puede ayudar a relajar los requisitos de calibración y, al mismo tiempo, proporcionar un mayor campo de visión y mayores capacidades de ecualización. Para llevar esto a cabo, el trabajo ha girado en torno a tres aspectos fundamentales. El primero es la investigación bibliográfica del trabajo existente en este campo. El segundo es el modelado matemático del proceso de combinación y el desarrollo de nuevos algoritmos de estimación de fase y retardo. Y el tercero es la propuesta de nuevas aplicaciones en las que usar estas técnicas. La investigación bibliográfica se centra principalmente en los capítulos 1, 2, 4 y 5. El capítulo 1 da una breve introducción a la teoría de combinación de antenas de gran apertura. En este capítulo, los principales campos de aplicación son descritos y además se establece la necesidad de compensar retardos en subbandas. La teoría de bancos de filtros se expone en el capítulo 2; se selecciona y simula un banco de filtros modulado uniformemente con fase lineal. Las propiedades de convergencia de varios filtros adaptativos se muestran en el capítulo 4. Y finalmente, las técnicas de estimación de retardo son estudiadas y resumidas en el capítulo 5. Desde el punto de vista matemático, las principales contribución de esta disertación han sido: • Sección 3.1.4. Cálculo de la desviación de haz de un conformador de haz con compensación de retardo en pasos discretos en frecuencia intermedia. • Sección 3.2. Modelo matemático de un conformador de haz en subbandas. • Sección 3.2.2. Cálculo de la desviación de haz de un conformador de haz en subbandas con un buffer de retardo grueso. • Sección 3.2.4. Análisis de la influencia de los alias internos en la compensación en subbandas de retardo y fase. • Sección 3.2.4.2. Cálculo de la desviación de haz de un conformador de haz con compensación de retardo en subbandas. • Sección 3.2.6. Cálculo de la ganancia de relación señal a ruido de la agrupación de antenas en cada una de las subbandas. • Sección 3.3.2. Modelado de la función de transferencia de la agrupación de antenas bajo errores de estimación de retardo. • Sección 3.3.3. Modelado de los efectos de derivas de fase y retardo entre actualizaciones de las estimaciones. • Sección 3.4. Cálculo de la directividad de la agrupación de antenas con y sin compensación de retardos en subbandas. • Sección 5.2.6. Desarrollo de un algorimo para estimar la fase y el retardo entre dos señales a partir de su descomposición de subbandas bajo entornos estacionarios. • Sección 5.5.1. Desarrollo de un algorimo para estimar la fase, el retardo y la deriva de retardo entre dos señales a partir de su descomposición de subbandas bajo entornos no estacionarios. Las aplicaciones que se pueden beneficiar de estas técnicas son descritas en el capítulo 7: • Sección 6.2. Agrupaciones de antenas para comunicaciones de Espacio Profundo con capacidad multihaz y sin requisitos de calibración geométrica o de retardo de grupo. • Sección 6.2.6. Combinación en banda ancha de antenas con separaciones de miles de kilómetros, para recepción de sondas de espacio profundo. • Secciones 6.4 and 6.3. Combinación de estaciones remotas en banda Ka en escenarios de diversidad espacial, para recepción de satélites LEO o GEO. • Sección 6.3. Recepción de satélites GEO colocados con arrays de antenas multihaz. Las publicaciones a las que ha dado lugar esta tesis son las siguientes • A. Torre. Wideband antenna arraying over long distances. Interplanetary Progress Report, 42-194:1–18, 2013. En esta pulicación se resumen los resultados de las secciones 3.2, 3.2.2, 3.3.2, los algoritmos en las secciones 5.2.6, 5.5.1 y la aplicación destacada en 6.2.6. • A. Torre. Reception of wideband signals from geostationary collocated satellites with antenna arrays. IET Communications, Vol. 8, Issue 13:2229–2237, September, 2014. En esta segunda se muestran los resultados de la sección 3.2.4, el algoritmo en la sección 5.2.6.1 , y la aplicación mostrada en 6.3. ABSTRACT This dissertation is an attempt to solve some of the problems found nowadays in the reception of satellite signals under two particular challenging scenarios: Deep Space and Ka-band communications. Deep Space communications require from larger apertures on ground in order to increase the data rate. The option of using single dishes with diameters larger than 35 meters has severe drawbacks. Such antennas are expensive to maintain, prone to long downtimes, difficult to point and have a degraded performance in high frequency bands. The array solution, either with 12 meter or 35 meter antennas is deemed to be the most economically and technically feasible solution. Ka-band communications can also benefit from antenna arraying technology. The smaller aperture antennas that make up the array are easier to point and have a wider field of view allowing multiple simultaneous beams. Besides, site diversity techniques can be replaced by pure combination in order to increase link margin. Combination of far away antennas over a large bandwidth, either because a wideband signal or multiple narrowband signals are received, is a demanding task. This dissertation will show that the use of frequency domain beamformers with subband delay compensation can help to ease calibration requirements and, at the same time, provide with a wider field of view and enhanced equalization capabilities. In order to do so, the work has been focused on three main aspects. The first one is the bibliographic research of previous work on this subject. The second one is the mathematical modeling of the array combination process and the development of new phase/delay estimation algorithms. And the third one is the proposal of new applications in which these techniques can be used. Bibliographic research is mainly done in chapters 1, 2, 4 and 5. Chapter 1 gives a brief introduction to previous work in the field of large aperture antenna arraying. In this chapter, the main fields of application are described and the need for subband delay compensation is established. Filter bank theory is shown in chapter 2; a linear phase uniform modulated filter bank is selected and simulated under diverse conditions. The convergence properties of several adaptive filters are shown in chapter 4. Finally, delay estimation techniques are studied and summarized in chapter 5. From a mathematical point of view, the main contributions of this dissertation have been: • Section 3.1.4. Calculation of beam squint of an IF beamformer with delay compensation at discrete time steps. • Section 3.2. Establishment of a mathematical model of a subband beamformer. • Section 3.2.2. Calculation of beam squint in a subband beamformer with a coarse delay buffer. • Section 3.2.4. Analysis of the influence of internal aliasing on phase and delay subband compensation. • Section 3.2.4.2. Calculation of beam squint of a beamformer with subband delay compensation. • Section 3.2.6. Calculation of the array SNR gain at each of the subbands. • Section 3.3.2. Modeling of the transfer function of an array subject to delay estimation errors. • Section 3.3.3. Modeling of the effects of phase and delay drifts between estimation updates. • Section 3.4. Calculation of array directivity with and without subband delay compensation. • Section 5.2.6. Development of an algorithm to estimate relative delay and phase between two signals from their subband decomposition in stationary environments. • Section 5.5.1. Development of an algorithm to estimate relative delay rate, delay and phase between two signals from their subband decomposition in non stationary environments. The applications that can benefit from these techniques are described in chapter 7: • Section 6.2. Arrays of antennas for Deep Space communications with multibeam capacity and without geometric or group delay calibration requirement. • Section 6.2.6. Wideband antenna arraying over long distances, in the range of thousands of kilometers, for reception of Deep Space probes. • Sections 6.4 y 6.3. Combination of remote stations in Ka-band site diversity scenarios for reception of LEO or GEO satellites. • Section 6.3. Reception of GEO collocated satellites with multibeam antenna arrays. The publications that have been made from the work in this dissertation are • A. Torre. Wideband antenna arraying over long distances. Interplanetary Progress Report, 42-194:1–18, 2013. This article shows the results in sections 3.2, 3.2.2, 3.3.2, the algorithms in sections 5.2.6, 5.5.1 and the application in section 6.2.6. • A. Torre. Reception of wideband signals from geostationary collocated satellites with antenna arrays. IET Communications, Vol. 8, Issue 13:2229–2237, September, 2014. This second article shows among others the results in section 3.2.4, the algorithm in section 5.2.6.1 , and the application in section 6.3.

Molecular heterochrony in the early development of Drosophila

Heterochrony, the relative change of developmental timing, is one of the major modes of macroevolutionary change; it identifies temporally disassociated units of developmental evolution. Here, we report the results of a fine-scale temporal study for the expression of the developmental gene hairy and morphological development in three species of Drosophila, D. melanogaster, D. simulans, and D. pseudoobscura. The results suggest that between and among closely related species, temporal displacement of ontogenetic trajectory is detected even at the earliest stage of development. Overall, D. simulans shows the earliest expression, followed by D. melanogaster, and then by D. pseudoobscura. Setting D. melanogaster as the standard, we find the approximate time to full expression is accelerated by 13 min, 48 s in D. simulans and retarded by 24 min in D. pseudoobscura. Morphologically, again with D. melanogaster setting the standard, initiation of cellularization is faster in D. simulans by 15 min, 42 s; and initiation of morphogenesis is faster in D. simulans by 18 min, 7 s. These results seem to be consistent with the finding that the approximate time to full expression of hairy is accelerated by 13 min, 48 s in D. simulans. On the other hand, the same morphological events are delayed by 5 min, 32 s, and by 11 min, 32 s, respectively, in D. pseudoobscura. These delays are small, compared with the 24-min delay in full expression. The timing changes, in total, seem consistent with continuous phyletic evolution of temporal trajectories. Finally, we speculate that epigenetic interactions of hairy expression timing and cell-cycle timing may have led to morphological differences in the terminal system of the larvae.

Research on vehicle-based driver status/performance monitoring; development, validation, and refinement of algorithms for detection of driver drowsiness.

National Highway Traffic Safety Administration, Washington, D.C.

Sobering agent: effectiveness measurement and development. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Development and field test of psychophysical tests for DWI arrest. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Visual detection of driving while intoxicated. Project interim report: identification of visual cues and development of detection methods.

National Highway Traffic Safety Administration, Washington, D.C.

Development of a visibility research capability. Final report.

National Highway Traffic Safety Administration, Washington, D.C.