Mouse model of human beta zero thalassemia: targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells.

beta zero-Thalassemia is an inherited disorder characterized by the absence of beta-globin polypeptides derived from the affected allele. The molecular basis for this deficiency is a mutation of the adult beta-globin structural gene or cis regulatory elements that control beta-globin gene expression. A mouse model of this disease would enable the testing of therapeutic regimens designed to correct the defect. Here we report a 16-kb deletion that includes both adult beta-like globin genes, beta maj and beta min, in mouse embryonic stem cells. Heterozygous animals derived from the targeted cells are severely anemic with dramatically reduced hemoglobin levels, abnormal red cell morphology, splenomegaly, and markedly increased reticulocyte counts. Homozygous animals die in utero; however, heterozygous mice are fertile and transmit the deleted allele to progeny. The anemic phenotype is completely rescued in progeny derived from mating beta zero-thalassemic animals with transgenic mice expressing high levels of human hemoglobin A. The beta zero-thalassemic mice can be used to test genetic therapies for beta zero-thalassemia and can be bred with transgenic mice expressing high levels of human hemoglobin HbS to produce an improved mouse model of sickle cell disease.

Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium.

Mutations in Cu/Zn superoxide dismutase (SOD), a hallmark of familial amyotrophic lateral sclerosis (FALS) in humans, are shown here to confer striking neuropathology in Drosophila. Heterozygotes with one wild-type and one deleted SOD allele retain the expected 50% of normal activity for this dimeric enzyme. However, heterozygotes with one wild-type and one missense SOD allele show lesser SOD activities, ranging from 37% for a heterozygote carrying a missense mutation predicted from structural models to destabilize the dimer interface, to an average of 13% for several heterozygotes carrying missense mutations predicted to destabilize the subunit fold. Genetic and biochemical evidence suggests a model of dimer dysequilibrium whereby SOD activity in missense heterozygotes is reduced through entrapment of wild-type subunits into unstable or enzymatically inactive heterodimers. This dramatic impairment of the activity of wild-type subunits in vivo has implications for our understanding of FALS and for possible therapeutic strategies.

A multimer model for P680, the primary electron donor of photosystem II.

We consider a model of the photosystem II (PS II) reaction center in which its spectral properties result from weak (approximately 100 cm-1) excitonic interactions between the majority of reaction center chlorins. Such a model is consistent with a structure similar to that of the reaction center of purple bacteria but with a reduced coupling of the chlorophyll special pair. We find that this model is consistent with many experimental studies of PS II. The similarity in magnitude of the exciton coupling and energetic disorder in PS II results in the exciton states being structurally highly heterogeneous. This model suggests that P680, the primary electron donor of PS II, should not be considered a dimer but a multimer of several weakly coupled pigments, including the pheophytin electron acceptor. We thus conclude that even if the reaction center of PS II is structurally similar to that of purple bacteria, its spectroscopy and primary photochemistry may be very different.

Comparison of the defensive elicitation activity of cerato-populin and cerato-platanin: a structural model

Plants can defend themselves from potential pathogenic microorganisms relying on a complex interplay of signaling pathways: activation of the MAPK cascade, transcription of defense related genes, production of reactive oxygen species, nitric oxide and synthesis of other defensive compounds such as phytoalexins. These events are triggered by the recognition of pathogen’s effectors (effector-triggered immunity) or PAMPs (PAMP-triggered immunity). The Cerato Platanin Family (CPF) members are Cys-rich proteins secreted and localized on fungal cell walls, involved in several aspects of fungal development and pathogen-host interactions. Although more than hundred genes of the CPF have been identified and analyzed, the structural and functional characterization of the expressed proteins has been restricted only to few members of the family. Interestingly, those proteins have been shown to bind chitin with diverse affinity and after foliar treatment they elicit defensive mechanisms in host and non-host plants. This property turns cerato platanins into interesting candidates, worth to be studied to develop new fungal elicitors with applications in sustainable agriculture. This study focus on cerato-platanin (CP), core member of the family and on the orthologous cerato-populin (Pop1). The latter shows an identity of 62% and an overall homology of 73% with respect to CP. Both proteins are able to induce MAPKs phosphorylation, production of reactive oxygen species and nitric oxide, overexpression of defense’s related genes, programmed cell death and synthesis of phytoalexins. CP, however, when compared to Pop1, induces a faster response and, in some cases, a stronger activity on plane leaves. Aim of the present research is to verify if the dissimilarities observed in the defense elicitation activity of these proteins can be associated to their structural and dynamic features. Taking advantage of the available CP NMR structure, Pop1’s 3D one was obtained by homology modeling. Experimental residual dipolar couplings and 1H, 15N, 13C resonance assignments were used to validate the model. Previous works on CPF members, addressed the highly conserved random coil regions (loops b1-b2 and b2-b3) as sufficient and necessary to induce necrosis in plants’ leaves: that region was investigated in both Pop1 and CP. In the two proteins the loops differ, in their primary sequence, for few mutations and an insertion with a consequent diversification of the proteins’ electrostatic surface. A set of 2D and 3D NMR experiments was performed to characterize both the spatial arrangement and the dynamic features of the loops. NOE data revealed a more extended network of interactions between the loops in Pop1 than in CP. In addition, in Pop1 we identified a salt bridge Lys25/Asp52 and a strong hydrophobic interaction between Phe26/Trp53. These structural features were expected not only to affect the loops’ spatial arrangement, but also to reduce the degree of their conformational freedom. Relaxation data and the order parameter S2 indeed highlighted reduced flexibility, in particular for loop b1-b2 of Pop1. In vitro NMR experiments, where Pop1 and CP were titrated with oligosaccharides, supported the hypothesis that the loops structural and dynamic differences may be responsible for the different chitin-binding properties of the two proteins: CP selectively binds tetramers of chitin in a shallow groove on one side of the barrel defined by loops b1-b2, b2-b3 and b4-b5, Pop1, instead, interacts in a non-specific fashion with oligosaccharides. Because the region involved in chitin-binding is also responsible for the defense elicitation activity, possibly being recognized by plant's receptors, it is reasonable to expect that those structural and dynamic modifications may also justify the different extent of defense elicitation. To test that hypothesis, the initial steps of a protocol aimed to the identify a receptor for CP, in silico, are presented.

MINLP Model for the Synthesis of Heat Exchanger Networks with Handling Pressure of Process Streams

This paper introduces a new mathematical model for the simultaneous synthesis of heat exchanger networks (HENs), wherein the handling pressure of process streams is used to enhance the heat integration. The proposed approach combines generalized disjunctive programming (GDP) and mixed-integer nonlinear programming (MINLP) formulation, in order to minimize the total annualized cost composed by operational and capital expenses. A multi-stage superstructure is developed for the HEN synthesis, assuming constant heat capacity flow rates and isothermal mixing, and allowing for streams splits. In this model, the pressure and temperature of streams must be treated as optimization variables, increasing further the complexity and difficulty to solve the problem. In addition, the model allows for coupling of compressors and turbines to save energy. A case study is performed to verify the accuracy of the proposed model. In this example, the optimal integration between the heat and work decreases the need for thermal utilities in the HEN design. As a result, the total annualized cost is also reduced due to the decrease in the operational expenses related to the heating and cooling of the streams.

Testing a new Free Core Nutation empirical model

The Free Core Nutation (FCN) is a free mode of the Earth's rotation caused by the different material characteristics of the Earth's core and mantle. This causes the rotational axes of those layers to slightly diverge from each other, resulting in a wobble of the Earth's rotation axis comparable to nutations. In this paper we focus on estimating empirical FCN models using the observed nutations derived from the VLBI sessions between 1993 and 2013. Assuming a fixed value for the oscillation period, the time-variable amplitudes and phases are estimated by means of multiple sliding window analyses. The effects of using different a priori Earth Rotation Parameters (ERP) in the derivation of models are also addressed. The optimal choice of the fundamental parameters of the model, namely the window width and step-size of its shift, is searched by performing a thorough experimental analysis using real data. The former analyses lead to the derivation of a model with a temporal resolution higher than the one used in the models currently available, with a sliding window reduced to 400 days and a day-by-day shift. It is shown that this new model increases the accuracy of the modeling of the observed Earth's rotation. Besides, empirical models determined from USNO Finals as a priori ERP present a slightly lower Weighted Root Mean Square (WRMS) of residuals than IERS 08 C04 along the whole period of VLBI observations, according to our computations. The model is also validated through comparisons with other recognized models. The level of agreement among them is satisfactory. Let us remark that our estimates give rise to the lowest residuals and seem to reproduce the FCN signal in more detail.

Boston, Massachusetts Region, Digital Elevation Model with Bathymetry

This raster layer represents surface elevation and bathymetry data for the Boston Region, Massachusetts. It was created by merging portions of MassGIS Digital Elevation Model 1:5,000 (2005) data with NOAA Estuarine Bathymetric Digital Elevation Models (30 m.) (1998). DEM data was derived from the digital terrain models that were produced as part of the MassGIS 1:5,000 Black and White Digital Orthophoto imagery project. Cellsize is 5 meters by 5 meters. Each cell has a floating point value, in meters, which represents its elevation above or below sea level.

Robotic Outcome Assessment in a Non-Human Primate Model of Middle Cerebral Artery Stroke

Stroke is a prevalent disorder with immense socioeconomic impact. A variety of chronic neurological deficits result from stroke. In particular, sensorimotor deficits are a significant barrier to achieving post-stroke independence. Unfortunately, the majority of pre-clinical studies that show improved outcomes in animal stroke models have failed in clinical trials. Pre-clinical studies using non-human primate (NHP) stroke models prior to initiating human trials are a potential step to improving translation from animal studies to clinical trials. Robotic assessment tools represent a quantitative, reliable, and reproducible means to assess reaching behaviour following stroke in both humans and NHPs. We investigated the use of robotic technology to assess sensorimotor impairments in NHPs following middle cerebral artery occlusion (MCAO). Two cynomolgus macaques underwent transient MCAO for 90 minutes. Approximately 1.5 years following the procedure these NHPs and two non-stroke control monkeys were trained in a reaching task with both arms in the KINARM exoskeleton. This robot permits elbow and shoulder movements in the horizontal plane. The task required NHPs to make reaching movements from a centrally positioned start target to 1 of 8 peripheral targets uniformly distributed around the first target. We analyzed four movement parameters: reaction time, movement time (MT), initial direction error (IDE), and number of speed maxima to characterize sensorimotor deficiencies. We hypothesized reduced performance in these attributes during a neurobehavioural task with the paretic limb of NHPs following MCAO compared to controls. Reaching movements in the non-affected limbs of control and experimental NHPs showed bell-shaped velocity profiles. In contrast, the reaching movements with the affected limbs were highly variable. We found distinctive patterns in MT, IDE, and number of speed peaks between control and experimental monkeys and between limbs of NHPs with MCAO. NHPs with MCAO demonstrated more speed peaks, longer MTs, and greater IDE in their paretic limb compared to controls. These initial results qualitatively match human stroke subjects’ performance, suggesting that robotic neurobehavioural assessment in NHPs with stroke is feasible and could have translational relevance in subsequent human studies. Further studies will be necessary to replicate and expand on these preliminary findings.

Repurposing of antiparasitic drugs: the hydroxy-naphthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model.

The three anti-malarial drugs artemiside, artemisone, and mefloquine, and the naphthoquinone buparvaquone known to be active against theileriosis in cattle and Leishmania infections in rodents, were assessed for activity against Neospora caninum infection. All four compounds inhibited the proliferation of N. caninum tachyzoites in vitro with IC50 in the sub-micromolar range, but artemisone and buparvaquone were most effective (IC50 = 3 and 4.9 nM, respectively). However, in a neosporosis mouse model for cerebral infection comprising Balb/c mice experimentally infected with the virulent isolate Nc-Spain7, the three anti-malarial compounds failed to exhibit any activity, since treatment did not reduce the parasite burden in brains and lungs compared to untreated controls. Thus, these compounds were not further evaluated in pregnant mice. On the other hand, buparvaquone, shown earlier to be effective in reducing the parasite load in the lungs in an acute neosporosis disease model, was further assessed in the pregnant mouse model. Buparvaquone efficiently inhibited vertical transmission in Balb/c mice experimentally infected at day 7 of pregnancy, reduced clinical signs in the pups, but had no effect on cerebral infection in the dams. This demonstrates proof-of-concept that drug repurposing may lead to the discovery of an effective compound against neosporosis that can protect offspring from vertical transmission and disease.

Effects of exercise training on intrauterine growth restriction in an animal model

Résumé Selon l'OMS, la retard de croissance intra-utérine (RCIU; 10% en dessous du poids normal pendant la grossesse) affecte 5-10% des grossesses et est une cause principale de la morbidité et de la mortalité périnatales. Dans notre étude précédente sur un modèle de souris transgénique de prééclampsie (R+A+), nous avons constaté que l’entraînement physique (ExT) avant et pendant la grossesse réduisait la pression artérielle maternelle et empêchait la RCIU en améliorant le développement placentaire. Dans le cadre de mon projet, nous avons confirmé les bénifices de l’ExT dans un modèle de RCIU (souris déficiente en p57Kip2 (p57-/+). Ainsi, nous avons observé la présence de RCIU, d’une masse placentaire réduite, d’une augmentation de la pathologie placentaire ainsi qu’une plus petite taille des portées chez les souris p57-/+ sédentaire. L’ExT prévient la RCIU ainsi que tous les paramètres mentionnés ci-haut. Nous avons observé que l'expression du facteur de croissance de l’endothélium vasculaire, un régulateur clé de l'angiogenèse lors de la croissance placentaire, était réduite dans le placenta des souris p57-/+ et normalisée par l’ExT. Nous avons également trouvé que l'expression en ARN dans le placenta de 2 facteurs inflammatoires (interleukine-1β et MCP-1) était augmenté chez les souris sédentaires p57-/+ alors que ceci n’était pas présent chez les souris entraînées, ce qui suggère que l'inflammation placentaire peut contribuer à la pathologie placentaire. Toutefois, contrairement aux souris R+A+, le système rénine-angiotensine placentaire chez les souris p57-/+ était normale et aucun effet de l’ExT a été observé. Ces résultats suggèrent que l’ExT prévient la RCIU en normalisant la pathologie placentaire, l’angiogenèse et l’inflammation placentaire.

Geochemistry and age model of sediment core BDP96-1

Within the framework of the Baikal Drilling Project (BDP), a 192 m long sediment core (BDP-96-1) was recovered from the Academician Ridge, a submerged topographic high between the North and Central Basins of Lake Baikal. Sedimentological, clay mineralogical and geochemical investigations were carried out on the core interval between 90 and 124 m depth, corresponding to ca. 2.4-3.4 Ma. The aim was to reconstruct the climatic and tectonic history of the continental region during the intensification of Northern Hemisphere glaciation in Late Pliocene time. A major climate change occurred in the Lake Baikal area at about 2.65 Ma. Enhanced physical weathering in the catchment, mirrored in the illite to smectite ratio, and temporarily reduced bioproduction in the lake, reflected by the diatom abundance, evidence a change towards a colder and more arid climate, probably associated with an intensification of the Siberian High. In addition, the coincident onset of distinct fluctuations in these parameters and in the Zr/Al ratio suggests the beginning of the Late Cenozoic high amplitude climate cycles at about 2.65 Ma. Fluctuations in the Zr/Al ratio are traced back to changes in the aeolian input, with high values in warmer, more humid phases due to a weaker Siberian High. Assuming that the sand content in the sediment reflects tectonic pulses, the Lake Baikal area was tectonically active during the entire investigated period, but in particular around 2.65 Ma. Tectonic movements have likely led to a gradual catchment change since about 3.15 Ma from the western towards the eastern lake surroundings, as indicated in the geochemistry and clay mineralogy of the sediments. The strong coincidence between tectonic and climatic changes in the Baikal area hints at the Himalayan uplift being one of the triggers for the Northern Hemisphere Glaciation.

Sedimentological investigations and age model on sediment core PS2561-2

Benthic d13C values (F. wuellerstorfi), kaolinite/chlorite ratios and sortable silt median grain sizes in sediments of a core from the abyssal Agulhas Basin record the varying impact of North Atlantic Deep Water (NADW) and Antarctic Bottom Water (AABW) during the last 200 ka. The data indicate that NADW influence decreased during glacials and increased during interglacials, in concert with the global climatic changes of the late Quaternary. In contrast, AABW displays a much more complex behaviour. Two independent modes of deep-water formation contributed to the AABW production in the Weddell Sea: 1) brine rejection during sea ice formation in polynyas and in the sea ice zone (Polynya Mode) and 2) super-cooling of Ice Shelf Water (ISW) beneath the Antarctic ice shelves (Ice Shelf Mode). Varying contributions of the two modes lead to a high millennial-scale variability of AABW production and export to the Agulhas Basin. Highest rates of AABW production occur during early glacials when increased sea ice formation and an active ISW production formed substantial amounts of deep water. Once full glacial conditions were reached and the Antarctic ice sheet grounded on the shelf, ISW production shut down and only brine rejection generated moderate amounts of deep water. AABW production rates dropped to an absolute minimum during Terminations I and II and the Marine Isotope Transition (MIS) 4/3 transition. Reduced sea ice formation concurrent with an enhanced fresh water influx from melting ice lowered the density of the surface water in the Weddell Sea, thus further reducing deep water formation via brine rejection, while the ISW formation was not yet operating again. During interglacials and the moderate interglacial MIS 3 both brine formation and ISW production were operating, contributing various amounts to AABW formation in the Weddell Sea.

Development of a Conceptual Site Model for Evaluating Seawater Intrusion under Sea Level Rise Scenarios Using Analytical Methods

Island County is located in the Puget Sound of Washington State and includes several islands, the largest of which is Whidbey Island. Central Whidbey Island was chosen as the project site, as residents use groundwater for their water supply and seawater intrusion near the coast is known to contaminate this resource. In 1989, Island County adopted a Saltwater Intrusion Policy and used chloride concentrations in existing wells in order to define and map “risk zones.” In 2005, this method of defining vulnerability was updated with the use of water level elevations in conjunction with chloride concentrations. The result of this work was a revised map of seawater intrusion vulnerability that is currently in use by Island County. This groundwater management strategy is defined as trigger-level management and is largely a reactive tool. In order to evaluate trends in the hydrogeologic processes at the site, including seawater intrusion under sea level rise scenarios, this report presents a workflow where groundwater flow and discharge to the sea are quantified using a revised conceptual site model. The revised conceptual site model used several simplifying assumptions that allow for first-order quantitative predictions of seawater intrusion using analytical methods. Data from water well reports included lithologic and well construction information, static water levels, and aquifer tests for specific capacity. Results from specific capacity tests define the relationship between discharge and drawdown and were input for a modified Theis equation to solve for transmissivity (Arihood, 2009). Components of the conceptual site model were created in ArcGIS and included interpolation of water level elevation, creation of groundwater basins, and the calculation of net recharge and groundwater discharge for each basin. The revised conceptual site model was then used to hypothesize regarding hydrogeologic processes based on observed trends in groundwater flow. Hypotheses used to explain a reduction in aquifer thickness and hydraulic gradient were: (1) A large increase in transmissivity occurring near the coast. (2) The reduced aquifer thickness and hydraulic gradient were the result of seawater intrusion. (3) Data used to create the conceptual site model were insufficient to resolve trends in groundwater flow. For Hypothesis 2, analytical solutions for groundwater flow under Dupuit assumptions were applied in order to evaluate seawater intrusion under projected sea level rise scenarios. Results indicated that a rise in sea level has little impact on the position of a saltwater wedge; however, a reduction in recharge has significant consequences. Future work should evaluate groundwater flow using an expanded monitoring well network and aquifer recharge should be promoted by reducing surface water runoff.

Salivary Gland and Tooth Abnormalities Massively Increase Caries Susceptibility in A Mouse Model of Cleft Lip/Palate

Thesis (Ph.D.)--University of Washington, 2016-04

An Electromechanical Coupled Model of the Probe of a Scanning Fiber Imaging System

Thesis (Master's)--University of Washington, 2016-06