Aberrant Ras/MAPK signaling in skeletal development

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis. Vita.Includes bibliographical references.The Mitogen-activated protein kinase(MAPK) signaling pathway has been studied intensively in the context of neoplastic transformation. Other studies have focused on the roles of this pathway during development and have modeled syndromes, such as Noonan Syndrome (NS), that are caused by aberrant germline Ras/MAPK signaling. One hallmark of these developmental syndromes is a defect in the skeletal development of the patients. However, the in vivo role of MAPK signaling during bone development is still controversial, with some studies supporting a positive role of the MAPK pathway during this process, and others arguing for a suppressive role. To analyze in depth the bone defects caused by aberrant K-ras/MAPK signaling during development and to understand how bone mass is regulated via K-ras/MAPK control of osteoblast differentiation we have generated two novel mouse models. Specifically, to investigate how hyperactive K-ras affects skeletal development when expressed in a spatial and temporal specific manner we activated K-rasG12D at various stages during limb development, either in all mesenchymal lineages (Prx1-Cre;K-rasG12D mutants), or specifically in the osteoblasts (Osx1-Cre;K-rasG12D). The Prx1-Cre;K-rasG12D mutant mice display greatly shortened and thickened bones that mirror the defects found in patients with NS. We determined that this bone defect appears during embryogenesis around E14.5 and is distinguished by impairment in formation of the bone collar. Furthermore, we describe a treatment strategy that rescues the skeletal phenotype, and identify a narrow developmental time window, where in utero treatment with MEK inhibitor is sufficient to completely rescue the bone phenotype. The Osx1-Cre;K-rasG12D mutant mice allowed us to activate expression of hyperactive K-ras in the osteoblast population at different times during the development of the organism. Using this model, we defined two time windows when expression of active K-ras results in opposing bone phenotypes in the mutant mice. In the first time window (approximately between E11.5 and E14.5) induction of active K-rasG12D in osteoblasts precursors impairs terminal differentiation and leads to profound bone loss. In the second time window, activation of K-rasG12D at birth acts to promote osteoblast differentiation and consequently long bone mineralization. Taken together, our data has revealed a critical involvement of K-ras/MAPK signaling in osteoblast differentiation during skeletal development, in embryogenesis and after birth. They also show how the MAPK pathway can be modulated chemically to rescue the skeletal defects seen in a mouse model for NS. These findings yield insight into diseases of the bone, including both developmental syndromes caused by aberrant MAPK activation, such as NS, and diseases characterized by an imbalance in the bone mass, such as osteoporosis.by Simona Nedelcu.Ph.D

Transient hepatic attenuation difference (THAD) or fat sparing? Aberrant right gastric vein (ARGV) determining a pseudolesion at the border of the IInd/IIIrd liver segments. Review of developmental concepts

The aberrant right gastric vein (ARGV) is a rare anatomical variation. It can be responsible for unexplained hyperdensities in the hepatic parenchyma on CT scans, also known as third inflow effects. We present two cases sharing similar vascular pattern and slightly different imagistic findings on ultrasound and computer-tomography performed studies. Both ultrasonographies showed a nodular-geographic hypoechoic area within a hyperechogenic fatty liver. Further CT evaluation showed in both cases a hyperattenuating homogenous area clearly visible on all four phases at the border of the IInd/IIIrd hepatic segment, that enhance especially on the portal venous phase, with no slow-fill, wash-out, central scar or rim-like features. The areas were considered to be focal fat sparing areas in diffuse fatty liver or a perfusion disorder due to the presence of an aberrant right gastric vein. The aim of this paper is to discuss the embryological aspects which are the groundwork for this vascular anomaly and to correlate the findings with imagistic aspects. These two ARGV produced pseudolesions, understood as focal fat sparing areas within diffuse fatty livers. These pseudolesions mimic liver tumours, therefore it is important to look for such an aberrant vessel in order to rule out other diagnoses

Rb regulates fate choice and lineage commitment in vivo

February 1, 2011Mutation of the retinoblastoma gene (RB1) tumour suppressor occurs in one-third of all human tumours and is particularly associated with retinoblastoma and osteosarcoma[superscript 1]. Numerous functions have been ascribed to the product of the human RB1 gene, the retinoblastoma protein (pRb). The best known is pRb’s ability to promote cell-cycle exit through inhibition of the E2F transcription factors and the transcriptional repression of genes encoding cell-cycle regulators[superscript 1]. In addition, pRb has been shown in vitro to regulate several transcription factors that are master differentiation inducers[superscript 2]. Depending on the differentiation factor and cellular context, pRb can either suppress or promote their transcriptional activity. For example, pRb binds to Runx2 and potentiates its ability to promote osteogenic differentiation in vitro[superscript 3]. In contrast, pRb acts with E2F to suppress peroxisome proliferator-activated receptor γ subunit (PPAR-γ), the master activator of adipogenesis[superscript 4, 5]. Because osteoblasts and adipocytes can both arise from mesenchymal stem cells, these observations suggest that pRb might play a role in the choice between these two fates. However, so far, there is no evidence for this in vivo. Here we use mouse models to address this hypothesis in mesenchymal tissue development and tumorigenesis. Our data show that Rb status plays a key role in establishing fate choice between bone and brown adipose tissue in vivo.National Cancer Institute (U.S.) (Grant)National Institutes of Health (U.S.) (Grant

Abstract Number ‐ 53: Endovascular therapy versus medical management for mild strokes due to medium and distal vessel occlusions

Introduction Distal and medium vessel occlusion (DMVO) strokes account for 25–40% of all acute ischemic strokes1 and are associated with long‐term disability in as many as 77% of patients2. Approximately one third of DMVO stroke patients have a low National Institute of Stroke Scale (NIHSS) (≤6)3. Multiple randomized controlled trials showed superiority of endovascular therapy (EVT) over best medical management (BMT) in patients presenting with proximal LVO involving the anterior circulation and NIHSS of >64. The efficacy and safety of EVT for DMVO patients is uncertain, especially in patients with low NIHSS. A better understanding of this issue is important because many patients with DMVO and low NIHSS scores suffer disabling deficits while procedural risk are increased due to the target vessels’ small caliber, tortuosity, and thinner walls. We conducted a retrospective single center study to compare clinical and safety outcomes of DMVO patients with NIHSS ≤ 6 that were treated with EVT versus BMT. Methods We retrospectively analyzed consecutive patients with acute mild (NIHSS ≤ 6) DMVO stroke that presented between January 2018 and December 2021. We defined DMVO stroke as stroke caused by an occlusion of the M2‐4 segments of the MCA, A2‐3 segments of the ACA, P1‐2 segments of the PCA. Outcomes of interest were the NIHSS at day one and at discharge, the change in NIHSS from admission to discharge (ΔNIHSS) and the mRS at 90‐days. Safety outcomes were clinical deterioration, defined as an increase in the NIHSS by 4 or more points and the occurrence of symptomatic intracranial hemorrhage (sICH), defined as any type of ICH causing an increase in the NIHSS by 4 or more points. Results Overall, we included 80 subjects that fulfilled our study criteria. Of these, 41 were treated with BMT and 39 were selected to undergo EVT. Clinical characteristics of subjects selected for BMT versus EVT were overall similar except for more frequent diabetes (p = 0.035) and antiplatelet use (p = 0.045) as well as less frequent anticoagulation (p = 0.019) in the BMT group. Moreover, subjects selected for EVT had a lower pre‐stroke mRS (p = 0.025) and a lower ASPECT score on the admission head CT (p = 0.044). Overall, there was no statistical difference between NIHSS at day 1 (p = 0.654), NIHSS at discharge (p = 0.244), and ΔNIHSS from Day 0 to Day 1 (p = 0.08). There was further no difference in the discharge (p = 0.895) and 3‐month (p = 0.957) mRS between groups. Regarding safety outcomes, there was no difference in the number of all types of ICHs combined between the two groups (p = 0.229) and there was no difference in the number of patients who had clinical deterioration (p = 0.258). Conclusions Our analysis shows that in patients who present with mild DMVO strokes, early and 3‐month clinical outcomes were comparable between EVT and BMT groups. Moreover, risk of intracranial hemorrhage, clinical deterioration, and death were similar between groups. Nevertheless, given the low number of subjects, our analysis did not reach statistical significance and further large‐scale studies are required to clarify efficacy and safety of EVT versus BMT in DMVO patients with low clinical deficit severit

Study of microstructure and mechanical properties of injection molded Arboform parts

534-540The industry of petroleum-based plastic materials is one of the major responsible for numerous unpleasant environmental impacts. The biggest negative effect is given by absence of plastics biodegradability and recyclability, leading to accumulation of huge amount of waste. The experimental study is planed over the Taguchi methodology with two levels and six input parameters. Experimental data show that the tensile strength reached 58.61±2.73 MPa at 23oC and 5.45±0.34 MPa at 60oC. The friction coefficient exhibits a slight decrease during the first 100 s, in case of disk rotation, and then the value is stable throughout the whole testing. Considering disk oscillation, there is an increase in the friction coefficient, which reaches a peak after about 50 s and then decreases within the range of 50-150 s. The material seems to have a completely amorphous structure, without being able to accurately determine the type of existing constituents. It includes C, O and other accompanying elements in very small proportions. The SEM analysis shows a randomly oriented structure. In the chemical elements spectrum, carbon and oxygen predominate, in both percentage and atomic mass. According to the analyses performed Arboform L, V3 Nature can replace plastic materials in many industries, such as automotive, toys, construction, electrical, computers, mobile phone cases, etc. </span

Dynamical Mechanical and Thermal Analyses of Biodegradable Raw Materials for Additive Manufacturing

In order to find new ways to ensure sustainable development on a global level, it is essential to combine current top technologies, such as additive manufacturing, with the economic, ecological, and social fields. One objective of this paper refers to wire manufacture such as Arboblend V2 Nature, Arbofill Fichte, and Arboblend V2 Nature reinforced with Extrudr BDP &ldquo;Pearl&rdquo; (BDP&mdash;Biodegradable Plastic) in order to replace the plastic materials. After wire manufacture by extrusion, the diameter accuracy was analyzed compared with the Fiber Wood wire using SEM analyses and also EDAX&mdash;Energy Dispersive X-ray Analysis and DSC&mdash;Differential Scanning Calorimetry analyses were done in order to identify their elemental composition and the phase transitions suffered by the materials during heating. Using the samples obtained through the Fused Deposition Modeling (FDM) method, both crystalline phases and chemical composition information (XRD analysis) were identified, as well was determined the visco-elastic behavior Dynamic Mechanical Analysis (DMA), for the reinforced material and Fiber Wood. The extruded wires have allowed size for the printing equipment, around 1.75 mm with tolerance of &plusmn; 0.05 mm. The wire material diagrams, Arboblend V2 Nature reinforced with Extrudr BDP &ldquo;Pearl&rdquo; and Fiber Wood following the calorimetric analysis, presented peaks corresponding to material crystallization, while Arbofill Fichte revealed only the melting temperature. The storage module was almost double in case of Arboblend V2 Nature reinforced with Extrudr BDP &ldquo;Pearl&rdquo; compared with Fiber Wood and materials&rsquo; melting temperatures were confirmed by the analyses carried out

Biodegradable polymer properties through ceramic coatings

Coating of bio-based polymers with ceramic layer has attracted interest recently, the research topic raising difficulties regarding the technology of obtaining layers that involve very high working temperatures. The study aims to analyses the mechanical, tribological and structural characteristics of the Arboblend V2 Nature biodegradable polymer after the deposition of ceramic microlayers. The micro powders used were Amdry 6420 (Cr2O3), Metco 143 (ZrO2 18TiO2 10Y2O3) and Metco 136F (Cr2O3-xSiO2-yTiO2). The coated samples were obtained by injection molding and the deposition was achieved by using Atmospheric Plasma Spray (APS) method. The results of the related analyses showed that, in general, the deposits of ceramic micro particles increased the material surface characteristics (hardness, scratch resistance, apparent friction coefficient), due to the uniformity of the ceramic coating on the polymeric substrate. Based on these, it was possible to recommend the use of coated bio-based polymer - Arboblend V2 Nature in harsh operating conditions, such as the automotive industry

Characterization of Arboblend V2 Nature Textured Surfaces Obtained by Injection Molding

Surface texturing is an engineering technology used in order to improve the surface characteristic of plastic parts obtained by injection molding. Applying this process not only changes the part surface properties, but also its topography. The novel functionalities of plastic products become useful when other materials make contact with the textured surface. Of course, these characteristics may vary depending on the laser positioning, dimensions, and geometry of the texture. The present paper presents the surface characteristics obtained after the laser texturing of the Arboblend V2 Nature biodegradable polymer. Three distinct geometries were studied: hexagonal, square, and triangular, and different behaviors of them were highlighted during surface free energy (SFE) and contact angle (WCA) measurements: a hydrophobic character for square and hexagonal geometry with distilled water as the measure liquid, and a hydrophilic character with diiodomethane as the measure liquid; for triangle geometry, the contact angle measurements were impossible to extract because the drop turns into a flat puddle. Additionally, the friction coefficient varied depending on the geometry texture, with the lowest value being recorded by the sample with hexagonal geometry. The micro-indentation tests highlighted increased surface micro-hardness compared to the basic material. The possibility of use in the practice of textured surfaces is viable; thus, based on the obtained results, there is even the possibility to replace non-biodegradable polymers from different sectors of activity