Synthesis of a Folate Conjugated Fluorescent Imaging Dye

In recent years methods of selective targeting of pathologic cells have become increasingly important for both detection and treatment of various cancers. The use of folic acid as a selective therapeutic and targeting agent for cancer cells is becoming ever more significant. This is due to the well characterized up regulation of folate receptors for cancer cells. In this experiment we focus on the development of a new folate conjugate dye by using a small fluorescent molecule known as Oregon Green 514 for fluorescent imaging of cancer cells. To obtain this objective we I inked folate with Oregon green 514 dye via a solid phase synthesis technique. Purification and identification of the newly created fluorescent probe was accomplished by High Performance Liquid Chromatography and Mass Spectrometry. The folate-conjugated imaging probe was tested on L1210 leukemia cells, and was internalized by binding selectively to over expressed folate receptors on the surface of cancer cells. The folate receptor was confirmed to be responsible for conjugate internalization by a competition study with free folic acid

Understanding barriers to efficient nucleic acid delivery with bioresponsive block copolymers

Thesis (Ph. D.)--Harvard-MIT Program in Health Sciences and Technology, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have been held back by immunogenicity and toxicity concerns, but synthetic vectors have lagged in transfection efficiency. This thesis describes the rational design and systematic study of three classes of bioresponsive polymers for nucleic acid delivery. A central theme of the study was understanding how the structure of the polymers impacted each of the intracellular steps of delivery, rather than solely the end result. A powerful tool for efficiently quantifying endosomal escape was developed and applied to each of the material systems described. First, a linear-dendritic poly(amido amine) -poly(ethylene glycol) (PAMAM-PEG) block copolymer system previously developed in our lab was evaluated and its ability to overcome the sequential barriers of uptake, endosomal escape, and nuclear import were characterized. Next, a class of crosslinked linear polyethyleimine (xLPEI) hyperbranched polymers, which can contain disulfideresponsive linkages, were synthesized and investigated. It was demonstrated that free polymer in solution, not the presence of a functional bioresponsive domain, was responsible for the highly efficient and relatively nontoxic DNA delivery of this promising class of crosslinked polyamines. Finally, this analysis was applied to siRNA delivery by a library of amine-functionalized synthetic polypeptides. The pH-responsive secondary structure, micelle formation, and ester hydrolysis were studied prior to the discrete barrier-oriented analysis of the siRNA delivery potential of this library. It is hoped that the tools, materials, and systemic analysis of structure-function relationships in this thesis will enhance the process of discovery and development of clinically relevant gene carriers.by Daniel Kenneth Bonner.Ph.D

Histone H2AX Is Phosphorylated at Sites of Retroviral DNA Integration but Is Dispensable for Postintegration Repair

The histone variant H2AX is rapidly phosphorylated (denoted {gamma}H2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for {gamma}H2AX in DNA repair; however, {gamma}H2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on {gamma}H2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish {gamma}H2AX functions. We found that integration promotes transient formation of {gamma}H2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of {gamma}H2AX in chromatin repair

Self-assembled RNA interference microsponges for efficient siRNA delivery

The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles1, 2, cationic complexes3, 4, inorganic nanoparticles5, 6, 7, 8, RNA nanoparticles9, 10 and dendrimers11. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic1, 12, 13. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell’s RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.National Institutes of Health (U.S.) (NIH) NIBIB Grant R01-EB008082)United States. American Recovery and Reinvestment Act of 2009 ((ARRA) grant)National Science Foundation (U.S.) (Division of Materials Research Polymers Program grant #0705234)David H. Koch Institute for Integrative Cancer Research at MIT (Nanotechnology grant

Alternating linear-chain antiferromagnetism in copper nitrate Cu(NO\u3csub\u3e3\u3c/sub\u3e)\u3csub\u3e2\u3c/sub\u3e.2.5 H\u3csub\u3e2\u3c/sub\u3eO

Current interest in the behavior of Heisenberg alternating antiferromagnetic quantum chains has been stimulated by the discovery of an unusual class of magnetoelastic spin-Peierls systems. Copper nitrate, Cu(NO3)2.2.5 H2O, does not display a spin-Peierls transition, but its dominant magnetic behavior is that of a strongly alternating antiferromagnetic chain with temperature-independent alternation. A remarkable, simultaneous fit is demonstrated between theoretical studies and a wide variety of zero- (low-) field experimental measurements, including susceptibility, magnetization, and specific heat. The fitting parameters are α(degree of alternation) = 0.27, J1/k=2.58 K, gb=2.31, and g⊥=2.11. Slight systematic discrepancies are attributed to weak interchain coupling. Theoretical studies also predict a rich variety of behavior in high fields, particularly in the region involving the lower and upper critical fields, Hc1 = 28 kOe and Hc2 = 44 kOe. Experimental specific-heat measurements at H = 28.2 and 35.7 kOe show quantitative agreement with theory in this interesting parameter region. The fitting parameters are the same as for zero field and, again, small discrepancies between theory and experiment may be attributed to interchain coupling. The exceptional magnetic characterization of copper nitrate suggests its use for further experimental study in the vicinity of the high-field ordering region

A plug-and-play ratiometric pH-sensing nanoprobe for high-throughput investigation of endosomal escape

An important aspect in the design of nanomaterials for delivery is an understanding of its uptake and ultimate release to the cytosol of target cells. Real-time chemical sensing using a nanoparticle-based platform affords exquisite insight into the trafficking of materials and their cargo into cells. This versatile and tunable technology provides a powerful tool to probe the mechanism of cellular entry and cytosolic delivery of a variety of materials, allowing for a simple and convenient means to screen materials towards efficient delivery of therapeutics such as nucleic acids

Spin gap in the Quasi-One-Dimensional S=1/2 Antiferromagnet: Cu2(1,4-diazacycloheptane)2Cl4

Cu_{2}(1,4-diazacycloheptane)_{2}Cl_{4} contains double chains of spin 1/2 Cu^{2+} ions. We report ac susceptibility, specific heat, and inelastic neutron scattering measurements on this material. The magnetic susceptibility, $\chi(T)$, shows a rounded maximum at T = 8 K indicative of a low dimensional antiferromagnet with no zero field magnetic phase transition. We compare the $\chi(T)$ data to exact diagonalization results for various one dimensional spin Hamiltonians and find excellent agreement for a spin ladder with intra-rung coupling $J_1 = 1.143(3)$ meV and two mutually frustrating inter-rung interactions: $J_2 = 0.21(3)$ meV and $J_3 = 0.09(5)$ meV. The specific heat in zero field is exponentially activated with an activation energy $\Delta = 0.89(1)$ meV. A spin gap is also found through inelastic neutron scattering on powder samples which identify a band of magnetic excitations for $0.8 < \hbar\omega < 1.5$ meV. Using sum-rules we derive an expression for the dynamic spin correlation function associated with non-interacting propagating triplets in a spin ladder. The van-Hove singularities of such a model are not observed in our scattering data indicating that magnetic excitations in Cu_{2}(1,4-diazacycloheptane)_{2}Cl_{4} are more complicated. For magnetic fields above $H_{c1} \simeq 7.2$ T specific heat data versus temperature show anomalies indicating a phase transition to an ordered state below T = 1 K.Comment: 9 pages, 8 postscript figures, LaTeX, Submitted to PRB 8/4/97, e-mail Comments to [email protected]

The Aeolus Mission Concept, an Innovative Mission to Study the Winds and Climate of Mars

Aeolus is a mission to provide the first set of global, seasonal, and diurnal data to characterize winds and study the climate of Mars. Aeolus measures surface and atmospheric temperatures, aerosol abundances, and Doppler shifts in atmospheric spectral lines. The payload includes a system of four of a new type of miniaturized Spatial Heterodyne Spectrometer (SHS) paired to two orthogonal viewing telescopes that can measure CO2 (daytime absorption) and O2 (day and night emission) lines in the Martian atmosphere. The Thermal Limb Sounder (TLS) instrument measures atmospheric temperature profiles and aerosol (H2O ice clouds, dust) profiles, and the Surface Radiometric Sensor Package (SuRSeP) measures the total reflected solar radiance, and surface temperatures down to 140K. These combined spectral and thermal measurements will provide a new understanding of the global energy balance, dust transport processes, and climate cycles in the Martian atmosphere. The mission concept for Aeolus consists of a single sub-100 kg secondary spacecraft in a highly inclined orbit, allowing it to pass over all local times. Aeolus attains global coverage of the surface for a mission duration of one Martian year, to capture climate patterns during each Martian season. This paper gives an overview of the Aeolus payload, spacecraft, and the methodology used to mature the Aeolus mission concept

Efficacy and safety of erenumab in women with a history of menstrual migraine

Background We performed a post hoc, subgroup analysis of a phase 3, randomized, double-blind, placebo-controlled study of erenumab for prevention of episodic migraine (STRIVE) to determine the efficacy and safety of erenumab in women with self-reported menstrual migraine. Methods Patients received placebo, erenumab 70 mg, or erenumab 140 mg subcutaneously once monthly during the 6-month double-blind treatment phase of STRIVE. Women who reported history of menstrual migraine and who were = 50% reduction from baseline in MMD, and incidence of adverse events. Results Among 814 women enrolled in STRIVE, 232 (28.5%) reported a history of menstrual migraine and were 5, suggesting a high proportion of women with attacks outside of the 5-day perimenstrual window (2 days before and 3 days after the start of menstruation). Information on "migraine days" includes (and does not discriminate between) perimenstrual and intermenstrual migraine attacks. Between-group differences from placebo over months 4-6 for erenumab 70 mg and 140 mg were - 1.8 (P = 0.001) and - 2.1 (P = 50% reduction from baseline in MMD over months 4-6 were 2.2 (P = 0.024) and 2.8 (P = 0.002) times greater for erenumab 70 mg and 140 mg, respectively, than for placebo. Erenumab had an overall safety profile comparable to placebo. Conclusion Data from this subgroup analysis of women with menstrual migraine are consistent with data from the overall STRIVE episodic migraine population, supporting the efficacy and safety of erenumab in women who experience menstrual migraine. Trial registration: ClinicalTrials.gov, NCT02456740. Registered 28 May 2015