Development of fluorescent semi-conductor nanocrystal conjugates for in vitro and in vivo imaging applications

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Semiconductor nanocrystals, also known as quantum dots (QDs), are promising imaging probes with characteristic optical properties: tunable bandgap from visible to infrared, narrow and symmetric emission features, broad absorption, high quantum yield (QY), excellent photostability, and a large two-photon absorption cross section. However, unlike other imaging probes, the surface ligands determine the solubility, stability, quantum yield (QY), biocompatibility, and derivatizability of QDs. Therefore, to use QDs for in vitro and in vivo imaging, QD ligands need to be elaborately engineered for each experiment. Single cell labeling in vivo requires extremely strict criteria for the QD conjugates to be used such as minimal nonspecific cell/serum binding, maintenance of high QY in complex in vivo environments, and compact size. The focus of this thesis is the synthesis of high quality QD conjugates that can be used for single molecule imaging in vivo and in vivo imaging studies that demonstrate the broad and powerful applicability of our new methods. We incorporated novel conjugation methods employing highly strained cycloolefins and a serum stable tetrazine derivative into newly developed polymeric imidazole ligands (PILs) to efficiently couple biomolecules on QDs. Unlike traditional conjugation methods, tetrazine-norbornene cycloaddition benefits from the non-interacting properties of the functional groups to the QD surface, and yields the high conjugation efficiencies on QDs. In addition, the rapid kinetics, absence of catalyst, and bio-orthogonality of the cycloaddition allowed us to achieve in situ conjugation of the norbornene-bearing QDs to tetrazine-bearing epidermal growth factor (EGF) proteins on the HeLa cell surface. On the in vivo front, we accomplished single endogenous cell imaging in live mice. The ability to target single cells using multiple biomarkers and track them for the extended periods of time allowed us to study the microenvironment of the endogenous hematopoietic stem cells (HSCs), which was not possible using conventional techniques engaging dye conjugated antibodies. Lastly, a new class of QD ligands containing betaine moieties was developed to reduce the size of QD conjugates, which we expect will be greatly beneficial for in vitro and in vivo targeting in dense environments. We successfully demonstrated functionalization of the sulfonate betaine poly imidazole ligands (SBPILs) with biomolecules, and the biocompatibility of SBPIL QDs both in vitro and in vivo.by Hee-Sun Han.Ph.D

HIF-1α mediates isoflurane-induced vascular protection in subarachnoid hemorrhage

OBJECTIVE: Outcome after aneurysmal subarachnoid hemorrhage (SAH) depends critically on delayed cerebral ischemia (DCI) – a process driven primarily by vascular events including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction. This study sought to determine the impact of postconditioning – the phenomenon whereby endogenous protection against severe injury is enhanced by subsequent exposure to a mild stressor – on SAH-induced DCI. METHODS: Adult male C57BL/6 mice were subjected to sham, SAH, or SAH plus isoflurane postconditioning. Neurological outcome was assessed daily via sensorimotor scoring. Contributors to DCI including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction were measured 3 days later. Isoflurane-induced changes in hypoxia-inducible factor 1alpha (HIF-1α)-dependent genes were assessed via quantitative polymerase chain reaction. HIF-1α was inhibited pharmacologically via 2-methoxyestradiol (2ME2) or genetically via endothelial cell HIF-1α-null mice (EC-HIF-1α-null). All experiments were performed in a randomized and blinded fashion. RESULTS: Isoflurane postconditioning initiated at clinically relevant time points after SAH significantly reduced cerebral vasospasm, microvessel thrombosis, microvascular dysfunction, and neurological deficits in wild-type (WT) mice. Isoflurane modulated HIF-1α-dependent genes – changes that were abolished in 2ME2-treated WT mice and EC-HIF-1α-null mice. Isoflurane-induced DCI protection was attenuated in 2ME2-treated WT mice and EC-HIF-1α-null mice. INTERPRETATION: Isoflurane postconditioning provides strong HIF-1α-mediated macro- and microvascular protection in SAH, leading to improved neurological outcome. These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF-1α as a critical mediator of this vascular protection. They also identify isoflurane postconditioning as a promising novel therapeutic for SAH

Exact soluble two-dimensional charged wormhole

We present an exactly soluble charged wormhole model in two dimensions by adding infalling chiral fermions on the static wormhole. The infalling energy due to the infalling charged matter requires the classical back reaction of the geometry, which is solved by taking into account of the nontrivial nonchiral exotic energy. Finally, we obtain the exact expression for the size of the throat depending on the total amount of the infalling net energy and discuss the interesting transition from the AdS spacetime to the wormhole geometry.Comment: 8 pages, no figure

Multilocus sequence typing (MLST) analysis of Vibrio cholerae O1 El Tor isolates from Mozambique that harbour the classical CTX prophage.

Vibrio cholerae O1 isolates belonging to the Ogawa serotype, El Tor biotype, harbouring the classical CTX prophage were first isolated in Mozambique in 2004. Multilocus sequence typing (MLST) analysis using nine genetic loci showed that the Mozambique isolates have the same sequence type (ST) as O1 El Tor N16961, a representative of the current seventh cholera pandemic. Analysis of the CTX prophage in the Mozambique isolates indicated that there is one type of rstR in these isolates: the classical CTX prophage. It was also found that the ctxB-rstR-rstA-rstB-phs-cep fragment was PCR-amplified from these isolates, which indicates the presence of a tandem repeat of the classical CTX prophage in the genome of the Mozambique isolates. The possible origin of these isolates and the presence of the tandem repeat of the classical prophage in them implicate the presence of the classical CTX phage

Phenotypic and Functional Changes of Peripheral Ly6C(+) T Regulatory Cells Driven by Conventional Effector T Cells

A relatively high affinity/avidity of T cell receptor (TCR) recognition for self-peptide bound to major histocompatibility complex II (self-pMHC) ligands is a distinctive feature of CD4 T regulatory (Treg) cells, including their development in the thymus and maintenance of their suppressive functions in the periphery. Despite such high self-reactivity, however, all thymic-derived peripheral Treg populations are neither homogenous in their phenotype nor uniformly immune-suppressive in their function under steady state condition. We show here that based on the previously defined heterogeneity in the phenotype of peripheral Treg populations, Ly6C expression on Treg marks a lower degree of activation, proliferation, and differentiation status as well as functional incompetence. We also demonstrate that Ly6C expression on Treg in a steady state is either up-or downregulated depending on relative amounts of tonic TCR signals derived from its contacts with self-ligands. Interestingly, peripheral appearance and maintenance of these Ly6C-expressing Treg cells largely differed in an age-dependent manner, with their proportion being continuously increased from perinatal to young adult period but then being gradually declined with age. The reduction of Ly6C(+) Treg in the aged mice was not due to their augmented cell death but rather resulted from downregulation of Ly6C expression. The Ly6C down-regulation was accompanied by proliferation of Ly6C(+) Treg cells and subsequent change into Ly6C-effector Treg with concomitant restoration of immune-suppressive activity. Importantly, we found that this phenotypic and functional change of Ly6C(+) Treg is largely driven by conventional effector T cell population. Collectively, these findings suggest a potential cross-talk between peripheral Treg subsets and effector T cells and provides better understanding for Treg homeostasis and function on maintaining self-tolerance.110Ysciescopu

Gomisin A Suppresses Colorectal Lung Metastasis by Inducing AMPK/p38-Mediated Apoptosis and Decreasing Metastatic Abilities of Colorectal Cancer Cells

Gomisin A (G.A) is a dietary lignan compound from Schisandra chinensis. In this study, the effect of G.A on the proliferation and metastasis of colorectal cancer (CRC) cells was investigated using several CRC cell lines and a lung metastasis mouse model. Both oral and intraperitoneal administration of G.A (50 mg/kg) inhibited lung metastasis of CT26 cells. Various concentrations of G.A were incubated with CRC cell lines and their viability was determined using a cell counting kit-8 assay. G.A significantly decreased the viability of various CRC cell lines, whereas it did not change the proliferation of normal colon cells. G.A induced G0/G1 phase arrest and apoptosis of CT26 and HT29 cells by regulating cyclin D1/cyclin-dependent kinase 4 (CDK4) expression and apoptotic proteins such as caspases and B-cell lymphoma-2 (Bcl-2) family proteins, respectively. G.A-induced apoptosis was mediated by AMPK/p38 activation in CRC cells. A non-cytotoxic concentration of G.A inhibited epithelial–mesenchymal transition of CRC cells by modulating E-cadherin and N-cadherin expression levels. Moreover, the migration and invasion of CRC cells were reduced by G.A treatment. Especially, G.A decreased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities. G.A ameliorated lung metastasis of CRC cells by decreasing cell survival and metastatic abilities of CRC cells. Thus, G.A might be a potential novel therapeutic agent for metastatic CRC

Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells

HSPG mitigates Aβ1-40-induced mitochondrial and cytosolic ROS production in VSMC under physiological oxygen concentration. To determine if differing levels oxygen impact ROS production in Aβ1-40 treated VSMC, cells were kept in 10 % oxygen (Panel A) or 1 % oxygen (conditions that are considered hypoxic; Panel B) in cell culture incubator with % 5 CO2. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H2XRos, washed, and treated with Aβ1-40. In some cases, cells were pre-treated with heat-inactivated (HI) enzyme. Fluorescence was measured after 30 minutes. Results are representative of 3 independent experiments performed in triplicate. *p < 0.05 vs. vehicle-treated control. #p < 0.05 vs. comparison group. (JPEG 70 kb

Far-ultraviolet Observations of the North Ecliptic Pole with SPEAR

We present SPEAR/FIMS far-ultraviolet observations near the North Ecliptic Pole. This area, at b~30 degrees and with intermediate HI column, seems to be a fairly typical line of sight that is representative of general processes in the diffuse ISM. We detect a surprising number of emission lines of many elements at various ionization states representing gas phases from the warm neutral medium (WNM) to the hot ionized medium (HIM). We also detect fluorescence bands of H2, which may be due to the ubiquitous diffuse H2 previously observed in absorption.Comment: 5 pages, 3 figures, Accepted for publication in ApJ Letter