PhD

dissertationUnderstanding the molecular and genetic mechanisms responsible for the development of the vertebrate inner ear, which mediates hearing and equilibrium, is an ongoing quest in developmental biology. Most of the cells found in the sensory tissue of the mature inner ear are derived from an ectodermal placode that is located adjacent to the developing hindbrain. This tissue undergoes complex morphogenesis to form auditory and vestibular compartments which have distinct morphologic characteristics along each of the developmental axes. Otic development involves signaling interactions within and between otic and non-otic tissues. However, the molecular basis of otic morphogenesis and axis formation remains relatively unexplored. This study uses the mouse model to assess the role of the Fibroblast Growth Factor (FGF) signaling system in otic development. The work described herein analyses expression patterns of the members of Fg/'gene family and examines specific roles of Fgf3 and Fgfl6 in inner ear morphogenesis. We show that Fgf'16 is one of the earliest regionally restricted transcripts expressed in otic tissue and is polarized along the antero-posterior otic axis. Simultaneous knock out of Fgfl6 function and expression of Cre recombinase in its place allowed us to determine the consequences of inactivating Fgfl6 and to examine the fate of mouse posterior otic cells. We show that Fgfl6 does not have a unique role in inner ear development and describe the fates of Fgf'16 expressing otic cells in anterior and posterior semicircular canals, the three ampullary cristae, and the cochlear stria vascularis. In addition, this thesis shows that Fgf3 is required for dorsal pattering and morphogenesis of the inner ear epithelium. The range of malformations observed in Fgf3 mutants has close parallels with those seen in hearing impaired patients. Through a series of conditional mutagenesis experiments, we also suggest that there is an early placode stage requirement for hindbrain-expressed Fgf3 to induce proper development of the endolymphatic duct. Taken together, this study provides new information on the normal development of the inner ear and the factors that regulate the process of otic morphogenesis, providing insights into genetic mechanisms responsible for the myriad of human inner ear malformations

Differential effects of alcohol and its metabolite acetaldehyde on vascular smooth muscle cell Notch signaling and growth

Alcohol (EtOH) consumption can variously affect cardiovascular disease. Our aim was to compare the effects of EtOH and its primary metabolite acetaldehyde (ACT) on vascular smooth muscle Notch signaling and cell growth, which are important for atherogenesis. Human coronary artery smooth muscle cells (HCASMCs) were treated with EtOH (25 mM) or ACT (10 or 25 μM). As previously reported, EtOH inhibited Notch signaling and growth of HCASMCs. In contrast, ACT treatment stimulated HCASMC proliferation (cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling, as determined by increased Notch receptor (N1 and N3) and target gene (Hairy-related transcription factor 1–3) mRNA levels. Interaction of the ligand with the Notch receptor initiates proteolytic cleavage by α- and γ-secretase, resulting in the release of the active Notch intracellular domain. Neither EtOH nor ACT had any significant effect on α-secretase activity. A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Delta-like ligand 4-stimulated γ-secretase activity in solubilized HCASMCs (similar to the effect of the control inhibitor DAPT) but no effect of ACT treatment. EtOH, but not ACT, affected the association and distribution of the γ-secretase catalytic subunit presenilin-1 with lipid rafts, as determined by dual fluorescent labeling and confocal microscopic visualization. In conclusion, ACT stimulates vascular smooth muscle cell Notch signaling and growth, effects opposite to those of EtOH. These differential actions on vascular smooth muscle cells of EtOH and its metabolite ACT may be important in mediating the ultimate effects of drinking on cardiovascular disease

Association between exposure to radioactive iodine after the Chernobyl accident and thyroid volume in Belarus 10-15 years later

Background: While there is a robust literature on environmental exposure to iodine-131 (131I) in childhood and adolescence and the risk of thyroid cancer and benign nodules, little is known about its effects on thyroid volume. Methods: To assess the effect of 131I dose to the thyroid on the volume of the thyroid gland, we examined the data from the baseline screening of the Belarusian-American Cohort Study of residents of Belarus who were exposed to the Chernobyl fallout at ages ≤18 years. Thyroid dose estimates were based on individual thyroid activity measurements made shortly after the accident and dosimetric data from questionnaires obtained 10-15 years later at baseline screening. During baseline screening, thyroid gland volume was assessed from thyroid ultrasound measurements. The association between radiation dose and thyroid volume was modeled using linear regression where radiation dose was expressed with power terms to address non-linearity. The model was adjusted for attained age, sex, and place of residence, and their modifying effects were examined. Results: The analysis was based on 10,703 subjects. We found a statistically significant positive association between radiation dose and thyroid volume (P \u3c 0.001). Heterogeneity of association was observed by attained age (P \u3c 0.001) with statistically significant association remaining only in the subgroup of ≥18 years at screening (P \u3c 0.001). For this group, increase in dose from 0.0005 to 0.15 Gy was associated with a 1.27 ml (95% CI: 0.46, 2.07) increase in thyroid volume. The estimated effect did not change with increasing doses above 0.15 Gy. Conclusions: This is the first study to examine the association between 131I dose to the thyroid gland and thyroid volume in a population of individuals exposed during childhood and systematically screened 10-15 years later. It provides evidence for a moderate statistically significant increase in thyroid volume among those who were ≥ 18 years at screening. Given that this effect was observed at very low doses and was restricted to a narrow dose range, further studies are necessary to better understand the effect

Multidifferential study of identified charged hadron distributions in ZZ-tagged jets in proton-proton collisions at s=\sqrt{s}=13 TeV

Jet fragmentation functions are measured for the first time in proton-proton collisions for charged pions, kaons, and protons within jets recoiling against a $Z$ boson. The charged-hadron distributions are studied longitudinally and transversely to the jet direction for jets with transverse momentum 20 $< p_{\textrm{T}} < 100$ GeV and in the pseudorapidity range $2.5 < \eta < 4$. The data sample was collected with the LHCb experiment at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 1.64 fb$^{-1}$. Triple differential distributions as a function of the hadron longitudinal momentum fraction, hadron transverse momentum, and jet transverse momentum are also measured for the first time. This helps constrain transverse-momentum-dependent fragmentation functions. Differences in the shapes and magnitudes of the measured distributions for the different hadron species provide insights into the hadronization process for jets predominantly initiated by light quarks.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-013.html (LHCb public pages

Measurement of the B0s→μ+μ− Branching Fraction and Effective Lifetime and Search for B0→μ+μ− Decays

A search for the rare decays Bs0→μ+μ- and B0→μ+μ- is performed at the LHCb experiment using data collected in pp collisions corresponding to a total integrated luminosity of 4.4  fb-1. An excess of Bs0→μ+μ- decays is observed with a significance of 7.8 standard deviations, representing the first observation of this decay in a single experiment. The branching fraction is measured to be B(Bs0→μ+μ-)=(3.0±0.6-0.2+0.3)×10-9, where the first uncertainty is statistical and the second systematic. The first measurement of the Bs0→μ+μ- effective lifetime, τ(Bs0→μ+μ-)=2.04±0.44±0.05  ps, is reported. No significant excess of B0→μ+μ- decays is found, and a 95% confidence level upper limit, B(B0→μ+μ-)<3.4×10-10, is determined. All results are in agreement with the standard model expectations.A search for the rare decays $B^0_s\to\mu^+\mu^-$ and $B^0\to\mu^+\mu^-$ is performed at the LHCb experiment using data collected in $pp$ collisions corresponding to a total integrated luminosity of 4.4 fb$^{-1}$. An excess of $B^0_s\to\mu^+\mu^-$ decays is observed with a significance of 7.8 standard deviations, representing the first observation of this decay in a single experiment. The branching fraction is measured to be ${\cal B}(B^0_s\to\mu^+\mu^-)=\left(3.0\pm 0.6^{+0.3}_{-0.2}\right)\times 10^{-9}$, where the first uncertainty is statistical and the second systematic. The first measurement of the $B^0_s\to\mu^+\mu^-$ effective lifetime, $\tau(B^0_s\to\mu^+\mu^-)=2.04\pm 0.44\pm 0.05$ ps, is reported. No significant excess of $B^0\to\mu^+\mu^-$ decays is found and a 95 % confidence level upper limit, ${\cal B}(B^0\to\mu^+\mu^-)<3.4\times 10^{-10}$, is determined. All results are in agreement with the Standard Model expectations

Perivascular Delivery of Notch 1 siRNA Inhibits Injury-Induced Arterial Remodeling

<div><p>Objectives</p><p>To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling.</p><p>Methods and Results</p><p>Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-X_L expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown.</p><p>Conclusion</p><p>These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.</p></div

Differential effects of alcohol and its metabolite acetaldehyde on vascular smooth muscle cell Notch signaling and growth

Alcohol (EtOH) consumption can variously affect cardiovascular disease. Our aim was to compare the effects of EtOH and its primary metabolite acetaldehyde (ACT) on vascular smooth muscle Notch signaling and cell growth, which are important for atherogenesis. Human coronary artery smooth muscle cells (HCASMCs) were treated with EtOH (25 mM) or ACT (10 or 25 μM). As previously reported, EtOH inhibited Notch signaling and growth of HCASMCs. In contrast, ACT treatment stimulated HCASMC proliferation (cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling, as determined by increased Notch receptor (N1 and N3) and target gene (Hairy-related transcription factor 1–3) mRNA levels. Interaction of the ligand with the Notch receptor initiates proteolytic cleavage by α- and γ-secretase, resulting in the release of the active Notch intracellular domain. Neither EtOH nor ACT had any significant effect on α-secretase activity. A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Delta-like ligand 4-stimulated γ-secretase activity in solubilized HCASMCs (similar to the effect of the control inhibitor DAPT) but no effect of ACT treatment. EtOH, but not ACT, affected the association and distribution of the γ-secretase catalytic subunit presenilin-1 with lipid rafts, as determined by dual fluorescent labeling and confocal microscopic visualization. In conclusion, ACT stimulates vascular smooth muscle cell Notch signaling and growth, effects opposite to those of EtOH. These differential actions on vascular smooth muscle cells of EtOH and its metabolite ACT may be important in mediating the ultimate effects of drinking on cardiovascular disease

Perivascular delivery of Notch 1 siRNA prevents injury-induced Vascular Remodeling.

<p>(A) Photomicrographs of confocal immunofluorescence staining for Notch 1, SMC α-actin or dual staining for α-actin/Notch 1 in carotid arteries 14 d after ligation in sham, ligated (scrambled siRNA) and ligated+Notch 1 siRNA vessels. (B) Photomicrographs of confocal immunofluorescence (60X) staining for Bax, Bcl-X_L and Caspase-3 expression in carotid arteries 14 d after ligation in sham, ligated control (scrambled siRNA) and ligated Notch 1 siRNA vessels. Data are mean ± SEM, 4 sections analyzed/animal, n = 6 animals for each group.</p

Perivascular delivery of Notch 1 siRNA in Injured Vessel inhibits Notch Signaling Component Expression.

<p>(A) Representative Western Blot and (B) cumulative protein data for Notch 1 IC and Notch target gene (Hrt-1 and Hrt-2) expression 14 days after carotid ligation in sham, ligated (scrambled siRNA) and ligated+Notch 1 siRNA vessels. (C) qRT-PCR analysis of Notch 1, Hrt-1 and Hrt-2 mRNA levels 14 days after carotid ligation in sham, ligated (scrambled siRNA) and ligated+Notch 1 siRNA vessels. Data are normalized to GAPDH and represent the mean ± SEM, n = 6.</p

Perivascular delivery of Notch 1 siRNA inhibits Medial SMC Notch Signaling Component Expression.

<p>(A) Representative image of medial SMC layer dissected by Laser Capture Microdissection (LCM) for mRNA analysis. (B) qRT-PCR analysis of medial SMC Notch 1, Hrt-1 and Hrt-2 mRNA levels 14 days after carotid ligation in sham, ligated (scrambled siRNA) and Ligated+Notch 1 siRNA vessels. Data are normalized to GAPDH and represent the mean ± SEM, n = 6.</p