Tailoring light for Raman microspectroscopy

Ziel dieser Arbeit ist die Weiterentwicklung einer kohärenten Raman Spektroskopiemethode für die Anwendung in der mikroskopischen Bildgebung und eine damit verbundene Entwicklung einer spezialisierten Lichtquelle. Die Lichtquelle wurde konzipiert, um mit einer hohen Impulsleistung effizient nichtlineare Prozesse treiben zu können und die erzeugten Signale gleichzeitig mit einer hohen Wiederholrate aufzunehmen. Besonders geeignet für die Anwendung in der mikroskopischen Bildgebung ist die kohärente Ramanstreuung, da sie einen chemisch selektiven Kontrast erzeugt. Die neue interferometrische Erweiterung von Femtosekunden stimulierter Ramanstreuung (FSRS) mithilfe eines Sagnac-Interferometers (iFSRS), beziehungsweise eines kollinearen Interferometers (II-FSRS) erhöht nicht nur deren Sensitivität, sondern ermöglicht nun auch den Zugriff auf spektrale Phaseninformationen. Insgesamt zeigt das entwickelte Gesamtkonzept eine vielversprechende Anwendbarkeit, um vielfältige Fragestellungen in den Lebenswissenschaften zu untersuchen.This thesis is about the development of coherent Raman scattering techniques to be applied to microspectroscopy and an associated development of a specialized light source. The light source is designed to deliver pulses with sufficient power to efficiently generate a nonlinear Raman response, which was exploited as a contrast mechanism for microscopy, at a high repetition frequency for fast signal acquisition. Indeed current broad-bandwidth femtosecond stimulated Raman scattering (FSRS) spectroscopy methods allow the extraction of the full Raman spectrum, but are still limited in sensitivity. Rectifying this drawback, the here presented novel interferometric advancements in FSRS, by the means of a Sagnac interferometer (iFSRS) or an in-line interferometer (II-FSRS), not only increase the sensitivity of the scheme, but also grant access to spectral phase information. These improvements in combination with the highly adapted light source enable the acquisition of hyperspectral images, applicable to a wide range of questions in the life-sciences

The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity

Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication

Structure Formation and Coupling Reactions of Hexaphenylbenzene and Its Brominated Analog

The on-surface coupling of the prototypical precursor molecule for graphene nanoribbon synthesis, 6,11-dibromo-1,2,3,4-tetraphenyltriphenylene (C42Br2H26, TPTP), and its non-brominated analog hexaphenylbenzene (C42H30, HPB), was investigated on coinage metal substrates as a function of thermal treatment. For HPB, which forms non-covalent 2D monolayers at room temperature, a thermally induced transition of the monolayer’s structure could be achieved by moderate annealing, which is likely driven by π-bond formation. It is found that the dibrominated carbon positions of TPTP do not guide the coupling if the growth occurs on a substrate at temperatures that are sufficient to initiate C H bond activation. Instead, similar one-dimensional molecular structures are obtained for both types of precursors, HPB and TPTP

Structure Formation and Coupling Reactions of Hexaphenylbenzene and Its Brominated Analog

The on-surface coupling of the prototypical precursor molecule for graphene nanoribbon synthesis, 6,11-dibromo-1,2,3,4-tetraphenyltriphenylene (C42Br2H26, TPTP), and its non-brominated analog hexaphenylbenzene (C42H30, HPB), was investigated on coinage metal substrates as a function of thermal treatment. For HPB, which forms non-covalent 2D monolayers at room temperature, a thermally induced transition of the monolayer’s structure could be achieved by moderate annealing, which is likely driven by π-bond formation. It is found that the dibrominated carbon positions of TPTP do not guide the coupling if the growth occurs on a substrate at temperatures that are sufficient to initiate C--H bond activation. Instead, similar one-dimensional molecular structures are obtained for both types of precursors, HPB and TPTP

Structure Formation and Coupling Reactions of Hexaphenylbenzene and Its Brominated Analog

The on‐surface coupling of the prototypical precursor molecule for graphene nanoribbon synthesis, 6,11‐dibromo‐1,2,3,4‐tetraphenyltriphenylene (C(42)Br(2)H(26), TPTP), and its non‐brominated analog hexaphenylbenzene (C(42)H(30), HPB), was investigated on coinage metal substrates as a function of thermal treatment. For HPB, which forms non‐covalent 2D monolayers at room temperature, a thermally induced transition of the monolayer's structure could be achieved by moderate annealing, which is likely driven by π‐bond formation. It is found that the dibrominated carbon positions of TPTP do not guide the coupling if the growth occurs on a substrate at temperatures that are sufficient to initiate C−H bond activation. Instead, similar one‐dimensional molecular structures are obtained for both types of precursors, HPB and TPTP

Intra- and interspecific polymorphisms ofLeishmania donovani andL. tropica minicircle DNA

A pair of degenerate polymerase chain reaction (PCR) primers (LEI-1, TCG GAT CC[C,T] [G,C]TG GGT AGG GGC GT; LEI-2, ACG GAT CC[G,C] [G,C][A,C]C TAT [A,T]TT ACA CC) defining a 0.15-kb segment ofLeishmania minicircle DNA was constructed. These primers amplified not only inter- but also intraspecifically polymorphic sequences. Individual sequences revealed a higher intraspecific than interspecific divergence. It is concluded that individual sequences are of limited relevance for species determination. In contrast, when a data base of 19 different sequences was analyzed in a dendrographic plot, an accurate species differentiation was feasible

Изменение вентиляционной функции легких в процессе формирования хронической обструктивной болезни легких и при ее сочетании с раком легкого

Aim. To study the ventilation function of the lungs in patients with varying degrees of severity of chronic obstructive pulmonary disease (COPD) and in patients with COPD combined with lung cancer (LC), as well as to establish the features of its disorders using spirography and body plethysmography.Materials and methods. A clinical and functional study of 57 individuals was carried out with 10 healthy patients (control group), 30 patients with COPD and 17 patients in whom LC was combined with COPD using the Masterlab Pro diagnostic complex (Erich Jaeger, Germany).Results. In patients with early COPD, a decrease in MEF75 (a ventilation parameter characterizing small airway patency) is the most informative. With the progression of bronchial obstruction, both restrictive and obstructive disorders, characterized by a decrease in FEV1, VC, a change in the structure of the total lung capacity in the form of an increase in the RV/TLC ratio such as an increase in the RV/TLC ratio and an increase in bronchial resistance were recorded. In patients with LC and mild COPD, pulmonary volumes, capacities, flow-volume loop and bronchial resistance parameters did not differ from patients with COPD with a similar bronchial obstruction. In patients with LC and more severe COPD, in contrast to patients suffering from a similar severity of COPD, a decrease in the patency of large, medium and small diameter bronchi (PEF, MEF25, MEF50, MEF75) was detected, which indicated development of generalized bronchial obstruction.Conclusion. Modern diagnostics of pulmonary ventilation disorders in patients with LC and COPD should be aimed at identifying the disease, and drug therapy should target maximum leveling of reversible components of bronchial obstruction in order to increase the functional reserve of the respiratory system and reduce the risk of postoperative complications caused by COPD.Цель. Изучение вентиляционной функции легких у пациентов с различной степенью тяжести хронической обструктивной болезни легких (ХОБЛ) и при ее сочетании с раком легкого (РЛ), а также установление особенностей респираторных нарушений с помощью спирографии и бодиплетизмографии.Материалы и методы. Проведено клинико-функциональное исследование 57 лиц (10  здоровых (группа контроля), 30 больных с ХОБЛ и 17 больных, у которых РЛ сочетался  с ХОБЛ) при помощи диагностического комплекса Masterlab Pro (Erich Jaeger, Германия).Результаты. При начальной стадии ХОБЛ наиболее информативно снижение максимального объема скорости на уровне 75% от форсированной жизненной емкости легких (МОС75) – вентиляционного показателя, характеризующего проходимость мелких дыхательных путей. При прогрессировании бронхиальной обструкции отмечались как обструктивные, так и рестриктивные нарушения, характеризующиеся снижением объема форсированного выдоха за первую секунду, жизненной емкости легких, изменением структуры общей емкости легких в виде увеличения отношения остаточного объема легких к общей емкости легких и повышения бронхиального сопротивления. У пациентов, страдающих РЛ в сочетании с нетяжелой ХОБЛ, показатели вентиляции легких и бронхиальное сопротивление не отличались от пациентов с ХОБЛ, имеющих аналогичную степень бронхиальной обструкции. При РЛ в сочетании с более тяжелой ХОБЛ, в отличие от пациентов, страдающих аналогичной тяжестью ХОБЛ,  установлено снижение проходимости бронхов крупного, среднего и мелкого диаметра (пиковая  объемная скорость, МОС25, МОС50, МОС75), свидетельствующее о развитии генерализованной бронхиальной обструкции. Заключение. Современная диагностика нарушений вентиляции легких у больных РЛ в  сочетании с ХОБЛ должна быть направлена на выявление, а медикаментозная терапия – на максимальное нивелирование обратимых компонентов бронхиальной обструкции с целью повышения функционального резерва системы дыхания и снижения риска послеоперационных осложнений, вызванных ХОБЛ

ОТДАЛЕННЫЕ РЕЗУЛЬТАТЫ ИНТРАОПЕРАЦИОННОЙ ЛУЧЕВОЙ ТЕРАПИИ ПРИ НЕМЕЛКОКЛЕТОЧНОМ РАКЕ ЛЕГКОГО

The purpose of the study was to analyze long-term outcomes of intraoperative radiation therapy (IORT) in patients with stage III non-small cell lung cancer (NS CLC).Material and Methods. The study included 103 patients with stage III NS CLC treated at the Cancer Research Institute (Tomsk, Russia). All patients were divided into two groups. Group I consisted of 51 patients, who underwent radical surgery and IORT at a single dose of 15 Gy. Group II (control group) comprised 52 patients, who underwent radical surgery alone. There were 34 (33 %) pneumonectomies, 39 (37.9 %) lob-, bilobectomies, 9 (8.7 %) reconstructive surgeries and 21 (20.4 %) combined surgeries. A compact pulsed betatron MIB-6E with the average electron energy of 6 MeV, located directly in the operating unit, was used for performing IORT. The Kaplan-Meier method was used for survival analysis. The significance of differences in survival between groups was assessed using the log rank test.Results. Excluding the cases lost to follow-up and deaths from concomitant non-malignant diseases, treatment outcomes were followed up in 97 of 103 patients with NS CLC for 3, 5, and 10 years. The 3-year and disease-free survival rates were significantly higher in the IORT group than in the control group (p<0.05). The IORT reduced the frequency of locoregional recurrence from 28.6 % to 20.8 % and increased recurrence-free survival from 12 to 17.1 months. In patients with stage III NS CLC, who received combined modality treatment including IORT, the 5and 10-year disease-free survival rates were 18.7 % and 12.5 %, respectively. In the control group patients, the corresponding values were 14.3 % and 6.1 %, respectively (р<0.05). The 5and 10-year overall survival rates were 29.2 % and 18.7 % versus 20.4 % and 8.2 %, respectively (р<0.05).Conclusion. In patients with stage III NS CLC, combined modality treatment, including radical surgery and IORT, results in a better local control and higher long-term survival rates compared to surgery alone.Цель исследования – изучить отдаленные результаты использования интраоперационной лучевой терапии (ИОЛТ) у больных немелкоклеточным раком легкого (НМРЛ) III стадии.Материал и методы. В исследование были включены 103 больных НМРЛ III стадии, которые проходили лечение в торако-абдоминальном отделении НИИ онкологии Томского НИМЦ. Больные были распределены на две группы: I группа (основная) – радикальная операция и ИОЛТ 15 Гр (51 больной); II группа (контрольная) – радикальная операция (52 больных). Хирургическое лечение в основной и контрольных группах было проведено в объеме: пневмонэктомии – 34 (33 %), лоб-, билобэктомии – 39 (37,9 %), реконструктивно-пластические операции – 9 (8,7 %) и комбинированные операции – 21 (20,4 %). В I группе после выполнения основного этапа оперативного вмешательства проводилась ИОЛТ в однократной дозе 15 Гр на пути регионарного лимфооттока. Для ИОЛТ в НИИ онкологии Томского НИМЦ применяется малогабаритный импульсный бетатрон МИБ-6Э со средней энергией электронов 6 МэВ, расположенный непосредственно в операционном блоке. Кривые выживаемости строились по методу Каплана – Майера. Значимость различий в выживаемости между группами оценивали при помощи «Log rank test».Результаты. С учетом выбывших из-под наблюдения и умерших от сопутствующих неонкологических заболеваний исходы лечения были прослежены у 97 из 103 больных НМРЛ в течение 3, 5 и 10 лет. Результаты лечения по показателю 3-летней безрецидивной и общей выживаемости были значимо (p<0,05) улучшены за счет использования высокой однократной дозы ИОЛТ, которая позволяет существенно снизить количество локорегионарных рецидивов – с 28,6 до 20,8 % и увеличить длительность безрецидивного периода с 12 до 17,1 мес. При комбинированном лечении с ИОЛТ безрецидивная 5и 10-летняя выживаемость больных НМРЛ III стадии составила 18,7 и 12,5 %, в группе контроля – 14,3 и 6,1 % (р<0,05), общая 5и 10-летняя выживаемость – 29,2 и 18,7 % против 20,4 и 8,2 % соответственно (р<0,05).Заключение. Комбинированное лечение, включающее радикальную операцию и ИОЛТ, обеспечивает надежный локальный контроль и позволяет значимо улучшить отдаленную выживаемость больных НМРЛ III стадии по сравнению с группой хирургического лечения

Extensive transmission of isoniazid resistant M. tuberculosis and its association with increased multidrug-resistant TB in two rural counties of eastern China: A molecular epidemiological study

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to investigate the molecular characteristics of isoniazid resistant <it>Mycobacterium tuberculosis </it>(MTB), as well as its contribution to the dissemination of multi-drug resistant TB (MDR-TB) in rural areas of eastern China.</p> <p>Methods</p> <p>A population-based epidemiological study was conducted in two rural counties of eastern China from 2004 to 2005. In total, 131 isoniazid resistant MTB isolates were molecularly characterized by DNA sequencing and genotyped by IS<it>6110 </it>restriction fragment length polymorphism (RFLP) and spoligotyping.</p> <p>Results</p> <p>The <it>katG</it>315Thr mutation was observed in 74 of 131 isoniazid resistant isolates and more likely to be MDR-TB (48.6%) and have mutations in <it>rpoB </it>gene (47.3%). Spoligotyping identified 80.2% of isoniazid resistant MTB isolates as belonging to the Beijing family. Cluster analysis by genotyping based on IS<it>6110 </it>RFLP, showed that 48.1% isoniazid resistant isolates were grouped into 26 clusters and <it>katG</it>315Thr mutants had a significantly higher clustering proportion compared to those with <it>katG </it>wild type (73%.vs.18%; OR, 12.70; 95%CI, 6.357-14.80). Thirty-one of the 53 MDR-TB isolates were observed in 19 clusters. Of these clusters, isoniazid resistance in MDR-TB isolates was all due to the <it>katG</it>315Thr mutation; 18 clusters also contained mono-isoniazid resistant and other isoniazid resistant isolates.</p> <p>Conclusions</p> <p>These results highlighted that isoniazid resistant MTB especially with <it>katG</it>315Thr is likely to be clustered in a community, develop extra resistance to rifampicin and become MDR-TB in Chinese rural settings.</p