Cytogenetic and molecular analyses in troglobitic and epigean species of Pimelodella (Siluriformes: Heptapteridae) from Brazil

Samples from seven different locations of the genus Pimelodella were genetically examined, two caves (exclusively subterranean, upper Tocantins River and Sao Francisco River) and five epigean (from upper Parana River basin). Cytogenetic analyses revealed the same diploid number (2n=46) for all species besides similarities in both number and location of nucleolar organizer regions and C bands. FISH with 5S rDNA probes and CMA(3) staining indicated significant differences among the studied species. Application of PCR-RFLP in ATPase 6 and 8 mitochondrial genes allowed building a minimum evolution phenogram identifying the close evolutionary relationship among groups. Both chromosomal and molecular data were useful to infer the relationships among studied Pimelodella species.

When size makes a difference: allometry, life-history and morphological evolution of capuchins (Cebus) and squirrels (Saimiri) monkeys (Cebinae, Platyrrhini)

Abstract Background How are morphological evolution and developmental changes related? This rather old and intriguing question had a substantial boost after the 70s within the framework of heterochrony (changes in rates or timing of development) and nowadays has the potential to make another major leap forward through the combination of approaches: molecular biology, developmental experimentation, comparative systematic studies, geometric morphometrics and quantitative genetics. Here I take an integrated approach combining life-history comparative analyses, classical and geometric morphometrics applied to ontogenetic series to understand changes in size and shape which happen during the evolution of two New World Monkeys (NWM) sister genera. Results Cebus and Saimiri share the same basic allometric patterns in skull traits, a result robust to sexual and ontogenetic variation. If adults of both genera are compared in the same scale (discounting size differences) most differences are small and not statistically significant. These results are consistent using both approaches, classical and geometric Morphometrics. Cebus is a genus characterized by a number of peramorphic traits (adult-like) while Saimiri is a genus with paedomorphic (child like) traits. Yet, the whole clade Cebinae is characterized by a unique combination of very high pre-natal growth rates and relatively slow post-natal growth rates when compared to the rest of the NWM. Morphologically Cebinae can be considered paedomorphic in relation to the other NWM. Geometric morphometrics allows the precise separation of absolute size, shape variation associated with size (allometry), and shape variation non-associated with size. Interestingly, and despite the fact that they were extracted as independent factors (principal components), evolutionary allometry (those differences in allometric shape associated with intergeneric differences) and ontogenetic allometry (differences in allometric shape associated with ontogenetic variation within genus) are correlated within these two genera. Furthermore, morphological differences produced along these two axes are quite similar. Cebus and Saimiri are aligned along the same evolutionary allometry and have parallel ontogenetic allometry trajectories. Conclusion The evolution of these two Platyrrhini monkeys is basically due to a size differentiation (and consequently to shape changes associated with size). Many life-history changes are correlated or may be the causal agents in such evolution, such as delayed on-set of reproduction in Cebus and larger neonates in Saimiri.

C 3-symmetric discotic liquid crystalline materials for molecular electronics: versatile synthesis and self-organization

This thesis presents the versatile synthesis and self-organization of C3-symmetric discotic nanographene molecules as well as their potential applications as materials in molecular electronics. The details can be described as follows: 1) A novel synthetic strategy towards properly designed C3 symmetric 1,3,5-tris-2’arylbenzene precursors has been developed. After the final planarization by treatment with FeCl3 under mild conditions, for the first time, it became possible to access a variety of new C3-symmetric hexa-peri-hexabenzocoronenes (HBCs) and a series of triangle-shaped nanographenes. D3 symmetric HBC with three alkyl substituents and C2 symmetric HBC with two alkyl substituents were synthesized and found to show the surprising decrease of isotropic points., the self-assembly at the liquid-solid interface displayed a unique zigzag and flower patterns. 2) Triangle-shaped discotics revealed a unique self-assembly behavior in solution, solid state as well as at the solution-substrate interface. A mesophase stability over the broad temperature range with helical supramoelcular arrangement were observed in the bulk state. The honeycomb pattern as the result of novel self-assembly was presented. Triangle-shaped discotics with swallow alkyl tails were fabricated into photovoltaic devices, the supramolecular arrangement upon thermal treatment was found to play a key role in the improvement of solar efficiency. 3) A novel class of C3 symmetric HBCs with alternating polar/apolar substituents was synthesized. Their peculiar self-assembly in solution, in the bulk and on the surface were investigated by NMR techniques, X-ray diffraction as well as different electron microscope techniques. 4) A novel concept for manipulating the intracolumnar stacking of discotics and thus for controlling the helical pitch was presented. A unique staggered stacking in the column was achieved for the first time. Theoretical simulations confirmed this self-organization and predicted that this packing should show the highest charge carrier mobility for all discotics.

Direct electron transfer to cytochrome c oxidase investigated by electrochemistry and time-resolved surface-enhanced infrared absorption spectroscopy

Cytochrom c Oxidase (CcO), der Komplex IV der Atmungskette, ist eine der Häm-Kupfer enthaltenden Oxidasen und hat eine wichtige Funktion im Zellmetabolismus. Das Enzym enthält vier prosthetische Gruppen und befindet sich in der inneren Membran von Mitochondrien und in der Zellmembran einiger aerober Bakterien. Die CcO katalysiert den Elektronentransfer (ET) von Cytochrom c zu O2, wobei die eigentliche Reaktion am binuklearen Zentrum (CuB-Häm a3) erfolgt. Bei der Reduktion von O2 zu zwei H2O werden vier Protonen verbraucht. Zudem werden vier Protonen über die Membran transportiert, wodurch eine elektrochemische Potentialdifferenz dieser Ionen zwischen Matrix und Intermembranphase entsteht. Trotz ihrer Wichtigkeit sind Membranproteine wie die CcO noch wenig untersucht, weshalb auch der Mechanismus der Atmungskette noch nicht vollständig aufgeklärt ist. Das Ziel dieser Arbeit ist, einen Beitrag zum Verständnis der Funktion der CcO zu leisten. Hierzu wurde die CcO aus Rhodobacter sphaeroides über einen His-Anker, der am C-Terminus der Untereinheit II angebracht wurde, an eine funktionalisierte Metallelektrode in definierter Orientierung gebunden. Der erste Elektronenakzeptor, das CuA, liegt dabei am nächsten zur Metalloberfläche. Dann wurde eine Doppelschicht aus Lipiden insitu zwischen die gebundenen Proteine eingefügt, was zur sog. proteingebundenen Lipid-Doppelschicht Membran (ptBLM) führt. Dabei musste die optimale Oberflächenkonzentration der gebundenen Proteine herausgefunden werden. Elektrochemische Impedanzspektroskopie(EIS), Oberflächenplasmonenresonanzspektroskopie (SPR) und zyklische Voltammetrie (CV) wurden angewandt um die Aktivität der CcO als Funktion der Packungsdichte zu charakterisieren. Der Hauptteil der Arbeit betrifft die Untersuchung des direkten ET zur CcO unter anaeroben Bedingungen. Die Kombination aus zeitaufgelöster oberflächenverstärkter Infrarot-Absorptionsspektroskopie (tr-SEIRAS) und Elektrochemie hat sich dafür als besonders geeignet erwiesen. In einer ersten Studie wurde der ET mit Hilfe von fast scan CV untersucht, wobei CVs von nicht-aktivierter sowie aktivierter CcO mit verschiedenen Vorschubgeschwindigkeiten gemessen wurden. Die aktivierte Form wurde nach dem katalytischen Umsatz des Proteins in Anwesenheit von O2 erhalten. Ein vier-ET-modell wurde entwickelt um die CVs zu analysieren. Die Methode erlaubt zwischen dem Mechanismus des sequentiellen und des unabhängigen ET zu den vier Zentren CuA, Häm a, Häm a3 und CuB zu unterscheiden. Zudem lassen sich die Standardredoxpotentiale und die kinetischen Koeffizienten des ET bestimmen. In einer zweiten Studie wurde tr-SEIRAS im step scan Modus angewandt. Dafür wurden Rechteckpulse an die CcO angelegt und SEIRAS im ART-Modus verwendet um Spektren bei definierten Zeitscheiben aufzunehmen. Aus diesen Spektren wurden einzelne Banden isoliert, die Veränderungen von Vibrationsmoden der Aminosäuren und Peptidgruppen in Abhängigkeit des Redoxzustands der Zentren zeigen. Aufgrund von Zuordnungen aus der Literatur, die durch potentiometrische Titration der CcO ermittelt wurden, konnten die Banden versuchsweise den Redoxzentren zugeordnet werden. Die Bandenflächen gegen die Zeit aufgetragen geben dann die Redox-Kinetik der Zentren wieder und wurden wiederum mit dem vier-ET-Modell ausgewertet. Die Ergebnisse beider Studien erlauben die Schlussfolgerung, dass der ET zur CcO in einer ptBLM mit größter Wahrscheinlichkeit dem sequentiellen Mechanismus folgt, was dem natürlichen ET von Cytochrom c zur CcO entspricht.

Molecular mechanisms of shikonin and its derivatives in cancer therapy

The identification of molecular processes involved in cancer development and prognosis opened avenues for targeted therapies, which made treatment more tumor-specific and less toxic than conventional therapies. One important example is the epidermal growth factor receptor (EGFR) and EGFR-specific inhibitors (i.e. erlotinib). However, challenges such as drug resistance still remain in targeted therapies. Therefore, novel candidate compounds and new strategies are needed for improvement of therapy efficacy. Shikonin and its derivatives are cytotoxic constituents in traditional Chinese herbal medicine Zicao (Lithospermum erythrorhizin). In this study, we investigated the molecular mechanisms underlying the anti-cancer effects of shikonin and its derivatives in glioblastoma cells and leukemia cells. Most of shikonin derivatives showed strong cytotoxicity towards erlotinib-resistant glioblastoma cells, especially U87MG.ΔEGFR cells which overexpressed a deletion-activated EGFR (ΔEGFR). Moreover, shikonin and some derivatives worked synergistically with erlotinib in killing EGFR-overexpressing cells. Combination treatment with shikonin and erlotinib overcame the drug resistance of these cells to erlotinib. Western blotting analysis revealed that shikonin inhibited ΔEGFR phosphorylation and led to corresponding decreases in phosphorylation of EGFR downstream molecules. By means of Loewe additivity and Bliss independence drug interaction models, we found erlotinb and shikonin or its derivatives corporately suppressed ΔEGFR phosphorylation. We believed this to be a main mechanism responsible for their synergism in U87MG.ΔEGFR cells. In leukemia cells, which did not express EGFR, shikonin and its derivatives exhibited even greater cytotoxicity, suggesting the existence of other mechanisms. Microarray-based gene expression analysis uncovered the transcription factor c-MYC as the commonly deregulated molecule by shikonin and its derivatives. As validated by Western blotting analysis, DNA-binding assays and molecular docking, shikonin and its derivatives bound and inhibited c-MYC. Furthermore, the deregulation of ERK, JNK MAPK and AKT activity was closely associated with the reduction of c-MYC, indicating the involvement of these signaling molecules in shikonin-triggered c-MYC inactivation. In conclusion, the inhibition of EGFR signaling, synergism with erlotinib and targeting of c-MYC illustrate the multi-targeted feature of natural naphthoquinones such as shikonin and derivatives. This may open attractive possibilities for their use in a molecular targeted cancer therapy.

Inference of Raja miraletus population structure using mitochondrial and nuclear DNA: comparing the resolution power of molecular markers in exploring species and population boundaries

This study focused on the role of oceanographic discontinuities and the presence of transitional areas in shaping the population structure and the phylogeography of the Raja miraletus species complex, coupled with the test of the effective occurrence of past speciation events. The comparisons between the Atlantic African and the North-Eastern Atlantic-Mediterranean geographic populations were unravelled using both Cytochrome Oxidase I and eight microsatellite loci. This approach guaranteed a robust dataset for the identification of a speciation event between the Atlantic African clade, corresponding to the ex Raja ocellifera nominal species, and the NE Atlantic-Mediterranean R. miraletus clade. As a matter of fact, the origin of the Atlantic Africa and the NE Atlantic-Mediterranean deep split dated about 11.74MYA and was likely due to the synergic influence currents and two upwelling areas crossing the Western African Waters. Within the Mediterranean Sea, particular attention was also paid to the transitional area represented by Adventura and Maltese Bank, that might have contributed in sustaining the connectivity of the Western and the Eastern Mediterranean geographical populations. Furthermore, the geology of the easternmost part of Sicily and the geo-morphological depression of the Calabrian Arc could have driven the differentiation of the Eastern Mediterranean Sea. Although bathymetric and oceanographic discontinuity could represent barriers to dispersal and migration between Eastern and Western Mediterranean samples, a clear and complete genetic separation among them was not detected. Results produced by this work identified a speciation event defining Raja ocellifera and R. miraletus as two different species, and describing the R. miraletus species complex as the most ancient cryptic speciation event in the family Rajidae, representing another example of how strictly connected the environment, the behavioural habits and the evolutionary and ecologic drivers are.

Can molecular markers stratify the diagnostic value of high-grade prostatic intraepithelial neoplasia?

The diagnostic performance of isolated high-grade prostatic intraepithelial neoplasia in prostatic biopsies has recently been questioned, and molecular analysis of high-grade prostatic intraepithelial neoplasia has been proposed for improved prediction of prostate cancer. Here, we retrospectively studied the value of isolated high-grade prostatic intraepithelial neoplasia and the immunohistochemical markers ?-methylacyl coenzyme A racemase, Bcl-2, annexin II, and Ki-67 for better risk stratification of high-grade prostatic intraepithelial neoplasia in our local Swiss population. From an initial 165 diagnoses of isolated high-grade prostatic intraepithelial neoplasia, we refuted 61 (37%) after consensus expert review. We used 30 reviewed high-grade prostatic intraepithelial neoplasia cases with simultaneous biopsy prostate cancer as positive controls. Rebiopsies were performed in 66 patients with isolated high-grade prostatic intraepithelial neoplasia, and the median time interval between initial and repeat biopsy was 3 months. Twenty (30%) of the rebiopsies were positive for prostate cancer, and 10 (15%) showed persistent isolated high-grade prostatic intraepithelial neoplasia. Another 2 (3%) of the 66 patients were diagnosed with prostate cancer in a second rebiopsy. Mean prostate-specific antigen serum levels did not significantly differ between the 22 patients with prostate cancer and the 44 without prostate cancer in rebiopsies, and the 30 positive control patients, respectively (median values, 8.1, 7.7, and 8.8 ng/mL). None of the immunohistochemical markers, including ?-methylacyl coenzyme A racemase, Bcl-2, annexin II, and Ki-67, revealed a statistically significant association with the risk of prostate cancer in repeat biopsies. Taken together, the 33% risk of being diagnosed with prostate cancer after a diagnosis of high-grade prostatic intraepithelial neoplasia justifies rebiopsy, at least in our not systematically prostate-specific antigen-screened population. There is not enough evidence that immunohistochemical markers can reproducibly stratify the risk of prostate cancer after a diagnosis of isolated high-grade prostatic intraepithelial neoplasia.

The IS/OS junction layer in the natural history of type 2 idiopathic macular telangiectasia

To document the progression of a break in the photoreceptor inner segment/outer segment (IS/OS) junction layer and its functional correlates over time in the natural history of type 2 idiopathic macular telangiectasia (type 2 MacTel).

Method for the determination of specific molecular markers of biomass burning in lake sediments

Fire has an influence on regional to global atmospheric chemistry and climate. Molecular markers of biomass burning archived in lake sediments are becoming increasingly important in paleoenvironmental reconstruction and may help determine the interaction between climate and fire activity. Here, we present a high performance anion exchange chromatography–mass spectrometry method to allow separation and analysis of levoglucosan, mannosan and galactosan in lake sediments, with implications for reconstructing past biomass burning events. Determining mannosan and galactosan in Lake Kirkpatrick, New Zealand (45.03°S, 168.57°E) sediment cores and comparing these isomers with the more abundant biomass burning markers levoglucosan and charcoal represents a significant advancement in our ability to analyze past fire activity. Levoglucosan, mannosan and galactosan concentrations correlated significantly with macroscopic charcoal concentration. Levoglucosan/mannosan and levoglucosan/(mannosan + galactosan) ratios may help determine not only when fires occurred, but also if changes in the primary burned vegetation occurred.

Molecular markers of human papillomavirus (HPV) infection and epidemiologic risk factors for squamous intraepithelial lesions (SIL) of the cervix

Infection with certain types of HPV is a necessary event in the development of cervical carcinoma; however, not all women who become infected will progress. While much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation, little is known about the additional factors needed to drive the process. Currently, conventional cervical screening is insufficient at identifying women who are likely to progress from premalignant lesions to carcinoma. Aneuploidy and chromatin texture from image cytometry have been suggested as quantitative measures of nuclear damage in premalignant lesions and cancer, and traditional epidemiologic studies have identified potential factors to aid in the discrimination of those lesions likely to progress. ^ In the current study, real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, LSIL, and HSIL. Quantitative cytometry was used to gather information about the DNA index and chromatin features of cells from the same women. Logistic regression modeling was used to establish predictor variables for histologic grade based on the traditional epidemiologic risk factors and molecular markers. ^ Prevalence of mRNA transcripts was lower among women with normal histology (27%) than for women with LSIL (40%) and HSIL (37%) with mean levels ranging from 2.0 to 4.2. The transcriptional activity of HPV 18 was higher than that of HPV 16 and increased with increasing level of dysplasia, reinforcing the more aggressive nature of HPV 18. DNA index and mRNA level increased with increasing histological grade. Chromatin score was not correlated with histology but was higher for HPV 18 samples and those with both HPV 18 and HPV 16. However, chromatin score and DNA index were not correlated with mRNA levels. The most predictive variables in the regression modeling were mRNA level, DNA index, parity, and age, and the ROC curves for LSIL and HSIL indicated excellent discrimination. ^ Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from cervical swabs. Epidemiological modeling of malignant progression in the cervix should include molecular markers, as well as the traditional epidemiological risk factors. ^