ROLE OF PROGESTERONE RECEPTOR MEMBRANE COMPONENT 1 (PGRMC1) IN CONTROLLING GERMINAL AND SOMATIC CELL DIVISION AND FUNCTION.

La sub-fertilit\ue0 rappresenta uno dei principali problemi che l'industria lattiero-casearia si trova ad affrontare attualmente. Si tratta di un problema multifattoriale che dipende da diversi aspetti della funzione riproduttiva. La qualit\ue0 dell'ovocita, determinata durante la follicologenesi, \ue8 sicuramente cruciale per ottenere risultati riproduttivi ottimali. Per migliorare la fertilit\ue0 femminile nel bovino \ue8 fondamentale definire i fattori e i meccanismi che determinano la qualit\ue0 del gamete femminile, in quanto il declino della funzione riproduttiva in questa specie \ue8 dovuto principalmente alla scarsa qualit\ue0 degli ovociti. In particolare, \ue8 importante conoscere quali proteine regolano la meiosi, la fecondabilit\ue0 e lo sviluppo embrionale dell'ovocita. In questo progetto, abbiamo considerato principalmente il ruolo del Progesterone Receptor Membrane Component-1 (PGRMC1) sia nell'ovocita che nel compartimento follicolare, considerato che il dialogo tra compartimento germinale e compartimento somatico \ue8 uno dei fattori chiave coinvolti nell'acquisizione della competenza allo sviluppo del gamete femminile. Il PGRMC1 \ue8 una proteina multifunzionale ed altamente conservata in specie anche filogeneticamente distanti. Nei mammiferi \ue8 espressa in diversi sistemi, compresi gli organi riproduttivi, e localizza in diversi compartimenti subcellulari. Tuttavia, il suo ruolo e il preciso meccanismo d'azione in ogni sistema e/o compartimento subcellulare non sono ancora del tutto conosciuti. La parte pi\uf9 considerevole di questo progetto di dottorato riguarda gli studi effettuati sul ruolo del PGRMC1 nella divisione cellulare, data la sua localizzazione a livello di fuso mitotico e meiotico. Abbiamo dimostrato che la deplezione del PGRMC1 determina difetti nella divisione cellulare e la sua interazione con l'Aurora chinasi B (AURKB) indica che potrebbe svolgere la sua azione durante la citodieresi, l'ultima fase della divisione. Durante la maturazione meiotica, abbiamo valutato anche il suo ruolo nel mediare l'azione del progesterone (P4) confrontando il suo effetto con quello del recettore nucleare del progesterone (nPGR). L'inibizione di entrambi i tipi di recettore determina lo stesso effetto sull'organizzazione della piastra metafasica e sulla competenza allo sviluppo dell'ovocita, ma in fasi differenti. Inoltre, abbiamo testato l'ipotesi che il PGRMC1 possa modulare la funzione del nucleolo. Studi di immunofluorescenza hanno confermato la presenza del PGRMC1 nel nucleolo di cellule della granulosa (bGC) e negli ovociti di bovino, nonch\ue8 la colocalizzazione con la nucleolina, la proteina nucleolare pi\uf9 abbondante che svolge importanti funzioni in questo compartimento subcellulare. Inoltre, la down-regolazione del PGRMC1 determina uno spostamento della nucleolina dal nucleolo al nucleoplasma suggerendo un'associazione funzionale tra queste due proteine. Questa interazione \ue8 probabilmente mediata dalla presenza di ulteriori molecole in quanto successivi esperimenti di "in-situ proximity ligation" mostrano che le due proteine non interagiscono direttamente. Oltre al tema principale di questo progetto, la divisione cellulare \ue8 uno dei processi pi\uf9 importanti anche nel cancro e l'espressione del PGRMC1 \ue8 elevata in molti tipi di tumore nell'uomo. Per questo abbiamo intrapreso uno studio preliminare per caratterizzare la sua espressione nei diversi tipi di tumore mammario di cane. In base ai nostri risultati, l'espressione del PGRMC1 diminuisce con l'aumentare della malignit\ue0 del tumore e le diverse isoforme di PGRMC1 sono presenti sia nel tessuto sano che in quello neoplastico. In conclusione, i nostri risultati suggeriscono che il PGRMC1 pu\uf2 avere un ruolo nel compartimento follicolare, in particolare sia nella mitosi che nella meiosi. La sua funzione potrebbe essere legata alla sua localizzazione nucleolare, nonch\ue8 alla sua capacit\ue0 di mediare l'azione del P4 durante la meiosi dell'ovocita. Il suo ruolo come regolatore della divisione cellulare pu\uf2 essere rilevante anche in alcune patologie quali il cancro. Ulteriori studi saranno diretti a valutare i meccanismi di azione molecolari tramite i quali il PGRMC1 agisce in questi processi al fine di ampliare la nostra conoscenza di questa proteina poliedrica.Subfertility is one of the major problems that dairy industry is facing nowadays. Subfertility is a multifactorial issue depending on different aspects of reproductive function. Oocyte quality, which is determined during folliculogenesis, is certainly crucial for optimal reproductive outcome. Since declining fertility of cattle is mainly due to the poor quality of the oocytes, defining the factors and mechanisms that affect oocyte quality is essential to improve female fertility. In particular, basic knowledge of which proteins within the oocyte regulate meiosis, oocyte fertilizability and early embryonic development would be advantageous. In this project, we primarily considered the role of Progesterone Receptor Membrane Component-1 (PGRMC1) in both the oocyte and follicular compartment since the dialogue between the two compartments is one of the key factors involved in oocyte competence acquisition. PGRMC1 is a highly conserved and multifunctional protein that is found expressed in multiple systems, including reproductive organs, and localizes in multiple sub-cellular compartments. However, its role(s) and precise mechanism(s) of action in each systems and/or subcellular compartment are not yet fully understood. The more considerable part of this PhD project consists of studies on PGRMC1's involvement as a cell division regulator, according to its localization to the mitotic and meiotic spindle. We showed that PGRMC1 depletion leads to defective cell division in both oocyte and somatic cells and its action could be exerted during cytokinesis, the very last mitotic phase, as demonstrated by its association with Aurora kinase B (AURKB). During meiotic maturation, we also evaluated PGRMC1 involvement as a possible mediator of progesterone (P4) action, by comparing the effects of inhibiting its function with that of nuclear progesterone receptor's (nPGR) inhibition. Our data suggests that both receptors have an effect on meiotic progression but possibly at different stages of oocyte maturation. Moreover, we tested the hypothesis that PGRMC1 might modulate the function of the nucleolus. Immunofluorescence experiments confirmed PGRMC1 nucleolar localization in bovine granulosa cells (bGC) and bovine oocytes and its co-localization with nucleolin, the most abundant nucleolar protein exerting important functions in this subcellular compartment. Moreover, a PGRMC1/nucleolin functional association is suggested by PGRMC1's downregulation determining nucleolin shift from the nucleolus to the nucleoplasm. However, in situ proximity ligation assay did not detect a direct interaction between these two proteins, suggesting the involvement of additional molecules that could mediate PGRMC1/nucleolin interaction. Besides the main theme of this project, cell division is one of the most important processes in cancer biology and PGRMC1 is known to be overexpressed in many types of tumors in humans. Therefore, we studied its expression in different types of canine mammary tumors. Our preliminary results showed PGRMC1 expression decreases with the malignancy of the tumor and different PGRMC1 isoforms are present both in normal and tumoral tissue. To conclude, these findings suggest a role of PGRMC1 in the follicular compartment, being implicated in both meiotic and mitotic process of the germinal and somatic compartment, respectively. This action it is likely mediated by PGRMC1 located at a particular site of the spindle, i.e the midzone and /or the midbody of dividing cells. PGRMC1 also localizes at the nucleolus of both oocytes and granulosa cells where it likely exerts additional function(s) in mediating cellular stress and/or other nucleolar-relates process. Clearly these findings have major implication in the overall process of folliculogenesis. Moreover, PGRMC1's role as a regulator of cell proliferation could be relevant also in some diseases such as cancer. Further investigations will aim at investigating PGRMC1's molecular mechanisms of action in these processes to widen our knowledge on this multifaceted protein

Remediation of Expansive Soils Using Agricultural Waste Bagasse Ash

© 2016 The Authors. Published by Elsevier B.V. Bagasse is a fibrous material remaining after crushing sugarcane to extract its juice; and bagasse ash is produced after burning bagasse. Improper disposal of this material can create environmental problems around sugar manufacturing plants. Bagasse ash, comprising a high percentage of silica (SiO2), is considered as a sensible pozzolanic material with non-reactive behaviour and has potential to be used in road subgrade stabilisation. One of the main challenges for transportation organisations in Australia is to treat subgrades including expansive soils. Expansive soils exhibit significant movements when the moisture content changes, and hence it causes substantial damage to road pavements constructed over these type of soils. Road engineers need to employ materials having acceptable strength, relatively low price and being eco-friendly. In order to demonstrate the potential ability of bagasse ash in curtailing the adverse effects of expansive soils on roads, an array of experimental tests using bagasse ash have been conducted. In this study to activate and improve the effectiveness of bagasse ash, hydrated lime was used and mixed with black soil samples, collected from Queensland Australia. Samples were prepared using different contents of bagasse ash and hydrated lime (0%, 6%, 10%, 18% and 25% by the dry mass of soil), at a ratio of 3:1, respectively. The results of free swell ratio (FSR) test, unconfined compression strength (UCS) and California bearing ratio (CBR) tests are presented for untreated and treated samples after various curing time periods of 3, 7 and 28 days. The outcomes of these tests clearly demonstrate that stabilisation of expansive soils using bagasse ash and hydrated lime not only improves the strength, but also facilitates to cope with environmental concerns through reduction of sugar industry waste material

Finite driving rate and anisotropy effects in landslide modeling

In order to characterize landslide frequency-size distributions and individuate hazard scenarios and their possible precursors, we investigate a cellular automaton where the effects of a finite driving rate and the anisotropy are taken into account. The model is able to reproduce observed features of landslide events, such as power-law distributions, as experimentally reported. We analyze the key role of the driving rate and show that, as it is increased, a crossover from power-law to non power-law behaviors occurs. Finally, a systematic investigation of the model on varying its anisotropy factors is performed and the full diagram of its dynamical behaviors is presented.Comment: 8 pages, 9 figure

PGRMC1 localization and putative function in the nucleolus of bovine granulosa cells and oocytes

Progesterone Receptor Membrane Component-1 (PGRMC1) is a highly conserved multifunctional protein that is found in numerous systems, including reproductive system. Interestingly, PGRMC1 is expressed at several intracellular locations, including the nucleolus. The aim of this study is to investigate the functional relationship between PGRMC1 and nucleolus. Immunofluorescence experiments confirmed PGRMC1's nucleolar localization in cultured bovine granulosa cells (bGC) and oocytes. Additional experiments conducted on bGC revealed that PGRMC1 co-localizes with nucleolin (NCL), a major nucleolar protein. Furthermore, small interfering RNA (RNAi) mediated gene-silencing experiments showed that when PGRMC1 expression was depleted, NCL translocated from the nucleolus to the nucleoplasm. Similarly, oxidative stress induced by hydrogen peroxide (H2O2) treatment, reduced PGRMC1 immunofluorescent signal in the nucleolus and increased NCL nucleoplasmic signal, when compared to non-treated cells. Although PGRMC1 influenced NCL localization, a direct interaction between these two proteins was not detected using in situ proximity ligation assay. This suggests the involvement of additional molecules in mediating the co-localization of PGRMC1 and nucleolin. Since nucleolin translocates into the nucleoplasm in response to various cellular stressors, PGRMC1's ability to regulate its localization within the nucleolus is likely an important component of mechanism by which cells response to stress. This concept is consistent with PGRMC1's well-described ability to promote ovarian cell survival and provides a rationale for future studies on PGRMC1, NCL and the molecular mechanism by which these two proteins protect against the adverse effect of cellular stressors, including oxidative stress

Neoplastic transformation of mouse C3H 10T1/2 and Syrian hamster embryo cells by heavy ions

C3H 10T1/2 mouse-embryo fibroblasts were used for transformation experiments to study the effectiveness of various heavy ions with energies up to 20 MeV/u and LET values from 170 to 16.000 keV/μm. The transformation frequency per unit absorbed dose decreased with increasing ionization density; at the highest values of LET we found a decrease even of the transformation efficiency per unit fluence. Uranium ions at energies of 5, 9, and 16.3 MeV/u did not induced any transformation. In additional studies piimary Syrian hamster embryo cells (SHE) were exposed to heavy ions in order to characterize cytological and molecular changes which may be correlated with neoplastic transformation. Growth behaviour, chromosomal status, tumorigenicity in nude mice, and expression of oncogenes of transformed cell lines were examined

Sleep-related epileptic behaviors and non-REM-related parasomnias: Insights from stereo-EEG

During the last decade, many clinical and pathophysiological aspects of sleep-related epileptic and non-epileptic paroxysmal behaviors have been clarified. Advances have been achieved in part through the use of intracerebral recording methods such as stereo-electroencephalography (S-EEG), which has allowed a unique "in vivo" neurophysiological insight into focal epilepsy. Using S-EEG, the local features of physiological and pathological EEG activity in different cortical and subcortical structures have been better defined during the entire sleep-wake spectrum. For example, S-EEG has contributed to clarify the semiology of sleep-related seizures as well as highlight the specific epileptogenic networks involved during ictal activity. Moreover, intracerebral EEG recordings derived from patients with epilepsy have been valuable to study sleep physiology and specific sleep disorders. The occasional co-occurrence of NREM-related parasomnias in epileptic patients undergoing S-EEG investigation has permitted the recordings of such events, highlighting the presence of local electrophysiological dissociated states and clarifying the underlying pathophysiological substrate of such NREM sleep disorders. Based on these recent advances, the authors review and summarize the current and relevant S-EEG literature on sleep-related hypermotor epilepsies and NREM-related parasomnias. Finally, novel data and future research hypothesis will be discussed

Arqueología en la cuenca inferior del Río Salado (Provincia de Santa Fe). Primeras aproximaciones al estudio de un sitio arqueológico con enterratorios múltiples

En este trabajo se da cuenta de las tareas llevadas a cabo en el sitio arqueológico Río Salado Coronda II, en la ciudad de Santo Tomé, provincia de Santa Fe. Los estudios realizados abren una nueva serie de expectativas arqueológicas para el área de la cuenca inferior del río Salado, la cual se mantuvo durante muchos años relegada en función de las investigaciones realizadas en los ambientes de influencia directa del río Paraná. El hallazgo por parte de vecinos en el barrio de Villa Adelina Este de la ciudad de Santo Tomé (provincia de Santa Fe) de ítems arqueológicos, derivó en el descubrimiento de un área de enterratorios múltiples, asociada a restos materiales que sugieren un uso particular de ese espacio por parte de las sociedades que lo ocuparon en el pasado, vinculado al sistema de creencias. Se presentan aquí los primeros resultados obtenidos del análisis del registro arqueológico del sitio mencionado. </p

Effect of pharmacological inhibition of Progesterone receptors PGRMC1 and nPR on bovine oocyte meiosis

Folliculogenesis is the fundamental process leading to oocyte maturation and its developmental competence, which are determined by oocyte and follicular cells interplay (Luciano et al., 2004). Recent studies in cattle describe Progesterone (P4) as a key molecule acting during follicle development through different signaling pathways involving different receptors (Aparicio et al., 2011, Nilsson et al., 2009). The aim of this study is to evaluate the effect on oocyte meiotic maturation of inhibiting two P4 receptors: Progesterone Receptor Membrane Component 1 (PGRMC1) and the classic nuclear Progesterone Receptor (nPR) respectively using the specific inhibitors AG205 and Aglepristone. Bovine cumulus cell-oocyte complexes (COCs) and denuded oocytes (DOs) were in vitro matured with different concentrations of AG205. Our results showed a decrease both in first polar body (PBI) extrusion and in the percentage of oocytes reaching MII stage in treated oocytes compared to controls (one way ANOVA, P<0.05); these effects were more marked in DOs, confirming PGRMC1 specific role in the oocyte. In AG205 treated oocytes aberrant meiotic figures were observed, including double metaphase plates or DNA scattered in the ooplasm. In addition, aberrant meiotic plates showed irregular co-localization of PGRMC1 and AURKB; the proteins didn\u2019t localize at the centromeric region of each chromosomes as previously described (Luciano et al., 2013). This results suggests a P4 role in meiotic division mediated by PGRMC1 receptor. By contrast, Aglepristone inhibition of nPR didn\u2019t affect dramatically the percentage of oocytes reaching MII stage of maturation. However, MII plates morphology analysis showed a significantly greater tubulin spindle length. This feature could account for the previously described reduced in vitro embryo development consequent to nPR inhibition (Aparicio et al., 2011). Thus, P4 driven nuclear maturation could act on different oocyte development stages. Further studies are in progress to elucidate P4 complex action in mammalian oocyte functio

The novel transcriptional regulator SczA mediates protection against Zn2+ stress by activation of the Zn2+-resistance gene czcD in Streptococcus pneumoniae

Maintenance of the intracellular homeostasis of metal ions is important for the virulence of many bacterial pathogens. Here, we demonstrate that the czcD gene of the human pathogen Streptococcus pneumoniae is involved in resistance against Zn2+, and that its transcription is induced by the transition-metal ions Zn2+, Co2+ and Ni2+. Upstream of czcD a gene was identified, encoding a novel TetR family regulator, SczA, that is responsible for the metal ion-dependent activation of czcD expression. Transcriptome analyses revealed that in a sczA mutant expression of czcD, a gene encoding a MerR-family transcriptional regulator and a gene encoding a zinc-containing alcohol dehydrogenase (adhB) were downregulated. Activation of the czcD promoter by SczA is shown to proceed by Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. This is the first example of a metalloregulatory protein belonging to the TetR family that has been described. The presence in S. pneumoniae of the Zn2+-resistance system characterized in this study might reflect the need for adjustment to a fluctuating Zn2+ pool encountered by this pathogen during infection of the human body