Variations in maternal care alter corticosterone and 17beta-estradiol levels, estrous cycle and folliculogenesis and stimulate the expression of estrogen receptors alpha and beta in the ovaries of UCh rats

Background: Variations in maternal care are associated with neonatal stress, hormonal disturbances and reproductive injuries during adulthood. However, the effects of these variations on sex hormones and steroid receptors during ovary development remain undetermined. This study aimed to investigate whether variations in maternal care are able to influence the hormonal profile, follicular dynamics and expression of AR, ER-alpha and ER-beta in the ovaries of UCh rat offspring. Methods: Twenty-four adult UCh rats, aged 120 days, were randomly divided into two groups (UChA and UChB) and mated. Maternal care was assessed from birth (day 0) to the 10th postnatal day (PND). In adulthood, twenty adult female rats (UChA and UChB offspring; n = 10/group), aged 120 days, were euthanized by decapitation during the morning estrus. Results: UChA females (providing high maternal care) more frequently displayed the behaviors of carrying pups, as well as licking/grooming and arched back nursing cares. Also, mothers providing high care had elevated corticosterone levels. Additionally, offspring receiving low maternal care showed the highest estrous cycle duration, increased corticosterone and 17beta-estradiol levels, overexpression of receptors ER-alpha and ER-beta, increased numbers of primordial, antral and mature follicles and accentuated granulosa cell proliferation. Conclusions: Our study suggests that low maternal care alters corticosterone and 17beta-estradiol levels, disrupting the estrous cycle and folliculogenesis and differentially regulating the expression of ER-alpha and ER-beta in the ovaries of adult rats.

Colonization of gastropods on subtidal reefs depends on density in adjacent habitats, not on disturbance regime

[EN] Habitats dominated by algal canopies are often altered by physical disturbances of varying severity, changing environmental conditions and biological processes. We used Artificial Seaweed Units (ASUs) to test whether severity of physical disturbances on algal canopies affects the post-disturbance colonization of gastropods on subtidal reefs. Specifically, we examined patterns of assemblage structure of gastropods to test the hypothesis that the extent and intensity of canopy removal affects the post-disturbance colonization of ASUs, testing the consistency of these effects among four regions encompassing a 68 latitudinal gradient in southwestern Australia. Because adjacent habitats can act as a source of new colonists (either as drifting migrants or as a source of propagules) from the perimeter surrounding perturbed areas, we also predicted that patterns of colonization (types and total abundances of colonizers) were influenced by the available pool of individuals at the scale of reefs. Three reefs were selected within each region. On each reef, ASUs were placed in the centre of circular canopy clearings of different size (0, 3, 13 and 50 m2) and intensity (50% vs 100%), and retrieved after 3 months. Resulting assemblages occupying the ASUs were quantitatively representative of the adjacent (undisturbed), algal-associated, assemblages at the scale of reef. Within reefs, recruited assemblages largely mimicked those associated with erect red algae. However, neither disturbance size nor intensity affected the colonization patterns across reefs and regions. These results suggest that algal-associated gastropods, regardless of the prevalent mode of dispersion, are resilient to physical disturbances to canopies across broad geographical regions as long as the pool of potential colonists is maintained. A high dispersal ability of gastropods likely ensures a quick colonization of recovering algal habitats.

Mechanism of colonization in the Mediterranean-sea. Asperarca nodulosa: morphological and genetic analysis

Il mar Mediterraneo è un bacino acquifero peculiare per la recente colonizzazione di specie aliene, per l’evento geologico legato alla Crisi di salinità del Messiniano e per l’ampio range di salinità. L’individuazione dei meccanismi di colonizzazione si è incentrata sullo studio morfologico, istologico e molecolare delle specie Asperarca nodulosa ed Anadara demiri (Arcidae-Bivalvia-Mollusca). La ricerca si è basata sulla caratterizzazione morfologica, con utilizzo del microscopio elettronico a scansione, al fine di individuare il tipo di sviluppo larvale. Successivamente i dati rilevati al S.E.M. sono stati supportati dall’indagine istologica che ha evidenziato la presenza di gonadi a sessi distinti e la non incubazione larvale. L’ulteriore analisi filogenetica ha permesso di evidenziare la netta suddivisione tra le tre popolazioni studiate, indagine effettuata tramite marcatori arbitrari (RAPDs) e nucleari specifici (ITS). I risultati ottenuti trovano supporto da quanto noto su base morfologica. I dati, nel complesso, mostrano una perdita delle capacità di diffusione della specie tramite sviluppo larvale plantotrofico a favore di quello lecitotrofico o diretto; tale tesi è ulteriormente supportata dai dati molecolari che mostrano una netta separazione delle popolazioni prese in esame ed un conseguente isolamento tra individui appartenenti a zone di profondità del Mediterraneo (sub-bacini abissali). La ricerca ha, inoltre,esaminato i meccanismi di introduzione attuali nel bacino acquifero che è soggetto ad una nuova invasione da parte specie aliene dovuta all’apertura del canale di Suez. L’analisi si è focalizzata sullo studio per l’ individuazione dell’origine della specie aliena A. demiri , di presunta derivazione Indo-Pacifica, ma rivelatasi, nei dati preliminari, di origine Atlantica.

Biomimetica per l'ingegneria tissutale dell'osso

Reconstruction of bone is needed for high bone loss due to congenital deformities, trauma or neoplastic diseases. Commonly, orthopaedic surgical treatments are autologus or allogenic bone implant or prosthetic implant. A choice to the traditional approaches could be represented by tissue engineering that use cells (and/or their products) and innovative biomaterials to perform bone substitutes biologically active as an alternative to artificial devices. In the last years, there was a wide improvement in biology on stem cells potential research and in biomedical engineering through development of new biomaterials designed to resemble the physiological tissues. Tissue engineering strategies and smart materials aim together to stimulate in vivo bone regeneration. This approaches drive at restore not only structure integrity and/or function of the original tissue, but also to induce new tissue deposition in situ. An intelligent bone substitute is now designed like not only a scaffold but also as carrier of regeneration biomolecular signals. Biomimetics has helped to project new tissue engineered devices to simulate the physiological substrates architecture, such extracellular matrix (ECM), and molecular signals that drive the integration at the interface between pre-existing tissue and scaffold. Biomimetic strategies want to increase the material surface biological activity with physical modifications (topography) o chemical ones (adhesive peptides), to improve cell adhesion to material surface and possibly scaffold colonization. This study evaluated the effects of biomimetic modifications of surgical materials surface, as poly-caprolattone (PCL) and titanium on bone stem cells behaviour in a marrow experimental model in vitro. Two biomimetic strategies were analyzed; ione beam irradiation, that changes the surface roughness at the nanoscale, and surface functionalization with specific adhesive peptides or Self Assembled Monolayers (SAMs). These new concept could be a mean to improve the early (cell adhesion, spreading..) and late phases (osteoblast differentiation) of cell/substrate interactions.

(De)colonization through topophilia: Marjorie Kinnan Rawlings’s life and work in Florida

(De)colonization Through Topophilia: Marjorie Kinnan Rawlings’s Life and Work in Florida attempts to reveal the author’s intimate connection to and mental growth through her place, namely the Cross Creek environs, and its subsequent effect on her writing. In 1928, Marjorie Kinnan Rawlings and her first husband Charles Rawlings came to Cross Creek, Florida. They bought the shabby farmhouse on Cross Creek Road, trying to be both, writers and farmers. However, while Charles Rawlings was unable to write in the backwoods of the Florida Interior, Rawlings found her literary voice and entered a symbiotic, reciprocal relationship with the natural world of the Cracker frontier. Her biographical preconditions – a childhood spent in the rural area of Rock Creek, outside of Washington D. C. - and a father who had instilled in her a sense of place or topophilia, enabled her to overcome severe marriage tensions and the hostile climate women writers faced during the Depression era. Nature as a helping ally and as an “undomesticated”(1) space/place is a recurrent motif throughout most of Rawlings’s Florida literature. At a time when writing the American landscape/documentary and the extraction of the self from texts was the prevalent literary genre, Marjorie Kinnan Rawlings inscribed herself into her texts. However, she knew that the American public was not yet ready for a ‘feminist revolt’, but was receptive of the longtime ‘inaudible’ voices from America’s regions, especially with regard to urban poverty and a homeward yearning during the Depression years. Fusing with the dynamic eco-consciousness of her Cracker friends and neighbors, Rawlings wrote in the literary category of regionalism enabling her to pursue three of her major aims: an individuated self, a self that assimilated with the ‘master narratives’ of her time and the recognition of the Florida Cracker and Scrub region. The first part of this dissertation briefly introduces the largely unknown and underestimated writer Marjorie Kinnan Rawlings, providing background information on her younger years, the relationship toward her family and other influential persons in her life. Furthermore, it takes a closer look at the literary category of regionalism and Rawlings’s use of ‘place’ in her writings. The second part is concerned with the ‘region’ itself, the state of Florida. It focuses on the natural peculiarities of the state’s Interior, the scrub and hammock land around her Cracker hamlet as well as the unique culture of the Florida Cracker. Part IV is concerned with the analysis of her four Florida books. The author is still widely related to the ever-popular novel The Yearling (1938). South Moon Under (1933) and Golden Apples (1935), her first two novels, have not been frequently republished and have subsequently fallen into oblivion. Cross Creek (1942), Rawlings’s last Florida book, however, has recently gained renewed popularity through its use in classes on nature writers and the non-fiction essay but it requires and is here re-evaluated as the author’s (relational) autobiography. The analysis through place is brought to completion in this work and seems to intentionally close the circle of Rawlings’s Florida writings. It exemplifies once more that detachment from place is impossible for Rawlings and that the intermingling of life and place in literature, is essential for the (re)creation of her identity. Cross Creek is therefore not only one of Rawlings’s greatest achievements; it is more importantly the key to understanding the author’s self and her fiction. Through the ‘natural’ interrelationship of place and self and by looking “mutually outward and inward,”(2) Marjorie Kinnan Rawlings finds her literary voice, a home and ‘a room of her own’ in which to write and come to consciousness. Her Florida literature is not only product but also medium and process in her assessment of her identity and self. _____________ (1) Alaimo, Stacy. Undomesticated Ground: Recasting Nature as Feminist Space (Ithaca: Cornell UP, 2000) 23. (2) Libby, Brooke. “Nature Writing as Refuge: Autobiography in the Natural World” Reading Under the Sign of Nature. New Essays in Ecocriticism. Ed. John Tallmadge and Henry Harrington. (Salt Lake City: The U of Utah P, 2000) 200.

Development of newly conceived biomimetic nano-structured biomaterials as scaffolds for bone and osteochondral regeneration

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

Nuove biotenologie per la produzione di piante micorrizate con tartufo

Il lavoro svolto durante questa tesi di dottorato pone le basi per lo sviluppo di nuove biotecnologie della micorrizazione di piante forestali con tartufi pregiati ed in particolare con Tuber magnatum. Durante questa tesi è stato possibile isolare e mantenere in coltura pura il micelio di T. magnatum, ad ottenere e descrivere le sue micorrize e quelle di altri tartufi “bianchi” (T. oligospermum, T. borchii) e a seguire l’evoluzione del micelio nel suolo utilizzando la tecnica della real time PCR. Sono stati disegnati primer specie specifici in grado di identificare T. oligospermum ed è stata verificata la possibiltà di utilizzare questi primers in PCR multiplex con quelli specifici di T. magnatum e di T. borchii già presenti in bibliografia, al fine di “scovare” sia frodi nella commercializzaione degli ascomi sia eventuali contaminazioni nelle piante micorrizate. Per migliorare lo sviluppo miceliare di tartufo abbiamo si è cercato di migliorare il mezzo nutritivo per la crescita del micelio utilizzando: fonti di carbonio diverse, estratti radicali di nocciolo e singole frazioni separate dagli stessi. Infine sono stati sviluppati protocolli di crioconservazione per miceli di tartufo. Gli estratti radicali sono in grado di stimolare le crescita miceliare del tartufo modello T. borchii e dimodificarne la morfologia ifale. Questo risultati sono stati confermati anche dall’aumento dell’espressione di geni CDC42 e Rho-GDI, due geni legati alla crescita apicale polarizzata delle ife dei funghi filamentosi. Inoltre è stato dimostrato che il mantenimento in coltura per numerosi anni dei miceli di tartufo provoca una perdita della capacità d’infettare le radici delle piante e quindi il loro potenziale utilizzo sia a scopo sperimentale sia a scopo colturale. Questo pone in risalto l’importanza della conservazione a lungo termine del materiale biologico a disposizione ed è stato dimostrato che la crioconservazione è applicabile con successo anche alle specie del genere Tuber.

Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium

The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.

The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

Molecular epidemiology of the nasal colonization by methicillin-susceptible Staphylococcus aureus in Swiss children

Nasal carriage of Staphylococcus aureus contributes to an increased risk of developing an infection with the same bacterial strain. Genetic regulatory elements and toxin-expressing genes are virulence factors associated with the pathogenic potential of S. aureus. We undertook an extensive molecular characterization of methicillin-susceptible S. aureus (MSSA) carried by children. MSSA were recovered from the nostrils of children. The presence of Panton-Valentine leukocidin (PVL), exfoliatins A and B (exfoA and exfoB), and the toxic-shock staphylococcal toxin (TSST-1) and agr group typing were determined by quantitative PCR. A multiple-locus variable-number of tandem repeat analysis (MLVA) assay was also performed for genotyping. Five hundred and seventy-two strains of MSSA were analysed. Overall, 30% were positive for toxin-expressing genes: 29% contained one toxin and 1.6% two toxins. The most commonly detected toxin gene was tst, which was present in 145 (25%) strains. The TSST-1 gene was significantly associated with the agr group 3 (OR 56.8, 95% CI 32.0-100.8). MLVA analysis revealed a large diversity of genetic content and no clonal relationship was demonstrated among the analysed MSSA strains. Multilocus sequence typing confirmed this observation of diversity and identified ST45 as a frequent colonizer. This broad diversity in MSSA carriage strains suggests a limited selection pressure in our geographical area.

Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses

The lower intestine of adult mammals is densely colonized with nonpathogenic (commensal) microbes. Gut bacteria induce protective immune responses, which ensure host-microbial mutualism. The continuous presence of commensal intestinal bacteria has made it difficult to study mucosal immune dynamics. Here, we report a reversible germ-free colonization system in mice that is independent of diet or antibiotic manipulation. A slow (more than 14 days) onset of a long-lived (half-life over 16 weeks), highly specific anticommensal immunoglobulin A (IgA) response in germ-free mice was observed. Ongoing commensal exposure in colonized mice rapidly abrogated this response. Sequential doses lacked a classical prime-boost effect seen in systemic vaccination, but specific IgA induction occurred as a stepwise response to current bacterial exposure, such that the antibody repertoire matched the existing commensal content.

Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM(con21)). 16S rRNA sequence analysis comparing LCM, LCM(con21) and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri(RR) strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.