Optical memory with TNLC devices (1982)

In this paper we describe a twisted nematic liquid crystal (TNLC) device structure with optical feedback capable of bistable operation and optical memory. Its structure is the conventional one as employed in hybrid optical bistability.

MIA - a free and open source software for gray scale medical image analysis

Background Gray scale images make the bulk of data in bio-medical image analysis, and hence, the main focus of many image processing tasks lies in the processing of these monochrome images. With ever improving acquisition devices, spatial and temporal image resolution increases, and data sets become very large. Various image processing frameworks exists that make the development of new algorithms easy by using high level programming languages or visual programming. These frameworks are also accessable to researchers that have no background or little in software development because they take care of otherwise complex tasks. Specifically, the management of working memory is taken care of automatically, usually at the price of requiring more it. As a result, processing large data sets with these tools becomes increasingly difficult on work station class computers. One alternative to using these high level processing tools is the development of new algorithms in a languages like C++, that gives the developer full control over how memory is handled, but the resulting workflow for the prototyping of new algorithms is rather time intensive, and also not appropriate for a researcher with little or no knowledge in software development. Another alternative is in using command line tools that run image processing tasks, use the hard disk to store intermediate results, and provide automation by using shell scripts. Although not as convenient as, e.g. visual programming, this approach is still accessable to researchers without a background in computer science. However, only few tools exist that provide this kind of processing interface, they are usually quite task specific, and don’t provide an clear approach when one wants to shape a new command line tool from a prototype shell script. Results The proposed framework, MIA, provides a combination of command line tools, plug-ins, and libraries that make it possible to run image processing tasks interactively in a command shell and to prototype by using the according shell scripting language. Since the hard disk becomes the temporal storage memory management is usually a non-issue in the prototyping phase. By using string-based descriptions for filters, optimizers, and the likes, the transition from shell scripts to full fledged programs implemented in C++ is also made easy. In addition, its design based on atomic plug-ins and single tasks command line tools makes it easy to extend MIA, usually without the requirement to touch or recompile existing code. Conclusion In this article, we describe the general design of MIA, a general purpouse framework for gray scale image processing. We demonstrated the applicability of the software with example applications from three different research scenarios, namely motion compensation in myocardial perfusion imaging, the processing of high resolution image data that arises in virtual anthropology, and retrospective analysis of treatment outcome in orthognathic surgery. With MIA prototyping algorithms by using shell scripts that combine small, single-task command line tools is a viable alternative to the use of high level languages, an approach that is especially useful when large data sets need to be processed.

Contribución a la caracterización espacial de canales con sistemas MIMO-OFDM en la banda de 2,45 Ghz

La tecnología de múltiples antenas ha evolucionado para dar soporte a los actuales y futuros sistemas de comunicaciones inalámbricas en su afán por proporcionar la calidad de señal y las altas tasas de transmisión que demandan los nuevos servicios de voz, datos y multimedia. Sin embargo, es fundamental comprender las características espaciales del canal radio, ya que son las características del propio canal lo que limita en gran medida las prestaciones de los sistemas de comunicación actuales. Por ello surge la necesidad de estudiar la estructura espacial del canal de propagación para poder diseñar, evaluar e implementar de forma más eficiente tecnologías multiantena en los actuales y futuros sistemas de comunicación inalámbrica. Las tecnologías multiantena denominadas antenas inteligentes y MIMO han generado un gran interés en el área de comunicaciones inalámbricas, por ejemplo los sistemas de telefonía celular o más recientemente en las redes WLAN (Wireless Local Area Network), principalmente por la mejora que proporcionan en la calidad de las señales y en la tasa de transmisión de datos, respectivamente. Las ventajas de estas tecnologías se fundamentan en el uso de la dimensión espacial para obtener ganancia por diversidad espacial, como ya sucediera con las tecnologías FDMA (Frequency Division Multiplexing Access), TDMA (Time Division Multiplexing Access) y CDMA (Code Division Multiplexing Access) para obtener diversidad en las dimensiones de frecuencia, tiempo y código, respectivamente. Esta Tesis se centra en estudiar las características espaciales del canal con sistemas de múltiples antenas mediante la estimación de los perfiles de ángulos de llegada (DoA, Direction-of- Arrival) considerando esquemas de diversidad en espacio, polarización y frecuencia. Como primer paso se realiza una revisión de los sistemas con antenas inteligentes y los sistemas MIMO, describiendo con detalle la base matemática que sustenta las prestaciones ofrecidas por estos sistemas. Posteriormente se aportan distintos estudios sobre la estimación de los perfiles de DoA de canales radio con sistemas multiantena evaluando distintos aspectos de antenas, algoritmos de estimación, esquemas de polarización, campo lejano y campo cercano de las fuentes. Así mismo, se presenta un prototipo de medida MIMO-OFDM-SPAA3D en la banda ISM (Industrial, Scientific and Medical) de 2,45 Ghz, el cual está preparado para caracterizar experimentalmente el rendimiento de los sistemas MIMO, y para caracterizar espacialmente canales de propagación, considerando los esquemas de diversidad espacial, por polarización y frecuencia. Los estudios aportados se describen a continuación. Los sistemas de antenas inteligentes dependen en gran medida de la posición de los usuarios. Estos sistemas están equipados con arrays de antenas, los cuales aportan la diversidad espacial necesaria para obtener una representación espacial fidedigna del canal radio a través de los perfiles de DoA (DoA, Direction-of-Arrival) y por tanto, la posición de las fuentes de señal. Sin embargo, los errores de fabricación de arrays así como ciertos parámetros de señal conlleva un efecto negativo en las prestaciones de estos sistemas. Por ello se plantea un modelo de señal parametrizado que permite estudiar la influencia que tienen estos factores sobre los errores de estimación de DoA, tanto en acimut como en elevación, utilizando los algoritmos de estimación de DOA más conocidos en la literatura. A partir de las curvas de error, se pueden obtener parámetros de diseño para sistemas de localización basados en arrays. En un segundo estudio se evalúan esquemas de diversidad por polarización con los sistemas multiantena para mejorar la estimación de los perfiles de DoA en canales que presentan pérdidas por despolarización. Para ello se desarrolla un modelo de señal en array con sensibilidad de polarización que toma en cuenta el campo electromagnético de ondas planas. Se realizan simulaciones MC del modelo para estudiar el efecto de la orientación de la polarización como el número de polarizaciones usadas en el transmisor como en el receptor sobre la precisión en la estimación de los perfiles de DoA observados en el receptor. Además, se presentan los perfiles DoA obtenidos en escenarios quasiestáticos de interior con un prototipo de medida MIMO 4x4 de banda estrecha en la banda de 2,45 GHz, los cuales muestran gran fidelidad con el escenario real. Para la obtención de los perfiles DoA se propone un método basado en arrays virtuales, validado con los datos de simulación y los datos experimentales. Con relación a la localización 3D de fuentes en campo cercano (zona de Fresnel), se presenta un tercer estudio para obtener con gran exactitud la estructura espacial del canal de propagación en entornos de interior controlados (en cámara anecóica) utilizando arrays virtuales. El estudio analiza la influencia del tamaño del array y el diagrama de radiación en la estimación de los parámetros de localización proponiendo, para ello, un modelo de señal basado en un vector de enfoque de onda esférico (SWSV). Al aumentar el número de antenas del array se consigue reducir el error RMS de estimación y mejorar sustancialmente la representación espacial del canal. La estimación de los parámetros de localización se lleva a cabo con un nuevo método de búsqueda multinivel adaptativo, propuesto con el fin de reducir drásticamente el tiempo de procesado que demandan otros algoritmos multivariable basados en subespacios, como el MUSIC, a costa de incrementar los requisitos de memoria. Las simulaciones del modelo arrojan resultados que son validados con resultados experimentales y comparados con el límite de Cramer Rao en términos del error cuadrático medio. La compensación del diagrama de radiación acerca sustancialmente la exactitud de estimación de la distancia al límite de Cramer Rao. Finalmente, es igual de importante la evaluación teórica como experimental de las prestaciones de los sistemas MIMO-OFDM. Por ello, se presenta el diseño e implementación de un prototipo de medida MIMO-OFDM-SPAA3D autocalibrado con sistema de posicionamiento de antena automático en la banda de 2,45 Ghz con capacidad para evaluar la capacidad de los sistemas MIMO. Además, tiene la capacidad de caracterizar espacialmente canales MIMO, incorporando para ello una etapa de autocalibración para medir la respuesta en frecuencia de los transmisores y receptores de RF, y así poder caracterizar la respuesta de fase del canal con mayor precisión. Este sistema incorpora un posicionador de antena automático 3D (SPAA3D) basado en un scanner con 3 brazos mecánicos sobre los que se desplaza un posicionador de antena de forma independiente, controlado desde un PC. Este posicionador permite obtener una gran cantidad de mediciones del canal en regiones locales, lo cual favorece la caracterización estadística de los parámetros del sistema MIMO. Con este prototipo se realizan varias campañas de medida para evaluar el canal MIMO en términos de capacidad comparando 2 esquemas de polarización y tomando en cuenta la diversidad en frecuencia aportada por la modulación OFDM en distintos escenarios. ABSTRACT Multiple-antennas technologies have been evolved to be the support of the actual and future wireless communication systems in its way to provide the high quality and high data rates required by new data, voice and data services. However, it is important to understand the behavior of the spatial characteristics of the radio channel, since the channel by itself limits the performance of the actual wireless communications systems. This drawback raises the need to understand the spatial structure of the propagation channel in order to design, assess, and develop more efficient multiantenna technologies for the actual and future wireless communications systems. Multiantenna technologies such as ‘Smart Antennas’ and MIMO systems have generated great interest in the field of wireless communications, i.e. cellular communications systems and more recently WLAN (Wireless Local Area Networks), mainly because the higher quality and the high data rate they are able to provide. Their technological benefits are based on the exploitation of the spatial diversity provided by the use of multiple antennas as happened in the past with some multiaccess technologies such as FDMA (Frequency Division Multiplexing Access), TDMA (Time Division Multiplexing Access), and CDMA (Code Division Multiplexing Access), which give diversity in the domains of frequency, time and code, respectively. This Thesis is mainly focus to study the spatial channel characteristics using schemes of multiple antennas considering several diversity schemes such as space, polarization, and frequency. The spatial characteristics will be study in terms of the direction-of-arrival profiles viewed at the receiver side of the radio link. The first step is to do a review of the smart antennas and MIMO systems technologies highlighting their advantages and drawbacks from a mathematical point of view. In the second step, a set of studies concerning the spatial characterization of the radio channel through the DoA profiles are addressed. The performance of several DoA estimation methods is assessed considering several aspects regarding antenna array structure, polarization diversity, and far-field and near-field conditions. Most of the results of these studies come from simulations of data models and measurements with real multiantena prototypes. In the same way, having understand the importance of validate the theoretical data models with experimental results, a 2,4 GHz MIMO-OFDM-SPAA2D prototype is presented. This prototype is intended for evaluating MIMO-OFDM capacity in indoor and outdoor scenarios, characterize the spatial structure of radio channels, assess several diversity schemes such as polarization, space, and frequency diversity, among others aspects. The studies reported are briefly described below. As is stated in Chapter two, the determination of user position is a fundamental task to be resolved for the smart antenna systems. As these systems are equipped with antenna arrays, they can provide the enough spatial diversity to accurately draw the spatial characterization of the radio channel through the DoA profiles, and therefore the source location. However, certain real implementation factors related to antenna errors, signals, and receivers will certainly reduce the performance of such direction finding systems. In that sense, a parameterized narrowband signal model is proposed to evaluate the influence of these factors in the location parameter estimation through extensive MC simulations. The results obtained from several DoA algorithms may be useful to extract some parameter design for directing finding systems based on arrays. The second study goes through the importance that polarization schemes can have for estimating far-field DoA profiles in radio channels, particularly for scenarios that may introduce polarization losses. For this purpose, a narrowband signal model with polarization sensibility is developed to conduct an analysis of several polarization schemes at transmitter (TX) and receiver (RX) through extensive MC simulations. In addition, spatial characterization of quasistatic indoor scenarios is also carried out using a 2.45 GHz MIMO prototype equipped with single and dual-polarized antennas. A good agreement between the measured DoA profiles with the propagation scenario is achieved. The theoretical and experimental evaluation of polarization schemes is performed using virtual arrays. In that case, a DoA estimation method is proposed based on adding an phase reference to properly track the DoA, which shows good results. In the third study, the special case of near-field source localization with virtual arrays is addressed. Most of DoA estimation algorithms are focused in far-field source localization where the radiated wavefronts are assume to be planar waves at the receive array. However, when source are located close to the array, the assumption of plane waves is no longer valid as the wavefronts exhibit a spherical behavior along the array. Thus, a faster and effective method of azimuth, elevation angles-of-arrival, and range estimation for near-field sources is proposed. The efficacy of the proposed method is evaluated with simulation and validated with measurements collected from a measurement campaign carried out in a controlled propagation environment, i.e. anechoic chamber. Moreover, the performance of the method is assessed in terms of the RMSE for several array sizes, several source positions, and taking into account the effect of radiation pattern. In general, better results are obtained with larger array and larger source distances. The effect of the antennas is included in the data model leading to more accurate results, particularly for range rather than for angle estimation. Moreover, a new multivariable searching method based on the MUSIC algorithm, called MUSA (multilevel MUSIC-based algorithm), is presented. This method is proposed to estimate the 3D location parameters in a faster way than other multivariable algorithms, such as MUSIC algorithm, at the cost of increasing the memory size. Finally, in the last chapter, a MIMO-OFDM-SPAA3D prototype is presented to experimentally evaluate different MIMO schemes regarding antennas, polarization, and frequency in different indoor and outdoor scenarios. The prototype has been developed on a Software-Defined Radio (SDR) platform. It allows taking measurements where future wireless systems will be developed. The novelty of this prototype is concerning the following 2 subsystems. The first one is the tridimensional (3D) antenna positioning system (SPAA3D) based on three linear scanners which is developed for making automatic testing possible reducing errors of the antenna array positioning. A set of software has been developed for research works such as MIMO channel characterization, MIMO capacity, OFDM synchronization, and so on. The second subsystem is the RF autocalibration module at the TX and RX. This subsystem allows to properly tracking the spatial structure of indoor and outdoor channels in terms of DoA profiles. Some results are draw regarding performance of MIMO-OFDM systems with different polarization schemes and different propagation environments.

A strategy for organ allografts without using immunosuppressants or irradiation

A strategy to achieve regular and long lasting organ and tissue allografts without using immunosuppressants and/or irradiation has been established for mice. One hundred percent of skin allografts can be induced to survive >350 days after transplantation if spleen cells from the same donors are first injected into the portal vein of the recipients. The mechanisms underlying this long-term tolerance induction can be described as follows: (i) donor T cells from the spleen of the donor facilitate the acceptance of the allogeneic engraftment, (ii) donor-specific anergy is induced in the cytotoxic T-lymphocytes of the recipients, (iii) T helper type 2 cells become the dominant T cells in the recipients that are accepting the skin transplants, and (iv) a lasting chimerism (microchimerism) is established in these recipients. This strategy, perhaps with minor modifications, might permit one also to overcome major barriers to organ allografting in humans. If this were the case, it could represent production of long lasting immunologic tolerance without need for irradiation or cytotoxic chemo-preparative regimen and as such could greatly facilitate allotransplantation free of episodes of chronic or acute rejection or toxic and damaging preparatory regimens.

Stress in telephone helpline nurses is associated with failures of concentration, attention and memory, and with more conservative referral decisions

Peer reviewed

Late memory-related genes in the hippocampus revealed by RNA fingerprinting

Although long-term memory is thought to require a cellular program of gene expression and increased protein synthesis, the identity of proteins critical for associative memory is largely unknown. We used RNA fingerprinting to identify candidate memory-related genes (MRGs), which were up-regulated in the hippocampus of water maze-trained rats, a brain area that is critically involved in spatial learning. Two of the original 10 candidate genes implicated by RNA fingerprinting, the rat homolog of the ryanodine receptor type-2 and glutamate dehydrogenase (EC 1.4.1.3), were further investigated by Northern blot analysis, reverse transcription���PCR, and in situ hybridization and confirmed as MRGs with distinct temporal and regional expression. Successive RNA screening as illustrated here may help to reveal a spectrum of MRGs as they appear in distinct domains of memory storage.

Deciphering a neural code for vision

Deciphering the information that eyes, ears, and other sensory organs transmit to the brain is important for understanding the neural basis of behavior. Recordings from single sensory nerve cells have yielded useful insights, but single neurons generally do not mediate behavior; networks of neurons do. Monitoring the activity of all cells in a neural network of a behaving animal, however, is not yet possible. Taking an alternative approach, we used a realistic cell-based model to compute the ensemble of neural activity generated by one sensory organ, the lateral eye of the horseshoe crab, Limulus polyphemus. We studied how the neural network of this eye encodes natural scenes by presenting to the model movies recorded with a video camera mounted above the eye of an animal that was exploring its underwater habitat. Model predictions were confirmed by simultaneously recording responses from single optic nerve fibers of the same animal. We report here that the eye transmits to the brain robust “neural images” of objects having the size, contrast, and motion of potential mates. The neural code for such objects is not found in ambiguous messages of individual optic nerve fibers but rather in patterns of coherent activity that extend over small ensembles of nerve fibers and are bound together by stimulus motion. Integrative properties of neurons in the first synaptic layer of the brain appear well suited to detecting the patterns of coherent activity. Neural coding by this relatively simple eye helps explain how horseshoe crabs find mates and may lead to a better understanding of how more complex sensory organs process information.

Cellular responses to psychomotor stimulant and neuroleptic drugs are abnormal in mice lacking the D1 dopamine receptor

Stimulation of dopamine D1 receptors has profound effects on addictive behavior, movement control, and working memory. Many of these functions depend on dopaminergic systems in the striatum and D1–D2 dopamine receptor synergies have been implicated as well. We show here that deletion of the D1 dopamine receptor produces a neural phenotype in which amphetamine and cocaine, two addictive psychomotor stimulants, can no longer stimulate neurons in the striatum to express cFos or JunB or to regulate dynorphin. By contrast, haloperidol, a typical neuroleptic that acts preferentially at D2-class receptors, remains effective in inducing catalepsy and striatal Fos/Jun expression in the D1 mutants, and these behavioral and neural effects can be blocked by D2 dopamine receptor agonists. These findings demonstrate that D2 dopamine receptors can function without the enabling role of D1 receptors but that D1 dopamine receptors are essential for the control of gene expression and motor behavior by psychomotor stimulants.

Toward a molecular definition of long-term memory storage

The storage of long-term memory is associated with a cellular program of gene expression, altered protein synthesis, and the growth of new synaptic connections. Recent studies of a variety of memory processes, ranging in complexity from those produced by simple forms of implicit learning in invertebrates to those produced by more complex forms of explicit learning in mammals, suggest that part of the molecular switch required for consolidation of long-term memory is the activation of a cAMP-inducible cascade of genes and the recruitment of cAMP response element binding protein-related transcription factors. This conservation of steps in the mechanisms for learning-related synaptic plasticity suggests the possibility of a molecular biology of cognition.

Muscarinic acetylcholine receptor expression in memory circuits: Implications for treatment of Alzheimer disease

Cholinergic transmission at muscarinic acetylcholine receptors (mAChR) has been implicated in higher brain functions such as learning and memory, and loss of synapses may contribute to the symptoms of Alzheimer disease. A heterogeneous family of five genetically distinct mAChR subtypes differentially modulate a variety of intracellular signaling systems as well as the processing of key molecules involved in the pathology of the disease. Although many muscarinic effects have been identified in memory circuits, including a diversity of pre- and post-synaptic actions in hippocampus, the identities of the molecular subtypes responsible for any given function remain elusive. All five mAChR genes are expressed in hippocampus, and subtype-specific antibodies have enabled identification, quantification, and localization of the encoded proteins. The m1, m2, and m4 mAChR proteins are most abundant in forebrain regions and they have distinct cellular and subcellular localizations suggestive of various pre- and postsynaptic functions in cholinergic circuits. The subtypes are also differentially altered in postmortem brain samples from Alzheimer disease cases. Further understanding of the molecular pharmacology of failing synapses in Alzheimer disease, together with the development of new subtype-selective drugs, may provide more specific and effective treatments for the disease.

Location, location, location: The many addresses of memory formation

Memory formation, like real estate, can be summarized succinctly—location, location, location. It is an emergent property involving different anatomical regions in the brain, sets of neuronal circuits, and cellular and molecular interactions between and within those neurons. At each of these levels of description, location continues to be a major organizing principle guiding researchers. The difficulty in the field is the integration of information between the various levels of analyses, and it is proposed that molecular reporters may help to fill that void.

A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis.

Homeobox genes encode a large family of homeodomain proteins that play a key role in the pattern formation of animal embryos. By analogy, homeobox genes in plants are thought to mediate important processes in their embryogenesis, but there is very little evidence to support this notion. Here we described the temporal and spatial expression patterns of a rice homeobox gene, OSH1, during rice embryogenesis. In situ hybridization analysis revealed that in the wild-type embryo, OSH1 was first expressed at the globular stage, much earlier than organogenesis started, in a ventral region where shoot apical meristem and epiblast would later develop. This localized expression of OSH1 indicates that the cellular differentiation has already occurred at this stage. At later stages after organogenesis had initiated, OSH1 expression was observed in shoot apical meristem [except in the L1 (tunica) layer], epiblast, radicle, and their intervening tissues in descending strength of expression level with embryonic maturation. We also performed in situ hybridization analysis with a rice organless embryo mutant, orl1, that develops no embryonic organs. In the orl1 embryo, the expression pattern of OSH1 was the same as that in the wild-type embryo in spite of the lack of embryonic organs. This shows that OSH1 is not directly associated with organ differentiation, but may be related to a regulatory process before or independent of the organ determination. The results described here strongly suggest that, like animal homeobox genes, OSH1 plays an important role in regionalization of cell identity during early embryogenesis.

Organ-specific and Agamous-regulated expression and glycosylation of a pollen tube growth-promoting protein.

Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S-promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins.

Cellular aging, destabilization, and cancer.

Three major characteristics of aging in animals are a slowdown of cell proliferation, an increase in residual bodies associated with age pigments, and a marked increase in the likelihood of neoplastic transformation. The 28 L subline of the NIH 3T3 line of mouse embryo fibroblasts exhibits all these characteristics when held at confluence for extended periods. The impairment of proliferation is the first behavioral characteristic detected in low density subcultures from the confluent cultures, and it persists through many cell generations of exponential multiplication. There is an equal degree of growth impairment among replicate cultures (lineages) recovered after each of 2 successive rounds of confluence, although heterogeneity appears after the third round. The growth impairment pervades the entire cell population of each lineage. The degree and duration of impairment increase with repeated rounds of confluence. A marked increase of residual bodies characteristic of age pigments occurs in the cytoplasm of all the cells kept under prolonged confluence. Neoplastic transformation first appears as foci of multilayered cells on a monolayered background of nontransformed cells. The transformed cells arise at different times in the lineages and originate from a very small fraction of the population. The transformed cells selectively overgrow the entire population in successive rounds of confluence leading to an increase in saturation density of each lineage at different times. Under cloning conditions, isolated colonies of transformed cells develop more slowly than colonies of nontransformed cells but eventually reach a higher population density. The regularity of persistent growth impairment among the lineages and the appearance of large numbers of residual bodies in all the cells of each population are more characteristic of an epigenetic process than of specific local mutations. although random chromosomal lesions cannot be ruled out. By contrast, the low frequency and stochastic character of neoplastic transformation are consistent with a conventional genetic origin. The advent in long-term confluent NIH 3T3 cultures of three cardinal characteristics of cellular aging in vivo recommends it as a model for aging cells.

Valutazione della risposta immunitaria cellulare e fenotipizzazione linfocitaria nel suino in corso di infezione naturale da virus della PRRS (“Porcine Reproductive and Respiratory Syndrome”) e dopo stimolazione di PBMC (“Peripheral Blood Mononuclear Cells”) in vitro con Peptide Killer (KP)

SUMMARY The Porcine Reproductive and Respiratory Syndrome (PRRS) virus is one of the most spread pathogens in swine herds all over the world and responsible for a reproductive and respiratory syndrome that causes severe heath and economical problems. This virus emerged in late 1980’s but although about 30 years have passed by, the knowledge about some essential facets related to the features of the virus (pathogenesis, immune response, and epidemiology) seems to be still incomplete. Taking into account that the development of modern vaccines is based on how innate and acquire immunity react, a more and more thorough knowledge on the immune system is needed, in terms of molecular modulation/regulation of the inflammatory and immune response upon PRRSV infection. The present doctoral thesis, which is divided into 3 different studies, is aimed to increase the knowledge about the interaction between the immune system and the PRRS virus upon natural infection. The objective of the first study entitled “Coordinated immune response of memory and cytotoxic T cells together with IFN-γ secreting cells after porcine reproductive and respiratory syndrome virus (PRRSV) natural infection in conventional pigs” was to evaluate the activation and modulation of the immune response in pigs naturally infected by PRRSV compared to an uninfected control group. The course of viremia was evaluated by PCR, the antibody titres by ELISA, the number of IFN-γ secreting cells (IFN- SC) by an ELISPOT assay and the immunophenotyping of some lymphocyte subsets (cytotoxic cells, memory T lymphocytes and cytotoxic T lymphocytes) by flow cytometry. The results showed that the activation of the cell-mediated immune response against PRRSV is delayed upon infection and that however the levels of IFN-γ SC and lymphocyte subsets subsequently increase over time. Furthermore, it was observed that the course of the different immune cell subsets is time-associated with the levels of PRRSV-specific IFN-γ SC and this can be interpreted based on the functional role that such lymphocyte subsets could have in the specific production/secretion of the immunostimulatory cytokine IFN-γ. In addition, these data support the hypothesis that the age of the animals upon the onset of infection or the diverse immunobiological features of the field isolate, as typically hypothesized during PRRSV infection, are critical conditions able to influence the qualitative and quantitative course of the cell-mediated immune response during PRRSV natural infection. The second study entitled “Immune response to PCV2 vaccination in PRRSV viremic piglets” was aimed to evaluate whether PRRSV could interfere with the activation of the immune response to PCV2 vaccination in pigs. In this trial, 200 pigs were divided into 2 groups: PCV2-vaccinated (at 4 weeks of age) and PCV2-unvaccinated (control group). Some piglets of both groups got infected by PRRSV, as determined by PRRSV viremia detection, so that 4 groups were defined as follows: PCV2 vaccinated - PRRSV viremic PCV2 vaccinated - PRRSV non viremic PCV2 unvaccinated - PRRSV viremic PCV2 unvaccinated - PRRSV non viremic The following parameters were evaluated in the 4 groups: number of PCV2-specific IFN-γ secreting cells, antibody titres by ELISA and IPMA. Based on the immunological data analysis, it can be deduced that: 1) The low levels of antibodies against PCV2 in the PCV2-vaccinated – PRRSV-viremic group at vaccination (4 weeks of age) could be related to a reduced colostrum intake influenced by PRRSV viremia. 2) Independently of the viremia status, serological data of the PCV2-vaccinated group by ELISA and IPMA does not show statistically different differences. Consequently, it can be be stated that, under the conditions of the study, PRRSV does not interfere with the antibody response induced by the PCV2 vaccine. 3) The cell-mediated immune response in terms of number of PCV2-specific IFN-γ secreting cells in the PCV2-vaccinated – PRRSV-viremic group seems to be compromised, as demonstrated by the reduction of the number of IFN-γ secreting cells after PCV2 vaccination, compared to the PCV2-vaccinated – PRRSV-non-viremic group. The data highlight and further support the inhibitory role of PRRSV on the development and activation of the immune response and highlight how a natural infection at early age can negatively influence the immune response to other pathogens/antigens. The third study entitled “Phenotypic modulation of porcine CD14+ monocytes, natural killer/natural killer T cells and CD8αβ+ T cell subsets by an antibody-derived killer peptide (KP)” was aimed to determine whether and how the killer peptide (KP) could modulate the immune response in terms of activation of specific lymphocyte subsets. This is a preliminary approach also aimed to subsequently evaluate such KP with a potential antivural role or as adjuvant. In this work, pig peripheral blood mononuclear cells (PBMC) were stimulated with three KP concentrations (10, 20 and 40 g/ml) for three time points (24, 48 and 72 hours). TIME POINTS (hours) KP CONCENTRATIONS (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 By using flow cytometry, the qualitative and quantitative modulation of the following immune subsets was evaluated upon KP stimulation: monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and CD4+ and CD8α/β+ T lymphocyte subsets. Based on the data, it can be deduced that: 1) KP promotes a dose-dependent activation of monocytes, particularly after 24 hours of stimulation, by inducing a monocyte phenotypic and maturation shift mainly involved in sustaining the innate/inflammatory response. 2) KP induces a strong dose-dependent modulation of NK and NKT cells, characterized by an intense increase of the NKT cell fraction compared to NK cells, both subsets involved in the antibody-dependent cell cytotoxicity (ADCC). The increase is observed especially after 24 hours of stimulation. 3) KP promotes a significant activation of the cytotoxic T lymphocyte subset (CTL). 4) KP can modulate both the T helper and T cytotoxic phenotype, by inducing T helper cells to acquire the CD8α thus becoming doube positive cells (CD4+CD8+) and by inducing CTL (CD4-CD8+high) to acquire the double positive phenotype (CD4+CD8α+high). Therefore, KP may induce several effects on different immune cell subsets. For this reason, further research is needed aimed at characterizing each “effect” of KP and thus identifying the best use of the decapeptide for vaccination practice, therapeutic purposes or as vaccine adjuvant. RIASSUNTO Il virus della PRRS (Porcine Reproductive Respiratory Syndrome) è uno dei più diffusi agenti patogeni negli allevamenti suini di tutto il mondo, responsabile di una sindrome riproduttiva e respiratoria causa di gravi danni ad impatto sanitario ed economico. Questo virus è emerso attorno alla fine degli anni ’80 ma nonostante siano passati circa una trentina di anni, le conoscenze su alcuni punti essenziali che riguardano le caratteristiche del virus (patogenesi, risposta immunitaria, epidemiologia) appaiono ancora spesso incomplete. Considerando che lo sviluppo dei vaccini moderni è basato sui principi dell’immunità innata e acquisita è essenziale una sempre più completa conoscenza del sistema immunitario inteso come modulazione/regolazione molecolare della risposta infiammatoria e immunitaria in corso di tale infezione. Questo lavoro di tesi, suddiviso in tre diversi studi, ha l’intento di contribuire all’aumento delle informazioni riguardo l’interazione del sistema immunitario, con il virus della PRRS in condizioni di infezione naturale. L’obbiettivo del primo studio, intitolato “Associazione di cellule memoria, cellule citotossiche e cellule secernenti IFN- nella risposta immunitaria in corso di infezione naturale da Virus della Sindrome Riproduttiva e Respiratoria del Suino (PRRSV)” è stato di valutare l’attivazione e la modulazione della risposta immunitaria in suini naturalmente infetti da PRRSV rispetto ad un gruppo controllo non infetto. I parametri valutati sono stati la viremia mediante PCR, il titolo anticorpale mediante ELISA, il numero di cellule secernenti IFN- (IFN- SC) mediante tecnica ELISPOT e la fenotipizzazione di alcune sottopopolazioni linfocitarie (Cellule citotossiche, linfociti T memoria e linfociti T citotossici) mediante citofluorimetria a flusso. Dai risultati ottenuti è stato possibile osservare che l’attivazione della risposta immunitaria cellulo-mediata verso PRRSV appare ritardata durante l’infezione e che l’andamento, in termini di IFN- SC e dei cambiamenti delle sottopopolazioni linfocitarie, mostra comunque degli incrementi seppur successivi nel tempo. E’ stato inoltre osservato che gli andamenti delle diverse sottopopolazioni immunitarie cellulari appaiono temporalmente associati ai livelli di IFN- SC PRRSV-specifiche e ciò potrebbe essere interpretato sulla base del ruolo funzionale che tali sottopopolazioni linfocitarie potrebbero avere nella produzione/secrezione specifica della citochina immunoattivatrice IFN-. Questi dati inoltre supportano l’ipotesi che l’età degli animali alla comparsa dell’infezione o, come tipicamente ipotizzato nell’infezione da PRRSV, le differenti caratteristiche immunobiologiche dell’isolato di campo, sia condizioni critiche nell’ influenzare l’andamento qualitativo e quantitativo della risposta cellulo-mediata durante l’infezione naturale da PRRSV. Il secondo studio, dal titolo “Valutazione della risposta immunitaria nei confronti di una vaccinazione contro PCV2 in suini riscontrati PRRSV viremici e non viremici alla vaccinazione” ha avuto lo scopo di valutare se il virus della PRRS potesse andare ad interferire sull’attivazione della risposta immunitaria indotta da vaccinazione contro PCV2 nel suino. In questo lavoro sono stati arruolati 200 animali divisi in due gruppi, PCV2 Vaccinato (a 4 settimane di età) e PCV2 Non Vaccinato (controllo negativo). Alcuni suinetti di entrambi i gruppi, si sono naturalmente infettati con PRRSV, come determinato con l’analisi della viremia da PRRSV, per cui è stato possibile creare quattro sottogruppi, rispettivamente: PCV2 vaccinato - PRRSV viremico PCV2 vaccinato - PRRSV non viremico PCV2 non vaccinato - PRRSV viremico PCV2 non vaccinato - PRRSV non viremico Su questi quattro sottogruppi sono stati valutati i seguenti parametri: numero di cellule secernenti IFN- PCV2 specifiche, ed i titoli anticorpali mediante tecniche ELISA ed IPMA. Dall’analisi dei dati immunologici derivati dalle suddette tecniche è stato possibile dedurre che:  I bassi valori anticorpali nei confronti di PCV2 del gruppo Vaccinato PCV2-PRRSV viremico già al periodo della vaccinazione (4 settimane di età) potrebbero essere messi in relazione ad una ridotta assunzione di colostro legata allo stato di viremia da PRRSV  Indipendentemente dallo stato viremico, i dati sierologici del gruppo vaccinato PCV2 provenienti sia da ELISA sia da IPMA non mostrano differenze statisticamente significative. Di conseguenza è possibile affermare che in questo caso PRRSV non interferisce con la risposta anticorpale promossa dal vaccino PCV2.  La risposta immunitaria cellulo-mediata, intesa come numero di cellule secernenti IFN- PCV2 specifiche nel gruppo PCV2 vaccinato PRRS viremico sembra essere compromessa, come viene infatti dimostrato dalla diminuzione del numero di cellule secernenti IFN- dopo la vaccinazione contro PCV2, comparata con il gruppo PCV2 vaccinato- non viremico. I dati evidenziano ed ulteriormente sostengono il ruolo inibitorio del virus della PRRSV sullo sviluppo ed attivazione della risposta immunitaria e come un infezione naturale ad età precoci possa influenzare negativamente la risposta immunitaria ad altri patogeni/antigeni. Il terzo studio, intitolato “Modulazione fenotipica di: monociti CD14+, cellule natural killer (NK), T natural killer (NKT) e sottopopolazioni linfocitarie T CD4+ e CD8+ durante stimolazione con killer peptide (KP) nella specie suina” ha avuto come scopo quello di stabilire se e come il Peptide Killer (KP) potesse modulare la risposta immunitaria in termini di attivazione di specifiche sottopopolazioni linfocitarie. Si tratta di un approccio preliminare anche ai fini di successivamente valutare tale KP in un potenziale ruolo antivirale o come adiuvante. In questo lavoro, periferal blood mononuclear cells (PBMC) suine sono state stimolate con KP a tre diverse concentrazioni (10, 20 e 40 g/ml) per tre diversi tempi (24, 48 e 72 ore). TEMPI DI STIMOLAZIONE (ore) CONCENTRAZIONE DI KP (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 Mediante la citometria a flusso è stato dunque possibile analizzare il comportamento qualitativo e quantitativo di alcune sottopopolazioni linfocitarie sotto lo stimolo del KP, tra cui: monociti, cellule Natural Killer (NK), cellule T Natural Killer (NKT) e linfociti T CD4 e CD8+. Dai dati ottenuti è stato possibile dedurre che: 1) KP promuove un’attivazione dei monociti dose-dipendente in particolare dopo 24 ore di stimolazione, inducendo uno “shift” fenotipico e di maturazione monocitaria maggiormente coinvolto nel sostegno della risposta innata/infiammatoria. 2) KP induce una forte modulazione dose-dipendente di cellule NK e NKT con un forte aumento della frazione delle cellule NKT rispetto alle NK, sottopopolazioni entrambe coinvolte nella citotossicità cellulare mediata da anticorpi (ADCC). L’aumento è riscontrabile soprattutto dopo 24 ore di stimolazione. 3) KP promuove una significativa attivazione della sottopopolazione del linfociti T citotossici (CTL). 4) Per quanto riguarda la marcatura CD4+/CD8+ è stato dimostrato che KP ha la capacità di modulare sia il fenotipo T helper che T citotossico, inducendo le cellule T helper ad acquisire CD8 diventando quindi doppio positive (CD4+CD8+) ed inducendo il fenotipo CTL (CD4-CD8+high) ad acquisire il fenotipo doppio positivo (CD4+CD8α+high). Molti dunque potrebbero essere gli effetti che il decapeptide KP potrebbe esercitare sulle diverse sottopopolazioni del sistema immunitario, per questo motivo va evidenziata la necessità di impostare e attuare nuove ricerche che portino alla caratterizzazione di ciascuna “abilità” di KP e che conducano successivamente alla scoperta del migliore utilizzo che si possa fare del decapeptide sia dal punto di vista vaccinale, terapeutico oppure sotto forma di adiuvante vaccinale.