Regeneration from seeds of two Acer species in the face of climate change

The warming of the global system is an accepted phenomenon and it is considered unprecedented over decades to millennia. By the end of this century, depending on the concentration-driven scenario, an increase of the global mean temperature between 0.3 °C and 4.8 °C is projected. In Europe, the mean annual temperature will likely increase more in the northern than in central or southern regions. On the other hand, increased mean annual precipitation is expected in northern and central Europe, and decreases around the Mediterranean. Also the frequencies of rainfall will likely change, with more extreme rainfall events and longer dry intervals. Impacts of contemporary climate change are already visible in several ecosystems around the world. Climate influences plant population dynamics in many ways: not only species’ distributions and phenology are affected but also recruitment, growth and mortality. However, the sensitivity of the different life-cycle stages to climate change is expected to differ. One of the most sensitive phases is the sexual reproductive phase; therefore, this is a good indicator of species’ sensitivity to environmental change, often better than the response of other aspects of the plant life cycle such as growth and mortality of mature plants. Considering that the impacts of climate change on some key process of reproduction such as pollination, seed production, dormancy break, germination, seedling survival and subsequent seedling growth are not well known and are still being investigated, increasing our knowledge on this phase is very relevant. Moreover, models predicting changes in species’ performance, distribution and community composition urgently need to incorporate reliable information on species’ reproduction in the face of climate change. Considering that a compositional change due climate change is forecasted for European forests in favour of currently secondary tree species, increasing our understanding of the effects of climate change on these species’ recruitment is key to better understand and forecast population and community dynamics in forests. In this thesis we contribute to fill this knowledge gap by analysing the impacts of climate change on sexual reproduction of two important secondary forest tree species Acer platanoides (Norway maple) and A. pseudoplatanus (sycamore maple) along a latitudinal gradient from Italy to Norway. We analysed an important part of the sexual reproduction of these species from seed production (nutrient concentration, morphology and physiology of seeds) until interannual seedling survival and seedling growth simulating a wide range of warming intensities and changes in precipitation and biotic interactions (in this case, competition). We found that seed traits of A. platanoides were more influenced by the climatic conditions experienced by the mother tree than those of A. pseudoplatanus. Additionally, seed viability, germination percentage, and seedling biomass of A. platanoides were strongly related to the seed mass and seed nutrient concentrations. Additionally, A. platanoides seeds were more influenced by the environmental conditions (generally negatively affected by rising temperatures) than in A. pseudoplatanus, the former exhibited higher germination percentages and seedling biomass than A. pseudoplatanus. After seed production, the seed bed conditions were important for seed germination and seedlings performance. With three complementary full factorial experiments we assessed the effects of moderate and strong warming (2.7 °C to 7.2 °C), drought (80% and 60% soil moisture reduction of the field capacity) and changes in precipitation frequency (one, two and three times per week) on germination, seedling survival and growth of seeds and seedlings of different provenances sampled along a latitudinal gradient. The germination of A. platanoides was negatively affected by drought but increasing the frequency of precipitation enhanced germination. Moreover, the survival of both species decreased under (particularly strong) warming. The growth variables of both species were generally negatively affected by warming and drought, while the precipitation frequency did not change growth. The joint manipulation of different factors resulted for some variables in additive effects while for others in partial compensation. Often, A. platanoides exhibited higher sensitivity than A. pseudoplatanus to the treatments applied and especially to its joint manipulation. Finally, across these experiments, the temperature experienced by the mother tree during seed production and the seed provenance showed to play an important role on seed germination and seedling performance. The germination was mostly negatively related to the temperature experienced by the mother tree with seeds from central and northern provenances germinating better. However, the southern provenances generally performed better under simulated climate change. Finally, in an in situ transplant experiment, we observed that Acer platanoides was more sensitive to changes in competition with the other understorey plants while A. pseudoplatanus was affected by biotic changes and + 1°C warming and reduced precipitation. In general, competition reduced germination in A. platanoides and warming enhanced survival in A. pseudoplatanus. Reduced competition strongly increased the growth of A. platanoides seedlings. However, in this experiment, the seedlings’ responses were independent from the conditions experienced by the mother tree during seed production and maturation. Based on the findings in this work, we conclude that climate change will most likely have significant effects on the sexual reproduction of these two congeneric species. We clearly showed that the different aspects of plant reproduction (i.e., seed production, germination, seedling survival and growth) will be differently impacted by the projected changes in climatic conditions. Along this research often A. platanoides showed to be more responsive to the treatments applied than A. pseudoplatanus. Additionally, due the provenance specific responses observed it is possible that not only these two species will differently respond to climate change but also the impacts will be dissimilar across Europe. Future research with special focus on the subsequent life stages will be important to thoroughly predict the impacts of climate change on these species along their distribution range. Such information is key to soundly project changes in community’s composition and inform forest management and policy decisions

Dimorphic fruit colour is associated with differences in germination of calligonum comosum

© 2019, Canadian Science Publishing. Calligonum comosum L’Hér is a perennial evergreen shrub that produces dimorphic fruits of distinctly yellow or red colour. The species is found on sand dunes of the Middle East, is valued as a highly palatable livestock feed that survives extreme drought, and is suitable for desert restoration of non-saline sandy soils. The association of fruit colour with germination was assessed in laboratory conditions under two temperature (15/20 and 20/30 °C) and two light (0 and 12 h per day) treatments. Additionally, the association of fruit colour with emergence and seedling growth up to 90 days from sowing was assessed in shade house conditions. Germination percentage in the laboratory was the only variable that exhibited significant variation by seed colour. Germination was highest for red seeds grown in the dark at the higher temperature but the influence of seed colour on germination was not repeated in the shade house study. The dimorphic fruit colour of C. comosum is thus associated with differing germination rates, but further study is needed to determine whether morphs employ differing recruitment strategies

Regeneration after fire in the Monte tropical dry forest, northwest Argentina

Los bosques tropicales secos representan el 42% de la superficie de bosques tropicales y están fuertemente presionados por actividades humanas. Los disturbios antropogénicos redujeron la superficie de bosques de Prosopis en el Monte. Considerando su nivel de degradación, el proceso de regeneración es esencial para su mantenimiento a largo plazo. El fuego es el disturbio másimportante en estos bosques, pero el conocimiento de la dinámica de la vegetación post-fuego es limitado en el norte del Monte. En este trabajo se analizaron tres ambientes: bosque intacto, bosque quemado no inundado y bosque quemado inundado. Se analizó la riqueza y la estructura del sotobosque. Las estrategias de regeneración de las principales especies arbóreas (Prosopisspp. y Geoffroea decorticans) se estudiaron registrando: tipo de estrategia (año 2008), variación temporal en el establecimiento de regeneración por semillas (período 2008-2010), altura alcanzada por las plántulas establecidas a partir de semillas. La riqueza fue mayor y la estructura más compleja bajo la influencia del fuego e inundaciones. Las principales estrategias de regeneración fueron rebrotes de yemas basales para Prosopis sp. y de raíz para G. decorticans. El número de individuos establecidos a partir de semillas fue más del doble a los dos años de la primera medición, mientras que la altura fue influenciada por la disponibilidad de agua. Se concluyó que la recuperación del bosque está fuertemente condicionada por la naturaleza y el tiempo de ocurrencia de los disturbios. El manejo de los rebrotes puede ayudar a que alcancen más altura favoreciendo cambios en la composición y estructura del sotobosque. El control del pastoreo es importante para proteger la regeneración por semillas asegurando una alta diversidad genética.The tropical dry forest represents 42% of the global surface of tropical forest, but these unique ecosystems are under strong pressure of human activities. Anthropogenic disturbances resulted in a dramatic reduction of the extent of Prosopis forests in the Monte region (Argentina). Considering its degree of degradation, forest regeneration is essential for the long term maintenance of this system. The most important disturbance in these forests is fire, but the knowledge about post-fire vegetation dynamics is limited in the north of the Monte. We analyzed three co-existing forest conditions: intact forest, burned non-flooded forest, and burned flooded forest. The species richness and structure of the vegetation was analyzed. The regeneration strategies of the two main forest tree species (Prosopis spp. and Geoffroea decorticans) were analyzed recording the strategy type (year 2008), the temporal variation in seedling establishment (period 2008-2010), and height growth of the seedlings. The richness and understory structure were highly influenced by fire and floodings. The main strategies for regeneration were base sprout and root sprouts for Prosopis and G. decorticans, respectively. The number of seedlings was more than two times higher after two years from the first measurement, while the height growth seemed strongly influenced by water availability. We concluded that the recovery of the forest is influenced by the nature and timing of the disturbances. It is likely that the two disturbed environments will gradually evolve and ultimately show more similarities with the intact forest. However, the management of sprouts can help the sprouts to reach a considerable height in favor of shifts in the understory structure and composition. The control of grazing is important to protect the seedlings and ensure a high genetic variability.Fil: Carón, María Mercedes. University of Ghent; Bélgica. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dalmasso, Antonio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Ortin Vujovich, Adriana Elizabeth. Universidad Nacional de Salta; ArgentinaFil: Verheyen, Kris. University of Ghent; Bélgic

Effects of light, temperature, salinity, and maternal habitat on seed germination of aeluropus lagopoides (Poaceae): An economically important halophyte of arid Arabian deserts

In this study, salt tolerance during germination of Aeluropus lagopoides (L.) Trin. was tested using fresh seeds collected from three different maternal habitats under three thermoperiods and two light regimes. Additionally, we tested the ability of non-germinated seeds that had been exposed to different concentrations of NaCl to recover their germination in distilled water. The results showed a significant effect of seed source, temperature, and salinity, and some of their two-and three-way interactions on final germination and recovery percentage. The seeds from non-saline provenances had the highest percentages for germination (ca. 79%) under the 35/25 °C temperature regime, whereas the lowest percentages for germination (ca. 21%) was recorded for seeds from saline conditions under the 25/15 °C treatment. Additionally, percent germination was significantly lower for the seeds incubated in the saline solutions (100, 200, 400, and 600 mmol/L NaCl) and germinated under colder conditions (15/25 °C), compared with the seeds incubated in non-saline solutions (control group, 0 mmol/L NaCl) and germinated under warmer conditions (35/25 °C). The highest recovery percentage was recorded for seeds of the hyper-saline habitat incubated at 35/25 °C. Thus, seeds maintained their viability despite experiencing a range of saline conditions and were able to germinate upon the arrival of suitable conditions, which can be an adaptation to its saline arid desert habitat.Fil: Bhatt, Arvind. Botanical Garden; China. Kuwait Institute For Scientific Research; KuwaitFil: Gairola, Sanjay. Sharjah Research Academy; Emiratos Arabes UnidosFil: Carón, María Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Santo, Andrea. Università Degli Studi Di Cagliari.; ItaliaFil: Murru, Valentina. Università Degli Studi Di Cagliari.; ItaliaFil: El-Keblawy, Ali. University Of Sharjah; Emiratos Arabes UnidosFil: Mahmoud, Tamer. University Of Sharjah; Emiratos Arabes Unido

Analyzing individual drivers of global changes promotes inaccurate long-term policies in deforestation hotspots: The case of Gran Chaco

In the context of the global climate and biodiversity crises, forecasting the effectiveness of Protected Areas (PAs) and forest management to conserve biodiversity in the long-term is a high priority, especially in threatened environments. By combining distribution models and conservation planning protocols, we analyzed the effect of global climate and agriculture-linked activities in the long-term conservation opportunities of one most threatened deforestation hotspots: the South American Gran Chaco. We showed that assessing the effects of each driver of global change individually, promotes inaccurate long-term policies in deforestation hotspots. Our future scenarios indicated a low impact of climate change on the species distributions when it was analyzed individually. However, its effects were strongly exacerbated when both drivers of threat were combined in the same analyses, strongly diminishing conservation opportunities in the region: more than 50% of the remaining species' distribution and hotspot areas could be lost in the near future. In this dramatic context, we identified important opportunities to improve the level of long-term protection by increasing at least 5.6% the protection coverage and placing PAs strategically. It is imperative policymakers promote policies to generate a long-term improvement of conservation areas that are resilient to both threats as soon as possible for these threatened environments.Fil: Prieto Torres, David Alexander. Universidad Nacional Autonoma de Mexico. Facultad de Estudios Superiores de Iztacala; MéxicoFil: Díaz, Sandra Myrna. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Cordier, Javier Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Zoología Aplicada; ArgentinaFil: Torres, Ricardo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Carón, María Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Museo de Zoología; ArgentinaFil: Nori, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Zoología Aplicada; Argentin

Effects of mineral soil and forest floor on the regeneration of pedunculate oak, beech and red oak

Early regeneration is a critical life stage that affects the future species composition of forests. Knowledge about regeneration success under different environmental conditions allows better understanding of forest dynamics. We studied the effects of seedbed conditions on the establishment and performance of seedlings of pedunculate oak, beech and red oak. In 50 plots of a tree-diversity oriented research platform in mature forests in northern Belgium (TREEWEB), we installed a field experiment with three treatments (potting soil, mineral soil, mineral soil + forest floor), in which we sowed seeds of each species. We monitored early establishment and survival, height, root and shoot biomass of the seedlings after two growing seasons. Mineral soil negatively affected seedling establishment and performance relative to the potting soil. The negative soil effects did not vary with measured abiotic soil properties. In general, the forest floor did not deteriorate or mitigate the soil effects, and only for root biomass did the forest floor partly compensate the negative soil effects. Forest floor effects did not vary with the measured forest floor properties. In the studied forests, creating bare soil was not enough to promote regeneration; improving soil properties might be important for the success of natural regeneration.Fil: De Groote, Stefanie R. E.. University of Ghent; BélgicaFil: Vanhellemont, Margot. University of Ghent; BélgicaFil: Baeten, Lander. University of Ghent; BélgicaFil: Carón, María Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina. Universidad Nacional de Salta; ArgentinaFil: Martel, An. University of Ghent; BélgicaFil: Bonte, Dries. University of Ghent; BélgicaFil: Lens, Luc. University of Ghent; BélgicaFil: Verheyen, Kris. University of Ghent; Bélgic

Experimental hypothyroidism increases apoptosis in dimethylbenzanthracene-induced mammary tumors

Epidemiological and in vitro data have not provided conclusive evidence concerning the involvement of thyroid hormones (THs) on mammary carcinogenesis. We used an in vivo model to assess the relationship between THs, adipose tissue and breast cancer development. Female Sprague‑Dawley rats were treated with a dose of 7,12-dimethylbenz(a)anthracene (15 mg/rat) at 55 days of age and were then divided into four experimental groups: hypothyroid rats (HypoT, 0.01% 6-N-propyl-2-thiouracil in drinking water), untreated control (EUT); hyperthyroid rats (HyperT, 0.25 mg/kg/day T4 s.c.) and vehicle-treated control rats. The latency of tumor appearance and the incidence and progression of tumors were determined. At sacrifice, blood samples were collected for hormone determinations and samples of tumor and mammary glands were obtained for immunohistological studies. HypoT rats had retarded growth and an increase in mammary fat. The latency was longer (p<0.0001), the incidence rate was lower (p<0.05) and tumor growth was slower in HypoT rats compared to EUT and HyperT rats. Mitotic index and PCNA immunostaining were similar in all groups. HypoT rats showed increased apoptosis (p<0.05) as evaluated by the apoptotic index and TUNEL staining. No differences in serum prolactin and progesterone were observed. However, circulating estradiol (E2) was significantly lower in HypoT and HyperT rats. Serum leptin levels were reduced in HypoT rats even though the abdominal fat mass was similar in all groups. To note, the leptin level was higher in HypoT rats that developed mammary tumors than the level in non-tumoral HypoT rats. In conclusion, hypothyroidism altered animal growth, breast morphology, body composition, leptin secretion and serum E2 enhancing apoptosis and, consequently, retarding mammary carcinogenesis in rats.Fil: López Fontana, Constanza Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina;Fil: Sasso, Corina Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina;Fil: Maselli, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina;Fil: Santiano, Flavia Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina;Fil: Semino, Silvana Noemi. Universidad Nacional de Cuyo. Hospital Universitario. Laboratorio de Anatomía Patológica; Argentina;Fil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina;Fil: Jahn, Graciela Alma. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina;Fil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina

Patología cervical : valor predictivo de la técnica inmunocitoquímica p16/ki67 para detectar lesión CIN2+ subyacente

El carcinoma de cérvix representa la tercera neoplasia más frecuente en el mundo en las mujeres y una de las causas principales de muerte en países en vías de desarrollo. El virus del papiloma humano (VPH) es causa necesaria pero no suficiente para el desarrollo de esta neoplasia, puesto que casi todos los casos diagnosticados (99%) resultan positivos. Está totalmente asumido que el screening periódico con el test de Papanicolau es efectivo en reducir la incidencia y mortalidad del cáncer cervical, aunque en ocasiones puede presentar baja sensibilidad. La determinación de VPH-AR aporta baja especificidad, dado que la mayoría de infecciones por VPH son transitorias (hasta un 90% de las infecciones se aclaran en los dos primeros años). Nuestro estudio versará de cómo un marcador dual inmunocitoquímico (p16/ki67) puede indicar la desregulación del ciclo celular causado por oncoproteínas del VPH: E6 y E7. OBJETIVOS: El objetivo principal de esta tesis es: o Comprobar si la tinción dual con biomarcadores p16INK4a y Ki-67 que se expresan en las células de las citologías diagnosticadas de ASC y LSIL se asocia bien con lesión CIN2+ subyacente, o con progresión en un período determinado a CIN2+ Y los objetivos secundarios: o Comparar la tasa de positividad, sensibilidad y especificidad para citología con tinción dual versus determinaciónVPH-AR, para detectar lesiones CIN2+ o Ver qué relación guarda la positividad de la técnica dual p16/ki-67 con los distintos genotipos virales de alto riesgo, en los casos confirmados histológicamente como CIN2+. Frecuencia de los distintos genotipos en estas lesiones METODOLOGÍA GENERAL: • OBJETIVO PRINCIPAL: Se seleccionaron 306 pacientes a las que se les realizó técnica dual inmunocitoquímica p16/ki-67. Tras su realización se valoró como positiva o negativa. De las 306 muestras citológicas, el 50% de ellas se siguió de toma biópsica, con tres posibles diagnósticos: negativo para displasia (16), CIN1 (80) y CIN2+ (57). o El “gold standard” es el diagnóstico histológico de CIN2+ o El seguimiento de las pacientes fue de 1 día hasta 30 meses en algún caso • OBJETIVO Nº2: A 146 pacientes de las 306 se les realizó además tests de VPH: captura híbrida (HC2) y genotipado por PCR (Clart® HPV2). • OBJETIVO Nº3: De las 57 pacientes en las que se confirmaron histológicamente lesiones de CIN2+ se enviaron cortes de tejidos embebidos en parafina (TEP) a la unidad de virología para genotipado con PCR (método Clart®). RESULTADOS: OBJETIVO PRINCIPAL: o Se obtiene un aumento significativo de la sensibilidad para detectar lesión CIN2+ subyacente, manteniendo la alta especificidad que ya aportaba el test de papanicolau por sí solo. o Se observan leves diferencias en sensibilidad/especificidad entre los ASC y los LSIL OBJETIVO Nº 2: Se observa, tanto en ASC como en LSIL, una similar sensibilidad entre ambos tests (técnica dual y determinación de VPH) para detectar lesión CIN2+ subyacente cercana al 90%, existiendo sin embargo una especificidad mayor para la técnica dual, estadísticamente significativa. OBJETIVO Nº3: Se diagnosticó CIN2+ en 57 pacientes de las 306 incluídas en el estudio (18,6%). En más de la mitad de los casos (el 56%) la infección fue por un único genotipo, siendo el más frecuente el 16 (en 14 pacientes, el 50% de los casos positivos para VPH-AR), seguido del 31, y después con la misma frecuencia el 18, 45, 51 y 58. En los casos de infección múltiple también es el genotipo 16 el que más frecuentemente se asocia con otros genotipos (hasta en un 59% de los casos). El VPH 18 sólo aparece asociado en un 18% con otros genotipos. La frecuencia absoluta ha sido para el VPH16, seguido del 31 y posteriormente del 18. SUMMARY Cervical cancer is the third most common neoplasia in women and still is one of the main causes of death in less developed countries. Human papillomavirus (HPV) is necessary but not sufficient cause for this neoplasia as almost all the diagnosed cases (99%) were positive for cervical cancer. Regular screening together with the performance of a Pap test are believed to be effective in lowering cervical cancer incidence and mortality rates, although it can show low sensitivity. HR-HPV tests have a low specificity as most HPV infections are temporary (up to 90% of the infections resolve within the first two years). Our study will deal with how immunocytochemistry involving dual staining for p16 and Ki-67 can show cell cycle deregulation caused by HPV oncoproteins: E7 inactivates pRb, so p16 overexpression increases dramatically, and E6 stimulates the proliferative activity of Ki-67 by interfering with p53. OBJECTIVES: The main objective of this thesis is to: o Check that dual staining of ASC and LSIL cytology with p16INK4a and Ki-67 biomarkers is associated with an underlying CIN2+ lesion, or is progressing to CIN2+ over a specific period of time. And the following secondary objectives: o Comparing positivity, sensitivity and specificity rates for positive, dual stained results for dual staining cytology to determination of HR-HPV, in order to detect CIN2+ lesions. o Observing the connection of positive dual-stained results for p16 and Ki-67 with the different high-risk viral genotypes in histologically confirmed cases of CIN2+. Frequency with which different genotypes appear in these lesions. MATERIALS AND METHODS • MAIN OBJECTIVE: 306 selected patients underwent p16/Ki-67 dual immunostaining testing. The tests were positive or negative. Out of the 306 cytology samples, 50% were followed by bioptic sampling (histological analysis of 153 patients), with three possible diagnoses: negative for dysplasia (16), CIN1 (80) and CIN2+ (57). o The gold standard is the histologic diagnosis of CIN2+. o The Clinical Follow-up of patients took from 1 day up to 30 months in some cases. • OBJECTIVE No.2: 146 patients underwent HPV testing as well: Digene Hybrid Capture (HC 2) test and a PCR-based genotyping assay (Clart® HPV2). • OBJECTIVE No.3: Sections of paraffin-embedded tissues from the 57 patients were sent to the Department of Virology for a PCR-based genotyping assay (Clart® HPV2). RESULTS: MAIN OBJECTIVE: o Three hundred and forty-nine p16/Ki-67 dual-stained cytology tests were performed in 306 patients with ASC and LSIL diagnoses, resulting in a general and important rise in sensitivity for the detection of an underlying CIN2+ lesion, maintaining the same high specificity given only by the Pap test. o Some minor differences in sensitivity and specificity are found in ASC and LSIL. OBJECTIVE No. 2: In ASC and LSIL, both tests (dual staining and determination of HPV) have a similar sensitivity of 90% when detecting any underlying CIN2+ lesion, although a statistically significantly higher specificity of dual stained cytology has been observed. OBJECTIVE No. 3: CIN2+ has been diagnosed in 57 patients out of the 306 patients who participated in this study (18,6%). In more than half of the cases (56%), the infection was due to only one genotype, the most common being genotype 16 (in 14 patients or 50% of the HR-HPV-positive cases), followed by genotype 31 and then genotypes 18, 45, 51 and 58 with the same frequency. In cases of multiple infection, it is also genotype 16 which is most frequently associated with other genotypes (in up to 59% of the cases). HPV-18 is only associated with other genotypes in 18% of the cases. In order, the following absolute frequency was observed: HPV-16, 31 and finally 18