Low sclerostin levels: a predictive marker of persistent inflammation in ankylosing spondylitis during anti-tumor necrosis factor therapy?

Abstract Introduction Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy. Methods Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls. Results At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values. Conclusions Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.

Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all): from genetic alterations to impaired RNA editing

The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. Despite imatinib has led to significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases resistance developed quickly and disease progressed. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors, driving both the disease and resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were collected. We set up a fast, high-throughput method based on capillary electrophoresis technology to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6 failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a 60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line (SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6 strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a previously unknown link between specific molecular defects that involve the Ikaros gene and the resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of patients was also found. Other two different single nucleotide substitutions not recognized as SNP were observed. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.

Trasduzione del segnale inositide-dipendente nel carcinoma della mammella

Breast carcinoma, one of the most frequent malignancies in women, is a complex disease in which a number of different factors combine to drive pathogenesis. The biopathological characterization of these tumors is essential to determine their aggressiveness and to find the most appropriate therapy. As in others neoplasms, the deregulation of signal transduction pathways is frequently responsible for conferring selective biological advantages to the tumor. Phosphoinositides play an essential role in diverse cellular functions, their metabolism is highly active, and is tightly controlled. Among the enzymes implicated in this pathway, phospholipase C beta 1 (PLCβ1) is one of the key regulators, both at the cytoplasmic and the nuclear level. The PLCβ1 gene maps onto the short arm of chromosome 20, a region that has been shown to be altered in several solid tumors, including breast cancer. In the present study a FISH approach was used to investigate the genetic alterations of the PLCβ1 gene in various classes of breast cancer which differ in their invasiveness and proliferation status, according to their mitotic index. The overall aim was to find out whether this enzyme could be a suitable prognostic marker for this neoplasm. Our results show that 83% of cases had aneusomies at the 20p12 level, and the most frequent alteration is a gain in this specific locus. Indeed, we found that this amplification is not related to the invasion status since there were no differences in amplified tumor frequencies between in situ and invasive breast cancer. On the contrary, the gain of PLCβ1 was significantly related to the mitotic index (p = 0.001). To verify if the change in genetic dosage influences the expression of PLCβ1 we performed Real Time PCR and Immunohystochemical analysis. Our results confirmed that amplified tumors have higher levels of PLCβ1 mRNA, which is the sum of the two splicing isoforms 1a and 1b. On the other hand, even if protein levels were higher in the majority of cases compared to the nontumoral specimens, there were no significant associations between gain and overexpression. Finally, the significant association between the amplification of PLCβ1 and others important clinicopathological parameters, such as grading and hormonal receptors status, confirmed a correlation of this enzyme with the aggressiveness of breast cancer. This suggests that PLCβ1 has the potential to be a prognostic marker in these tumors. However, further work needs to be carried out to validate these preliminary findings.

Identificazione di un QTL principale per resistenza a ruggine bruna sul cromosoma 7B di frumento duro

Leaf rust caused by Puccinia triticina is a serious disease of durum wheat (Triticum durum) worldwide. However, genetic and molecular mapping studies aimed at characterizing leaf rust resistance genes in durum wheat have been only recently undertaken. The Italian durum wheat cv. Creso shows a high level of resistance to P. triticina that has been considered durable and that appears to be due to a combination of a single dominant gene and one or more additional factors conferring partial resistance. In this study, the genetic basis of leaf rust resistance carried by Creso was investigated using 176 recombinant inbred lines (RILs) from the cross between the cv. Colosseo (C, leaf rust resistance donor) and Lloyd (L, susceptible parent). Colosseo is a cv. directly related to Creso with the leaf rust resistance phenotype inherited from Creso, and was considered as resistance donor because of its better adaptation to local (Emilia Romagna, Italy) cultivation environment. RILs have been artificially inoculated with a mixture of 16 Italian P. triticina isolates that were characterized for virulence to seedlings of 22 common wheat cv. Thatcher isolines each carrying a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci, in order to determine their specialization with regard to the host species. The characterization of the leaf rust isolates was conducted at the Cereal Disease Laboratory of the University of Minnesota (St. Paul, USA) (Chapter 2). A genetic linkage map was constructed using segregation data from the population of 176 RILs from the cross CL. A total of 662 loci, including 162 simple sequence repeats (SSRs) and 500 Diversity Arrays Technology markers (DArTs), were analyzed by means of the package EasyMap 0.1. The integrated SSR-DArT linkage map consisted of 554 loci (162 SSR and 392 DArT markers) grouped into 19 linkage blocks with an average marker density of 5.7 cM/marker. The final map spanned a total of 2022 cM, which correspond to a tetraploid genome (AABB) coverage of ca. 77% (Chapter 3). The RIL population was phenotyped for their resistance to leaf rust under artificial inoculation in 2006; the percentage of infected leaf area (LRS, leaf rust susceptibility) was evaluated at three stages through the disease developmental cycle and the area under disease progress curve (AUDPC) was then calculated. The response at the seedling stage (infection type, IT) was also investigated. QTL analysis was carried out by means of the Composite Interval Mapping method based on a selection of markers from the CL map. A major QTL (QLr.ubo-7B.2) for leaf rust resistance controlling both the seedling and the adult plant response, was mapped on the distal region of chromosome arm 7BL (deletion bin 7BL10-0.78-1.00), in a gene-dense region known to carry several genes/QTLs for resistance to rusts and other major cereal fungal diseases in wheat and barley. QLr.ubo-7B.2 was identified within a supporting interval of ca. 5 cM tightly associated with three SSR markers (Xbarc340.2, Xgwm146 e Xgwm344.2), and showed an R2 and an LOD peak value for the AUDPC equal to 72.9% an 44.5, respectively. Three additional minor QTLs were also detected (QLr.ubo-7B.1 on chr. 7BS; QLr.ubo-2A on chr. 2AL and QLr.ubo-3A on chr. 3AS) (Chapter 4). The presence of the major QTL (QLr.ubo-7B.2) was validated by a linkage disequilibrium (LD)-based test using field data from two different plant materials: i) a set of 62 advanced lines from multiple crosses involving Creso and his directly related resistance derivates Colosseo and Plinio, and ii) a panel of 164 elite durum wheat accessions representative of the major durum breeding program of the Mediterranean basin. Lines and accessions were phenotyped for leaf rust resistance under artificial inoculation in two different field trials carried out at Argelato (BO, Italy) in 2006 and 2007; the durum elite accessions were also evaluated in two additional field experiments in Obregon (Messico; 2007 and 2008) and in a green-house experiment (seedling resistance) at the Cereal Disease Laboratory (St. Paul, USA, 2008). The molecular characterization involved 14 SSR markers mapping on the 7BL chromosome region found to harbour the major QTL. Association analysis was then performed with a mixed-linear-model approach. Results confirmed the presence of a major QTL for leaf rust resistance, both at adult plant and at seedling stage, located between markers Xbarc340.2, Xgwm146 and Xgwm344.2, in an interval that coincides with the supporting interval (LOD-2) of QLr.ubo-7B.2 as resulted from the RIL QTL analysis. (Chapter 5). The identification and mapping of the major QTL associated to the durable leaf rust resistance carried by Creso, together with the identification of the associated SSR markers, will enhance the selection efficiency in durum wheat breeding programs (MAS, Marker Assisted Selection) and will accelerate the release of cvs. with durable resistance through marker-assisted pyramiding of the tagged resistance genes/QTLs most effective against wheat fungal pathogens.

Marker molecolari relativi all'infezione da Papillomavirus umani: significato diagnostico e prognostico in lesioni virus-correlate

Introduzione: Il cervico-carcinoma è la seconda neoplasia maligna per incidenza e mortalità nelle donne in tutto il mondo dopo il cancro al seno. L’infezione persistente da Papillomavirus Umani (HPV) è causa necessaria dell’insorgenza del cervico-carcinoma e delle sue lesioni pre-cancerose. L’infezione da HPV si associa anche ad altri carcinomi del distretto ano-genitale (a livello anale, vulvare, vaginale e del pene) e a circa il 25% dei carcinomi squamosi dell’orofaringe. I circa 40 genotipi di HPV che infettano la mucosa genitale vengono suddivisi in alto rischio (HR-HPV) e basso rischio (LR-HPV) oncogeno a seconda della alta e bassa associazione con la neoplasia cervicale. I 13 genotipi a più alto rischio oncogeno sono 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 e 68. Di questi, otto (16, 18, 31, 33, 35, 45, 52 e 58) sono associati alla maggior parte dei carcinomi cervicali (circa 89%) e i genotipi 16 e 18 da soli sono riscontrati nel 70% circa delle neoplasie. L’insorgenza e progressione delle lesioni preneoplastiche cervicali è, però, associata non solo alla presenza di HPV ad alto rischio, ma, soprattutto, alla persistenza virale (> 18 mesi), alla capacità di integrazione degli HPV ad alto rischio e alla conseguente sovraespressione delle oncoproteine E6/E7. Inoltre, l’integrazione è spesso favorita da un’alta carica virale soprattutto per quanto riguarda alcuni genotipi (HPV16 e 18). L’infezione da HPV non interessa solo la cervice uterina ma tutto il distretto ano-genitale e quello testa-collo. L’HPV, in particolare il genotipo 16, è implicato, infatti, nell’insorgenza delle lesioni preneoplastiche della vulva (VIN) classificate come VIN classiche e nei carcinomi ad esse associati. L’incidenza del carcinoma vulvare in Europa è di 1.5/100.000 di cui circa il 45% è dovuto a HR-HPV (80% ad HPV16). Nonostante l’associazione tra HPV16 e carcinoma vulvare sia alta, ancora poco si conosce sul ruolo della carica virale e dell’integrazione in tali lesioni. Le lesioni che possono presentarsi nella regione testa-collo possono essere sia di natura benigna che maligna. I genotipi più frequentemente riscontrati in associazione a lesioni benigne (papillomi) sono HPV 6 e 11, quelli associati a forme tumorali (HNSCC) sono il genotipo 18 ma soprattutto il 16. Molti aspetti del coinvolgimento di HPV in queste patologie non sono ancora perfettamente conosciuti e spesso studi su tale argomento hanno mostrato risultati contraddittori, soprattutto perché vengono utilizzate metodiche con gradi diversi di sensibilità e specificità. Recenti dati di letteratura hanno tuttavia messo in evidenza che i pazienti affetti da HNSCC positivi ad HPV hanno una elevata risposta al trattamento chemioradioterapico rispetto ai pazienti HPV-negativi con un notevole impatto sul controllo locale e sulla sopravvivenza ma soprattutto sulla qualità di vita di tali pazienti, evitando di sottoporli a chirurgia sicuramente demolitiva. Scopo del lavoro: Sulla base di queste premesse, scopo di questo lavoro è stato quello di valutare l’importanza di marker quali la presenza/persistenza di HPV, la carica virale, la valutazione dello stato fisico del genoma virale e l’espressione degli mRNA oncogeni nella gestione di pazienti con lesioni preneoplastiche e neoplastiche di diverso grado, associate a papillomavirus mucosi. Per la valutazione dei markers virologici di progressione neoplastica abbiamo sviluppato dei saggi di real time PCR qualitativi e quantitativi studiati in modo da poter fornire, contemporaneamente e a seconda delle esigenze, risposte specifiche non solo sulla presenza e persistenza dei diversi genotipi di HPV, ma anche sul rischio di insorgenza, progressione e recidiva delle lesioni mediante lo studio di markers virologici quali carica virale, integrazione ed espressione degli mRNA. Abbiamo pertanto indirizzato la nostra attenzione verso tre popolazioni specifiche di pazienti: - donne con lesioni vulvari preneoplastiche (VIN) e neoplastiche, allo scopo di comprendere i complessi meccanismi patogenetici di tali patologie non sempre associate ad infezione da HPV; - pazienti con lesioni maligne a livello della regione testa-collo allo scopo di fornire informazioni utili all’elaborazione di un percorso terapeutico mirato (radiochemioterapico o chirurgico) a seconda o meno della presenza di infezione virale; - donne con lesioni cervicali di alto grado, trattate chirurgicamente per la rimozione delle lesioni e seguite nel follow-up, per stabilire l’importanza di tali marker nella valutazione della persistenza virale al fine di prevenire recidive di malattia.

A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects during the conversion of muscle to meat, due to their influence on meat texture and sensory characteristics, mainly in seasoned products. Their contribution is fundamental in flavour development of dry-curing hams. However, several authors have demonstrated that high cathepsin activity, in particular of cathepsin B, is correlated to defects of these products, such as an excessive meat softness together with abnormal free tyrosine content, astringent or metallic aftertastes and formation of a white film on the cut surface. Thus, investigation of their genetic variability could be useful to identify DNA markers associated with these dry cured hams parameters, but also with meat quality, production and carcass traits in Italian heavy pigs. Unfortunately, no association has been found between cathepsin markers and meat quality traits so far, in particular with cathepsin B activity, suggesting that other genes, besides these, affect meat quality parameters. Nevertheless, significant associations were observed with several carcass and production traits in pigs. A recent study has demonstrated that different single nucleotide polymorphisms (SNPs) localized in cathepsin D (CTSD), F (CTSF), H and Z genes were highly associated with growth, fat deposition and production traits in an Italian Large White pig population. The aim of this thesis was to confirm some of these results in other pig populations and identify new cathepsin markers in order to evaluate their effects on cathepsin activity and other production traits. Furthermore, starting from the data obtained in previous studies on CTSD gene, we also analyzed the known polymorphism located in the insulin-like growth factor 2 gene (IGF2 intron3-g.3072G>A). This marker is considered the causative mutation for the quantitative trait loci (QTL) affecting muscle mass and fat deposition in pigs. Since IGF2 maps very close to CTSD on porcine chromosome (SSC) 2, we wanted to clarify if the effects of the CTSD marker were due to linkage disequilibrium with the IGF2 intron3-g.3072G>A mutation or not. In the first chapter, we reported the results from these two SSC2 gene markers. First of all, we evaluated the effects of the IGF2 intron3-g.3072G>A polymorphism in the Italian Large White breed, for which no previous studies have analysed this marker. Highly significant associations were identified with all estimated breeding values for production and carcass traits (P<0.00001), while no effects were observed for meat quality traits. Instead, the IGF2 intron3-g.3072G>A mutation did not show any associations with the analyzed traits in the Italian Duroc pigs, probably due to the low level of variability at this polymorphic site for this breed. In the same Duroc pig population, significant associations were obtained for the CTSD marker for all production and carcass traits (P < 0.001), after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A polymorphism were also confirmed in a group of Italian Large White pigs homozygous for the IGF2 intron3-g.3072G allele G (IGF2 intron3-g.3072GG) and by haplotype analysis between the markers of the two considered genes. Taken together, all these data indicated that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition in pigs. In the second chapter, we reported the analysis of two new SNPs identified in cathepsin L (CTSL) and cathepsin S (CTSS) genes and the association results with meat quality parameters (including cathepsin B activity) and several production traits in an Italian Large White pig population. Allele frequencies of these two markers were evaluated in 7 different pig breeds. Furthermore, we mapped using a radiation hybrid panel the CTSS gene on SSC4. Association studies with several production traits, carried out in 268 Italian Large White pigs, indicated positive effects of the CTSL polymorphism on average daily gain, weight of lean cuts and backfat thickness (P<0.05). The results for these latter traits were also confirmed using a selective genotype approach in other Italian Large White pigs (P<0.01). In the 268 pig group, the CTSS polymorphism was associated with feed:gain ratio and average daily gain (P<0.05). Instead, no association was observed between the analysed markers and meat quality parameters. Finally, we wanted to verify if the positive results obtained for the cathepsin L and S markers and for other previous identified SNPs (cathepsin F, cathepsin Z and their inhibitor cystatin B) were confirmed in the Italian Duroc pig breed (third chapter). We analysed them in two groups of Duroc pigs: the first group was made of 218 performance-tested pigs not selected by any phenotypic criteria, the second group was made of 100 Italian Duroc pigs extreme and divergent for visible intermuscular fat trait. In the first group, the CTSL polymorphism was associated with weight of lean cuts (P<0.05), while suggestive associations were obtained for average daily gain and backfat thickness (P<0.10). Allele frequencies of the CTSL gene marker also differed positively among the visible intermuscular extreme tails. Instead, no positive effects were observed for the other DNA markers on the analysed traits. In conclusion, in agreement with the present data and for the biological role of these enzymes, the porcine CTSD and CTSL markers: a) may have a direct effect in the biological mechanisms involved in determining fat and lean meat content in pigs, or b) these markers could be very close to the putative functional mutation(s) present in other genes. These findings have important practical applications, in particular the CTSD and CTSL mutations could be applied in a marker assisted selection (MAS) both in the Italian Large White and Italian Duroc breeds. Marker assisted selection could also increase in efficiency by adding information from the cathepsin S genotype, but only in the Italian Large White breed.

Untersuchungen zur Verwendbarkeit von Purinnukleotidgehalten als prognostische Marker bei humanen Mammacarzinomzellen

Ein bei unterschiedlichen Zellinien mit NMR-Spektroskopie beobachteter vorübergehender Anstieg von ATP während Anthracyclinbehandlungen sollte in vitro an Monolayern bzw. Sphäroiden der humanen Brustkrebszellinien T47D, MCF-7, MCF-7adr und MCF-7mdr während 24stündigen Inkubationen mit 10 oder 0,1 µM Doxorubicin systematisch untersucht werden. Wachstumskurven sowie Clonogenitäts- und Vitalitätstests ergaben, daß T47D Zellen wesentlich empfindlicher auf das Medikament reagierten, als die MCF-7 Linie oder deren resistente Subklone. Eine zusätzliche Behandlung mit dem (-Tocopherol-Analogon CR-6 führte zu einer Verstärkung der Doxorubicinwirkung. Licht- oder elektronenmikroskopische Untersuchungen an Monolayern und Sphäroiden zeigten sowohl Apoptosen als auch Nekrosen, wobei Apoptosen vermehrt bei weniger empfindlichen Zellen oder niedriger Dosierung auftraten. Die Quantifizierung der Purinnukleotide erfolgte mit Hilfe der HPLC, bei T47D Sphäroiden zusätzlich mit abbildender Biolumineszenz. Bei sämtlichen Zellinien wurden während der Behandlung mit 10 µM vorübergehende Anstiege aller Purinnukleotide gemessen, wobei GTP höher stieg (78 % der unbehandelten Kontrolle) als ATP (30 %). Der Anstieg war bei T47D Zellen wesentlich höher als bei MCF-7, die MCF-7 Subklone zeigten untereinander keine deutlichen Unterschiede. Bei Sphäroiden war der Effekt etwas geringer als bei Monolayern. Die Inkubation mit 0,1 µM führte zu einem deutlich geringeren Anstieg. Der Effekt korrelierte mit einem massiven vorwiegend nekrotischen Zellsterben.

Transformation, Progression und Zellinienspezifität der chronischen myeloischen Leukämie

Die Analyse der CML-Zellinie K562 mittels Fluoreszenz in situ Hybridisierung (FISH), Multiplex-FISH (M-FISH) und comparativer genomischer Hybridisierung (CGH) ergab einen hypotriploiden Karyotyp mit 67 Chromosomen und 21 verschiedenen Marker-Chromosomen. Das bei über 90% der CML-Patienten nachgewiesene Ph-Chromosom entsteht durch die reziproke Translokation t(9;22)(q34;q11). Bei 5 - 10% der Patienten resultiert das Ph-Chromosom aus varianten Translokation. Anhand der Untersuchung dreier varianter Translokation mittels Bruchpunkt-übergreifender FISH-Proben für die BCR- und ABL-Gene werden drei verschiedene Mechanismen der Entstehung komplexer Translokationen dargestellt. Das Auftreten sekundärer Aberrationen wurde in 15 CML-Blastenkrisen untersucht. Zudem wurde anhand der CGH-Analyse von CD34-positiven Zellen, Monozyten, Granulozyten und T-Zellen die Zellinienspezifität sekundärer Aberrationen untersucht. In einem Fall wurde eine sekundäre Aberration in allen vier Fraktionen gefunden. In zwei Fällen traten sekundäre Aberrationen in allen untersuchten Fraktionen mit Ausnahme der T-Zellen auf. Aufgrund dieser Ergebnisse lassen sich zwei alternative Modelle der Tumor-Progression der CML ableiten: 1. Sekundäre Mutatonen treten vor der Differenzierung der hämatopoetischen Stammzelle auf. 2. Sekundäre Mutationen treten in einer hämatopoetischen Vorläuferzelle nach der T-Zell-Differenzierung auf.

Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.