Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

Genetic mechanisms of Na+, K+-ATPase regulation in hypertension

The spontaneously hypertensive rat (SHR) is a model of essential hypertension. During the early development of hypertension, the SHR demonstrates increased proximal tubule (PT) Na+ reabsorption. I hypothesized that the increased PT Na+ reabsorption exhibited by the young SHR was due to altered sub-cellular distribution of Na+, K +-ATPase compared to the normotensive Wistar Kyoto (WKY). The hypothesis is supported, herein, by observations of greater Na+, K +-ATPase α 1 abundance in PT plasma membrane and lower abundance in late endosomes of 4wk SHR despite no difference in total PT α 1 abundance. There is a greater amount of Ser-18 unphosphorylated α 1 in the 4wk SHR PT. Total PT Na+, K+-ATPase γ abundance is greater in SHR at 4wk and 16wk but γ abundance in plasma membrane is greater only at 4wk. The phosphatase, calcineurin, was chosen for study because it is involved in the stimulation of Na+, K +-ATPase. No difference in calcineurin coding sequence, expression, or activity was observed in SHR. Gene expression arrays were next used to find candidate genes involved in the regulation of Na+, K +-ATPase. The first candidate analyzed was soluble epoxide hydrolase (sEH). The gene encoding sEH (EPHX2) showed lower expression in SHR. There was also a reduction in sEH protein abundance but there was no correlation between protein abundance and blood pressure in F2 progeny. Two EPHX2 alleles were identified, an ancestral allele and a variant allele containing four polymorphisms. sEH activity was greater in animals carrying the variant allele but the inheritance of the variant allele did not correlate with blood pressure. Gene expression arrays also led to the examination of genes involved in redox balance/Na+, K+-ATPase regulation. A pattern of lower expression of genes involved in reactive radical detoxification in SHR was discerned. Six transcription factor binding sites were identified that occurred more often in these genes. Three transcription factors that bind to the HNF1 site were expressed at lower levels in SHR. This points to the HNF1 transcriptional complex as an important trans-acting regulator of a wide range of genes involved in altered redox balance in SHR. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

Persisting abdominal symptoms after travelers' diarrhea: Is there a genetic predisposition?

Functional gastrointestinal disorders (FGIDs) are defined as ailments of the mid or lower gastrointestinal tract which are not attributable to any discernable anatomic or biochemical defects.1 FGIDs include functional bowel disorders, also known as persisting abdominal symptoms (PAS). Irritable bowel syndrome (IBS) is one of the most common illnesses classified under PAS.2,3 This is the first prospective study that looks at the etiology and pathogenesis of post-infectious PAS in the context of environmental exposure and genetic susceptibility in a cohort of US travelers to Mexico. Our objective was to identify infectious, genetic and environmental factors that predispose to post infectious PAS. ^ Methods. This is a secondary data analysis of a prospective study on a cohort of 704 healthy North American tourists to Cuernavaca, Morelos and Guadalajara, Jalisco in Mexico. The subjects at risk for Travelers' diarrhea were assessed for chronic abdominal symptoms on enrollment and six months after the return to the US. ^ Outcomes. PAS was defined as disturbances of mid and lower gastrointestinal system without any known pathological or radiological abnormalities, or infectious, or metabolic causes. It refers to functional bowel disease, category C of functional gastrointestinal diseases as defined by the Rome II criterion. PAS was sub classified into Irritable bowel syndrome (IBS) and functional abdominal disease (FAD). ^ IBS is defined as recurrent abdominal pain or discomfort present at least 25% and associated with improvement with defecation, change in frequency and form of stool. FAD encompasses other abdominal symptoms of chronic nature that do not meet the criteria for IBS. It includes functional diarrhea, functional constipation, functional bloating: and unspecified bowel symptoms. ^ Results. Among the 704 travelers studied, there were 202 cases of PAS. The PAS cases included 175 cases of FAD and 27 cases of IBS. PAS was more frequent among subjects who developed traveler's diarrhea in Mexico compared to travelers who remained healthy during the short term visit to Mexico (52 vs. 38; OR = 1.8; CI, 1.3–2.5, P < 0.001). A statistically significant difference was noted in the mean age of subjects with PAS compared to healthy controls (28 vs. 34 yrs; OR = 0.97, CI, 0.95–0.98; P < 0.001). Travelers who experienced multiple episodes, a later onset of diarrhea in Mexico and passed greater numbers of unformed stools were more likely to be identified in PAS group at six months. Participants who developed TD caused by enterotoxigenic E.coli in Mexico showed a 2.6 times higher risk of developing FAD (P = 0.003). Infection with Providencia ssp. also demonstrated a greater risk to developing PAS. Subjects who sought treatment for diarrhea while in Mexico also displayed a significantly lower frequency of IBS at six months follow up (OR = 0.30; CI, 0.10–0.80; P = 0.02). ^ Forty six SNPs belonging to 14 genes were studied. Seven SNPs were associated with PAS at 6 months. These included four SNPs from the Caspase Recruitment Domain-Containing Protein 15 gene (CARD15), two SNPs from Surfactant Pulmonary-Associated Protein D gene (SFTPD) and one from Decay-Accelerating Factor For Complement gene (CD55). A genetic risk score (GRS) was composed based on the 7 SNPs that showed significant association with PAS. A 20% greater risk for PAS was noted for every unit increase in GRS. The risk increased by 30% for IBS. The mean GRS was high for IBS (2.2) and PAS (1.1) compared to healthy controls (0.51). These data suggests a role for these genetic polymorphisms in defining the susceptibility to PAS. ^ Conclusions. The study allows us to identify individuals at risk for developing post infectious IBS (PI-IBS) and persisting abdominal symptoms after an episode of TD. The observations in this study will be of use in developing measures to prevent and treat post-infectious irritable bowel syndrome among travelers including pre-travel counseling, the use of vaccines, antibiotic prophylaxis or the initiation of early antimicrobial therapy. This study also provides insights into the pathogenesis of post infectious PAS and IBS. (Abstract shortened by UMI.)^

Immune and genetic risk factors in glioma

Despite extensive research, the etiology of adult glioma remains largely unknown. We sought to further explore the role of immune and genetic factors in glioma etiology using data from the Harris County Brain Tumor Study and the first U.S. genome-wide association study of glioma. First, using a case-control study design, we examined the association between adult glioma risk and surrogates of the timing and frequency of common early childhood infections, birth order and sibship size, respectively. We found that each one-unit increase in birth order was associated with a 12% decreased risk of glioma development in adulthood (OR=0.88, 95% CI=0.81-0.96); however, sibship size was not associated with adult glioma risk (OR=0.96, 95% CI=0.91-1.02). Second, we used a multi-strategic approach to explore the relationships between glioma risk, history of asthma/allergies, and 23 functional SNPs in 11 inflammation genes. We found three inflammation gene SNPs to be significantly associated with glioma risk (COX2/PTGS2 rs20417 [OR=1.41]; IL10 rs1800896 [OR=1.57]; and IL13 rs20541 [OR=0.39]). Joint effects analysis of the risk-conferring alleles of these three SNPs revealed a trend of increasing risk with increasing number of adverse alleles among those without asthma/allergies (p<0.0001). Finally, we conducted a case-only study to explore pairwise SNP-SNP interactions in immune-related pathways among a population of 1304 non-Hispanic white glioma cases. After correction for multiple comparisons, we found 279 significant SNP-SNP interactions among polymorphisms of immune-related genes, many of which have not been previously examined. Our results, cumulatively, suggest an important role for immune and genetic factors in glioma etiology and provide several new hypotheses for future studies.^

ISOELECTRIC FOCUSING STUDIES OF STEROID BINDING PROTEINS

Steroid binding proteins are an obvious choice in the search for genetic factors in plasma that might predispose to upper body obesity, a risk factor for non-insulin dependent diabetes and cardiovascular disease. The two steroid binding proteins studied by isoelectric focusing were sex hormone binding globulin (SHBG), the transport protein for sex hormones and corticosteroid binding globulin (CBG), the transport protein for corticosteroids. Auto-radiography and immunoblotting on polyacrylamide gels were used to detect polymorphism in SHBG. Immunoblotting on agarose gels was used to visualize corticosteroid binding globulin. SHBG showed similar structural variation in American Caucasians, American Blacks and Canadian Indians. Two alleles (1, 2) were hypothesized with highly polymorphic frequencies in all three ethnic groups. CBG was not found to be polymorphic, but two variants were found in Caucasian male twins and in a Black individual. The finding of a good assay and a polymorphic system for SHBG are the first steps for additional studies into disease associations. ^

Detecting genetic and nutritional lung cancer risk factors related to folate metabolism using Bayesian generalized linear models

Complex diseases, such as cancer, are caused by various genetic and environmental factors, and their interactions. Joint analysis of these factors and their interactions would increase the power to detect risk factors but is statistically. Bayesian generalized linear models using student-t prior distributions on coefficients, is a novel method to simultaneously analyze genetic factors, environmental factors, and interactions. I performed simulation studies using three different disease models and demonstrated that the variable selection performance of Bayesian generalized linear models is comparable to that of Bayesian stochastic search variable selection, an improved method for variable selection when compared to standard methods. I further evaluated the variable selection performance of Bayesian generalized linear models using different numbers of candidate covariates and different sample sizes, and provided a guideline for required sample size to achieve a high power of variable selection using Bayesian generalize linear models, considering different scales of number of candidate covariates. ^ Polymorphisms in folate metabolism genes and nutritional factors have been previously associated with lung cancer risk. In this study, I simultaneously analyzed 115 tag SNPs in folate metabolism genes, 14 nutritional factors, and all possible genetic-nutritional interactions from 1239 lung cancer cases and 1692 controls using Bayesian generalized linear models stratified by never, former, and current smoking status. SNPs in MTRR were significantly associated with lung cancer risk across never, former, and current smokers. In never smokers, three SNPs in TYMS and three gene-nutrient interactions, including an interaction between SHMT1 and vitamin B12, an interaction between MTRR and total fat intake, and an interaction between MTR and alcohol use, were also identified as associated with lung cancer risk. These lung cancer risk factors are worthy of further investigation.^

Genetic variants within the Wnt/ B-catenin signaling pathway as indicators of bladder cancer clinical outcomes

The genetic factors that influence bladder cancer clinical outcomes are largely unknown. In this clinical outcomes study, I assessed genetic variations in the Wnt/β-catenin stem-cell pathway genes for association with recurrence and progression. A total of 230 SNPS in 40 genes from the Wnt/β-catenin pathway were genotyped in 419 histologically confirmed non-muscle invasive bladder cancer cases. Several significant associations were observed in the clinical outcomes analysis. Under the dominant model WNT8B: rs4919464 (HR: 1.55, 95% CI: 1.17-2.06, P=2.2x10-3) and WNT8B: rs3793771 (HR: 1.54, 95% CI: 1.09-1.62, P=4.6x10-3 ) were statistically significantly associated with an increase risk of recurrence while two other variants, APC2: rs11668593 (HR: 2.50, 95% CI: 1.43-4.35, P=1.2x10-3) and LRP5 : rs312778 (HR: 1.81, 95% CI: 1.23-2.65, P=2.7x10-3), were significantly associated with recurrence risk under the recessive model of inheritance. Four SNPs in the recessive model were associated with an increased risk of progression (AXIN2: rs1544427, LRP5: rs312778, AXIN1: rs370681, AXIN1: rs2301522). LRP5: rs312778 had the most significant increased risk of progression with a 2.68 (95% CI: 1.52-4.72, P=6.4x10-4)-fold increased risk. Stratification analysis based on treatment regimen (transurethral resection (TUR) and Bacillus Calmette-Guérin (BCG)) was also performed. Individuals with at least one variant in AXIN2: rs2007085 were found to have a 2.09 (95% CI: 1.24-3.52, P=5.4x10-3) -fold increased risk of recurrence in those that received TUR only, and no statistically significant effect was seen in those that received BCG. Individuals who received TUR with at least one variant in LEF1: rs10516550 were found to have a 2.26 (95% CI: 1.22-4.18, P=9.7x10-3)-fold increase risk of recurrence and no statistically significant effect was found in individuals who received BCG. Also, the recessive model of LRP6: rs2302684 in TUR only treatment was shown to have a 1.95 (95%CI: 1.18-3.21, P=8.8x10 -3)-fold increased risk of recurrence, and a suggested protective effect associated with a (HR: 0.83, 95% CI: 0.51-1.37, P=0.468) decreased risk of recurrence. Together, these findings implicate the Wnt/β-catenin stem-cell pathway as playing a role in bladder cancer clinical outcomes and have important implications for personalization of future treatment regimens. ^

Role of obesity, weight gain, physical activity, and polymorphisms in the mTOR pathway and the risk of renal cell carcinoma

The interplay between obesity, physical activity, weight gain and genetic variants in mTOR pathway have not been studied in renal cell carcinoma (RCC). We examined the associations between obesity, weight gain, physical activity and RCC risk. We also analyzed whether genetic variants in the mTOR pathway could modify the association. Incident renal cell carcinoma cases and healthy controls were recruited from the University of Texas MD Anderson Cancer Center in Houston, Texas. Cases and controls were frequency-matched by age (±5 years), ethnicity, sex, and county of residence. Epidemiologic data were collected via in-person interview. A total of 577 cases and 593 healthy controls (all white) were included. One hundred ninety-two (192) SNPs from 22 genes were available and their genotyping data were extracted from previous genome-wide association studies. Logistic regression and regression spline were performed to obtain odds ratios. Obesity at age 20, 40, and 3 years prior to diagnosis/recruitment, and moderate and large weight gain from age 20 to 40 were each significantly associated with increased RCC risk. Low physical activity was associated with a 4.08-fold (95% CI: 2.92-5.70) increased risk. Five single nucleotide polymorphisms (SNPs) were significantly associated with RCC risk and their cumulative effect increased the risk by up to 72% (95% CI: 1.20-2.46). Strata specific effects for weight change and genotyping cumulative groups were observed. However, no interaction was suggested by our study. In conclusion, energy balance related risk factors and genetic variants in the mTOR pathway may jointly influence susceptibility to RCC. ^

Exploration of genetic susceptibility to pancreatic cancer: A GWAS approach

Pancreatic cancer is the 4th most common cause for cancer death in the United States, accompanied by less than 5% five-year survival rate based on current treatments, particularly because it is usually detected at a late stage. Identifying a high-risk population to launch an effective preventive strategy and intervention to control this highly lethal disease is desperately needed. The genetic etiology of pancreatic cancer has not been well profiled. We hypothesized that unidentified genetic variants by previous genome-wide association study (GWAS) for pancreatic cancer, due to stringent statistical threshold or missing interaction analysis, may be unveiled using alternative approaches. To achieve this aim, we explored genetic susceptibility to pancreatic cancer in terms of marginal associations of pathway and genes, as well as their interactions with risk factors. We conducted pathway- and gene-based analysis using GWAS data from 3141 pancreatic cancer patients and 3367 controls with European ancestry. Using the gene set ridge regression in association studies (GRASS) method, we analyzed 197 pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Using the logistic kernel machine (LKM) test, we analyzed 17906 genes defined by University of California Santa Cruz (UCSC) database. Using the likelihood ratio test (LRT) in a logistic regression model, we analyzed 177 pathways and 17906 genes for interactions with risk factors in 2028 pancreatic cancer patients and 2109 controls with European ancestry. After adjusting for multiple comparisons, six pathways were marginally associated with risk of pancreatic cancer ( P < 0.00025): Fc epsilon RI signaling, maturity onset diabetes of the young, neuroactive ligand-receptor interaction, long-term depression (Ps < 0.0002), and the olfactory transduction and vascular smooth muscle contraction pathways (P = 0.0002; Nine genes were marginally associated with pancreatic cancer risk (P < 2.62 × 10−5), including five reported genes (ABO, HNF1A, CLPTM1L, SHH and MYC), as well as four novel genes (OR13C4, OR 13C3, KCNA6 and HNF4 G); three pathways significantly interacted with risk factors on modifying the risk of pancreatic cancer (P < 2.82 × 10−4): chemokine signaling pathway with obesity ( P < 1.43 × 10−4), calcium signaling pathway (P < 2.27 × 10−4) and MAPK signaling pathway with diabetes (P < 2.77 × 10−4). However, none of the 17906 genes tested for interactions survived the multiple comparisons corrections. In summary, our current GWAS study unveiled unidentified genetic susceptibility to pancreatic cancer using alternative methods. These novel findings provide new perspectives on genetic susceptibility to and molecular mechanisms of pancreatic cancer, once confirmed, will shed promising light on the prevention and treatment of this disease. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

Identification of genetic risk factors for cerebellar mutism in pediatric brain tumor patients

Following posterior fossa surgery for resection of childhood medulloblastoma and primitive neuroectodermal tumor (M/PNET), cerebellar mutism (CM) may develop. This is a condition of absent or diminished speech in a conscious patient with no evidence of oral apraxia, which can be accompanied by other symptoms of the posterior fossa syndrome complex, which includes ataxia and hypotonia. Little is known about the etiology. Therefore, we conducted a SNP, gene, and pathway-level analysis to assess the role of host genetic variation on the risk of CM in M/PNET subjects following treatment. Cases (n= 20) and controls (n= 53) were recruited from the Childhood Cancer Epidemiology and Prevention Center, in Houston, TX. DNA samples were genotyped using the Illumina Human 1M Quad SNP chip. Ten pathways were identified from logistic regression used to identify the marginal effect of each SNP on CM risk. The minP test was conducted to identify associations between SNPs categorized to genes and CM risk. Pathways were assessed to determine if there was a significant enrichment of genes in the pathway compared to all other pathways. There were 78 genes that reached the threshold of min P ≤0.05 in 948 genes. The Neurotoxicity pathway was the most significant pathway after adjusting for multiple comparisons (q=0.040 and q=0.005, using Fisher's exact test and a test of proportions, respectively). Most genes within the Neurotoxicity pathway that reached a threshold of minP ≤0.05 were known to have an apoptosis function, possibly inducing neuronal apoptosis in the dentatothalamocortical pathway, and may be important in CM etiology in this population. This is the first study to assess the potential role of genetic risk factors on CM. As an exploratory study, these results should be replicated in a larger sample. ^

Functional analysis of the Galpha protein, GNA -3, in the development of Neurospora crassa using biochemical and genetic studies

Extracellular signals regulate fungal development and, to sense and respond to these cues, fungi evolved signal transduction pathways similar to those in mammalian systems. In fungi, heterotrimeric G proteins, composed of α, β, and γ subunits, transduce many signals, such as pheromones and nutrients, intracellularly to alter adenylyl cyclase and MAPK cascades activity. ^ Previously, the Gα proteins GNA-1 and GNA-2 were characterized in regulating development in the fungus Neurospora crassa. R. A. Baasiri isolated a third Gα, gna-3, and P. S. Rowley generated Δgna-3 mutants. GNA-3 belongs to a fungal Gα family that regulates cAMP metabolism and virulence. The Δ gna-3 sexual cycle is defective in homozygous crosses, producing inviable spores. Δgna-3 mutants have reduced aerial hyphae formation and derepressed asexual sporulation (conidiation), causing accumulation of asexual spores (conidia). These defects are similar to an adenylyl cyclase mutant, cr-1; cAMP supplementation suppressed Δ gna-3 and cr-1. Inappropriate conidiation and expression of a conidiation gene, con-10, were higher in Δ gna-3 than cr-1 submerged cultures; peptone suppressed conidiation. Adenylyl cyclase activity and expression demonstrated that GNA-3 regulates enzyme levels. ^ A Δgna-1 cr-1 was analyzed with F. D. Ivey to differentiate GNA-1 roles in cAMP-dependent and -independent pathways. Δ gna-1 cr-1 defects were worse than cr-1 and refractory to cAMP, suggesting that GNA-1 is necessary for sensing extracellular CAMP. Submerged culture conidiation was highest in Δgna-1 cr-1, and only high cell density Δgna-1 cultures conidiated, which correlated with con-10 levels. Transcription of a putative heat shock cognate protein was highest in Δgna-1 cr-1. ^ Functional relationships between the three Gαs was analyzed by constructing Δgna-1 Δgna-2 Δ gna-3, Δgna-1 Δgna-3, and Δgna-2 Δgna-3 strains. Δ gna-2 Δgna-3 strains exhibited intensified Δ gna-3 phenotypes; Δgna-1 Δgna-2 Δgna-3 and Δgna-1 Δ gna-3 strains were identical to Δgna-1 cr-1 on plates and were non-responsive to cAMP. The highest levels of conidiation and con-10 were detected in submerged cultures of Δ gna-1 Δgna-2 Δgna-3 and Δgna-1 Δgna-3 mutants, which was partially suppressed by peptone supplementation. Stimulation of adenylyl cyclase is completely deficient in Δgna-1 Δ gna-2 Δgna-3 and Δgna-1 Δ gna-3 strains. Δgna-3 and Δ gna-1 Δgna-3 aerial hyphae and conidiation defects were suppressed by mutation of a PKA regulatory subunit. ^

Integration of socioeconomic and genetic aspects involved in the conservation of animal genetic resources = Integración de aspectos socioeconómicos y genéticos involucrados en la conservación de recursos genéticos animales

El sector ganadero está siendo gradualmente dominado por sistemas intensivos y especializados en los que los factores de producción están controlados y en los que los caracteres productivos son los criterios principales para la selección de especies y razas. Entretanto, muchos de los bienes y servicios que tradicionalmente suministraba el ganado, tales como los fertilizantes, la tracción animal o materias primas para la elaboración vestimenta y calzado están siendo reemplazados por productos industriales. Como consecuencia de ambos cambios, las razas seleccionadas intensivamente, las cuales están estrechamente ligadas a sistemas agrícolas de alta producción y altos insumos, han desplazado a muchas razas autóctonas, en las que la selección prácticamente ha cesado o es muy poco intensa. Actualmente existe una mayor conciencia social sobre la situación de las razas autóctonas y muchas funciones del ganado que previamente habían sido ignoradas están siendo reconocidas. Desde hace algunas décadas, se ha aceptado internacionalmente que las razas de ganado cumplen funciones económicas, socio-culturales, medioambientales y de seguridad alimentaria. Por ello, diferentes organismos internacionales han reconocido que la disminución de los recursos genéticos de animales domésticos (RGADs) es un problema grave y han recomendado su conservación. Aun así, la conservación de RGADs es un tema controvertido por la dificultad de valorar las funciones del ganado. Esta valoración es compleja debido que los RGADs tiene una doble naturaleza privada - pública. Como algunos economistas han subrayado, el ganado es un bien privado, sin embargo debido a algunas de sus funciones, también es un bien público. De esta forma, el aumento del conocimiento sobre valor de cada una de sus funciones facilitaría la toma de decisiones en relación a su conservación y desarrollo. Sin embargo, esta valoración es controvertida puesto que la importancia relativa de las funciones del ganado varía en función del momento, del lugar, de las especies y de las razas. El sector ganadero, debido a sus múltiples funciones, está influenciado por factores técnicos, medioambientales, sociales, culturales y políticos que están interrelacionados y que engloban a una enorme variedad de actores y procesos. Al igual que las funciones del ganado, los factores que afectan a su conservación y desarrollo están fuertemente condicionados por localización geográfica. Asimismo, estos factores pueden ser muy heterogéneos incluso dentro de una misma raza. Por otro lado, es razonable pensar que el ganadero es el actor principal de la conservación de razas locales. Actualmente, las razas locales están siendo Integration of socioeconomic and genetic aspects involved in the conservation of animal genetic resources 5 explotadas por ganaderos muy diversos bajo sistemas de producción también muy diferentes. Por todo ello, es de vital importancia comprender y evaluar el impacto que tienen las motivaciones, y el proceso de toma de decisiones de los ganaderos en la estructura genética de las razas. En esta tesis doctoral exploramos diferentes aspectos sociales, económicos y genéticos involucrados en la conservación de razas locales de ganado vacuno en Europa, como ejemplo de RGADs, esperando contribuir al entendimiento científico de este complejo tema. Nuestro objetivo es conseguir una visión global de los procesos subyacentes en la conservación y desarrollo de estas razas. Pretendemos ilustrar como se pueden utilizar métodos cuantitativos en el diseño y establecimiento de estrategias de conservación y desarrollo de RGADs objetivas y adecuadas. En primer lugar, exploramos el valor económico total (VET) del ganado analizando sus componentes públicos fuera de mercado usando como caso de estudio la raza vacuna Alistana-Sanabresa (AS). El VET de cualquier bien está formado por componentes de uso y de no-uso. Estos últimos incluyen el valor de opción, el valor de herencia y el valor de existencia. En el caso del ganado local, el valor de uso directo proviene de sus productos. Los valores de uso indirecto están relacionados con el papel que cumple las razas en el mantenimiento de los paisajes y cultura rural. El valor de opción se refiere a su futuro uso potencial y el valor de herencia al uso potencial de las generaciones venideras. Finalmente, el valor de existencia está relacionado con el bienestar que produce a la gente saber que existe un recurso específico. Nuestro objetivo fue determinar la importancia relativa que tienen los componentes fuera de mercado sobre el VET de la raza AS. Para ello evaluamos la voluntad de la gente a pagar por la conservación de la AS mediante experimentos de elección (EEs) a través de encuestas. Estos experimentos permiten valorar individualmente los distintos componentes del VET de cualquier bien. Los resultados los analizamos mediante de uso de modelos aleatorios logit. Encontramos que las funciones públicas de la raza AS tienen un valor significativo. Sus valores más importantes son el valor de uso indirecto como elemento cultural Zamorano y el valor de existencia (ambos representaron el 80% de VET). Además observamos que el valor que gente da a las funciones públicas de la razas de ganado dependen de sus características socioeconómicas. Los factores que condicionaron la voluntad a pagar para la conservación de la raza AS fueron el lugar de residencia (ciudad o pueblo), el haber visto animales de la raza o haber consumido sus productos y la actitud de los encuestados ante los conflictos entre el desarrollo económico y el medioambiente. Por otro lado, encontramos que no todo el mundo tiene una visión completa e integrada de todas las funciones públicas de la raza AS. Por este motivo, los programas o actividades de concienciación sobre su estado deberían hacer hincapié en este aspecto. La existencia de valores públicos de la raza AS implica que los ganaderos deberían recibir compensaciones económicas como pago por las funciones públicas que cumple su raza local. Las compensaciones asegurarían un tamaño de población que permitiría que la raza AS siga realizando estas funciones. Un mecanismo para ello podría ser el desarrollo del turismo rural relacionado con la raza. Esto aumentaría el valor de uso privado mientras que supondría un elemento añadido a las estrategias de conservación y desarrollo. No obstante, los ganaderos deben analizar cómo aprovechar los nichos de mercado existentes, así como mejorar la calidad de los productos de la raza prestando especial atención al etiquetado de los mismos. Una vez evaluada la importancia de las funciones públicas de las razas locales de ganado, analizamos la diversidad de factores técnicos, económicos y sociales de la producción de razas locales de ganado vacuno existente en Europa. Con este fin analizamos el caso de quince razas locales de ocho países en el contexto de un proyecto de colaboración internacional. Investigamos las diferencias entre los países para determinar los factores comunes clave que afectan a la viabilidad de las razas locales. Para ello entrevistamos mediante cuestionarios a un total de 355 ganaderos en las quince razas. Como indicador de viabilidad usamos los planes de los ganaderos de variación del tamaño de las ganaderías. Los cuestionarios incluían diferentes aspectos económicos, técnicos y sociales con potencial influencia en las dinámicas demográficas de las razas locales. Los datos recogidos los analizamos mediante distintas técnicas estadísticas multivariantes como el análisis discriminante y la regresión logística. Encontramos que los factores que afectan a la viabilidad de las razas locales en Europa son muy heterogéneos. Un resultado reseñable fue que los ganaderos de algunos países no consideran que la explotación de su raza tenga un alto valor social. Este hecho vuelve a poner de manifiesto la importancia de desarrollar programas Europeos de concienciación sobre la importancia de las funciones que cumplen las razas locales. Además los países analizados presentaron una alta variabilidad en cuanto a la importancia de los mercados locales en la distribución de los productos y en cuanto al porcentaje en propiedad del total de los pastos usados en las explotaciones. Este estudio reflejó la variabilidad de los sistemas y medios de producción (en el sentido socioeconómico, técnico y ecológico) que existe en Europa. Por ello hay que ser cautos en la implementación de las políticas comunes en los diferentes países. También encontramos que la variabilidad dentro de los países puede ser elevada debido a las diferencias entre razas, lo que implica que las políticas nacionales deber ser suficientemente flexibles para adaptarse a las peculiaridades de cada una de las razas. Por otro lado, encontramos una serie de factores comunes a la viabilidad de las razas en los distintos países; la edad de los ganaderos, la colaboración entre ellos y la apreciación social de las funciones culturales, medioambientales y sociales del ganado local. El envejecimiento de los ganaderos de razas locales no es solo un problema de falta de transferencia generacional, sino que también puede suponer una actitud más negativa hacia la inversión en las actividades ganaderas y en una menor capacidad de adaptación a los cambios del sector. La capacidad de adaptación de los ganaderos es un factor crucial en la viabilidad de las razas locales. Las estrategias y políticas de conservación comunes deben incluir las variables comunes a la viabilidad de las razas manteniendo flexibilidad suficiente para adaptarse a las especificidades nacionales. Estas estrategias y políticas deberían ir más allá de compensación económica a los ganaderos de razas locales por la menor productividad de sus razas. Las herramientas para la toma de decisiones ayudan a generar una visión amplia de la conservación y desarrollo de las razas locales. Estas herramientas abordan el diseño de estrategias de conservación y desarrollo de forma sistemática y estructurada. En la tercera parte de la tesis usamos una de estas herramientas, el análisis DAFO (Debilidades, Amenazas, Fortalezas y Oportunidades), con este propósito, reconociendo que la conservación de RGADs depende de los ganaderos. Desarrollamos un análisis DAFO cuantitativo y lo aplicamos a trece razas locales de ganado vacuno de seis países europeos en el contexto del proyecto de colaboración mencionado anteriormente. El método tiene cuatro pasos: 1) la definición del sistema; 2) la identificación y agrupación de los factores influyentes; 3) la cuantificación de la importancia de dichos factores y 4) la identificación y priorización de estrategias. Identificamos los factores utilizando multitud de agentes (multi-stakeholder appproach). Una vez determinados los factores se agruparon en una estructura de tres niveles. La importancia relativa de los cada uno de los factores para cada raza fue determinada por grupos de expertos en RGADs de los países integrados en el citado proyecto. Finalmente, desarrollamos un proceso de cuantificación para identificar y priorizar estrategias. La estructura de agrupación de factores permitió analizar el problema de la conservación desde el nivel general hasta el concreto. La unión de análisis específicos de cada una de las razas en un análisis DAFO común permitió evaluar la adecuación de las estrategias a cada caso concreto. Identificamos un total de 99 factores. El análisis reveló que mientras los factores menos importantes son muy consistentes entre razas, los factores y estrategias más relevantes son muy heterogéneos. La idoneidad de las estrategias fue mayor a medida que estas se hacían más generales. A pesar de dicha heterogeneidad, los factores influyentes y estrategias más importantes estaban ligados a aspectos positivos (fortalezas y oportunidades) lo que implica que el futuro de estas razas es prometedor. Los resultados de nuestro análisis también confirmaron la gran relevancia del valor cultural de estas razas. Las factores internos (fortalezas y debilidades) más importantes estaban relacionadas con los sistemas de producción y los ganaderos. Las oportunidades más relevantes estaban relacionadas con el desarrollo y marketing de nuevos productos mientras que las amenazas más importantes se encontraron a la hora de vender los productos actuales. Este resultado implica que sería fructífero trabajar en la motivación y colaboración entre ganaderos así como, en la mejora de sus capacidades. Concluimos que las políticas comunes europeas deberían centrarse en aspectos generales y ser los suficientemente flexibles para adaptarse a las singularidades de los países y las razas. Como ya se ha mencionado, los ganaderos juegan un papel esencial en la conservación y desarrollo de las razas autóctonas. Por ello es relevante entender que implicación puede tener la heterogeneidad de los mismos en la viabilidad de una raza. En la cuarta parte de la tesis hemos identificado tipos de ganaderos con el fin de entender cómo la relación entre la variabilidad de sus características socioeconómicas, los perfiles de las ganaderías y las dinámicas de las mismas. El análisis se ha realizado en un contexto sociológico, aplicando los conceptos de capital cultural y económico. Las tipologías se han determinado en función de factores socioeconómicos y culturales indicadores del capital cultural y capital económico de un individuo. Nuestro objetivo era estudiar si la tipología socioeconómica de los ganaderos afecta al perfil de su ganadería y a las decisiones que toman. Entrevistamos a 85 ganaderos de la raza Avileña-Negra Ibérica (ANI) y utilizamos los resultados de dichas entrevistas para ilustrar y testar el proceso. Definimos los tipos de ganaderos utilizando un análisis de clúster jerarquizado con un grupo de variables canónicas que se obtuvieron en función de cinco factores socioeconómicos: el nivel de educación del ganadero, el año en que empezó a ser ganadero de ANI, el porcentaje de los ingresos familiares que aporta la ganadería, el porcentaje de propiedad de la tierra de la explotación y la edad del ganadero. La tipología de los ganaderos de ANI resultó ser más compleja que en el pasado. Los resultados indicaron que los tipos de ganaderos variaban en muchos aspectos socioeconómicos y en los perfiles de sus Integration of socioeconomic and genetic aspects involved in the conservation of animal genetic resources 9 ganaderías. Los tipos de ganaderos determinados toman diferentes decisiones en relación a la modificación del tamaño de su ganadería y a sus objetivos de selección. Por otro lado, reaccionaron de forma diferente ante un hipotético escenario de reducción de las compensaciones económicas que les planteamos. En este estudio hemos visto que el capital cultural y el económico interactúan y hemos explicado como lo hacen en los distintos tipos de ganaderos. Por ejemplo, los ganaderos que poseían un mayor capital económico, capital cultural formal y capital cultural adquirido sobre la raza, eran los ganaderos cuyos animales tenían una mayor demanda por parte de otros ganaderos, lo cual podría responder a su mayor prestigio social dentro de la raza. Uno de los elementos claves para el futuro de la raza es si este prestigio responde a una superioridad genética de las animales. Esto ocurriría si los ganaderos utilizaran las herramientas que tienen a su disposición a la hora de seleccionar animales. Los tipos de ganaderos identificados mostraron también claras diferencias en sus formas de colaboración y en su reacción a una hipotética variación de las compensaciones económicas. Aunque algunos tipos de ganaderos mostraron un bajo nivel de dependencia a estas compensaciones, la mayoría se manifestaron altamente dependientes. Por ello cualquier cambio drástico en la política de ayudas puede comprometer el desarrollo de las razas autóctonas. La adaptación las políticas de compensaciones económicas a la heterogeneidad de los ganaderos podría aumentar la eficacia de las mismas por lo que sería interesante explorar posibilidades a este respecto. Concluimos destacando la necesidad de desarrollar políticas que tengan en cuenta la heterogeneidad de los ganaderos. Finalmente abordamos el estudio de la estructura genética de poblaciones ganaderas. Las decisiones de los ganaderos en relación a la selección de sementales y su número de descendientes configuran la estructura demográfica y genética de las razas. En la actualidad existe un interés renovado por estudiar las estructuras poblacionales debido a la influencia potencial de su estratificación sobre la predicción de valores genómicos y/o los análisis de asociación a genoma completo. Utilizamos dos métodos distintos, un algoritmo de clústeres basados en teoría de grafos (GCA) y un algoritmo de clustering bayesiano (STRUCTURE) para estudiar la estructura genética de la raza ANI. Prestamos especial atención al efecto de la presencia de parientes cercanos en la población y de la diferenciación genética entre subpoblaciones sobre el análisis de la estructura de la población. En primer lugar evaluamos el comportamiento de los dos algoritmos en poblaciones simuladas para posteriormente analizar los genotipos para 17 microsatélites de 13343 animales de 57 ganaderías distintas de raza ANI. La ANI es un ejemplo de raza con relaciones complejas. Por otro lado, utilizamos el archivo de pedigrí de la raza para estudiar el flujo de genes, calculando, entre otras cosas, la contribución de cada ganadería a la constitución genética de la raza. En el caso de las poblaciones simuladas, cuando el FST entre subpoblaciones fue suficientemente alto, ambos algoritmos, GCA y STRUCTURE, identificaron la misma estructura genética independientemente de que existieran o no relaciones familiares. Por el contrario, cuando el grado de diferenciación entre poblaciones fue bajo, el STRUCTURE identificó la estructura familiar mientras que GCA no permitió obtener ningún resultado concluyente. El GCA resultó ser un algoritmo más rápido y eficiente para de inferir la estructura genética en poblaciones con relaciones complejas. Este algoritmo también puede ser usado para reducir el número de clústeres a testar con el STRUTURE. En cuanto al análisis de la población de ANI, ambos algoritmos describieron la misma estructura, lo cual sugiere que los resultados son robustos. Se identificaron tres subpoblaciones diferenciadas que pudieran corresponderse con tres linajes distintos. Estos linajes estarían directamente relacionados con las ganaderías que han tenido una mayor contribución a la constitución genética de la raza. Por otro lado, hay un conjunto muy numeroso de individuos con una mezcla de orígenes. La información molecular describe una estructura estratificada de la población que se corresponde con la evolución demográfica de la raza. Es esencial analizar en mayor profundidad la composición de este último grupo de animales para determinar cómo afecta a la variabilidad genética de la población de ANI. SUMMARY Summary Livestock sector is gradually dominated by intensive and specialized systems where the production environment is controlled and the production traits are the main criteria for the selection of species and breeds. In the meantime, the traditional use of domestic animals for draught work, clothes and manure has been replaced by industrial products. As a consequence of both these changes, the intensively selected breeds closely linked with high-input highoutput production systems have displaced many native breeds where the selection has practically ceased or been very mild. People are now more aware of the state of endangerment among the native breeds and the previously ignored values of livestock are gaining recognition. For some decades now, the economic, socio-cultural, environmental and food security function of livestock breeds have been accepted worldwide and their loss has been recognized as a major problem. Therefore, the conservation of farm animal genetic resources (FAnGR) has been recommended. The conservation of FAnGR is controversial due to the complexity of the evaluation of its functions. This evaluation is difficult due to the nature of FAnGR both as private and public good. As some economists have highlighted, livestock animals are private goods, however, they are also public goods by their functions. Therefore, there is a need to increase the knowledge about the value of all livestock functions since to support the decision-making for the sustainable conservation and breeding of livestock. This is not straightforward since the relative importance of livestock functions depends on time, place, species and breed. Since livestock play a variety of roles, their production is driven by interrelated and everchanging economic, technical, environmental, social, cultural and political elements involving an enormous range of stakeholders. Not only FAnGR functions but also the importance of factors affecting the development and conservation of FAnGR can be very different across geographical areas. Furthermore, heterogeneity can be found even within breeds. Local breeds are nowadays raised by highly diverse farmers in equally diverse farms. It is quite reasonable to think that farmer is the major actor in the in situ conservation of livestock breeds. Thus, there is a need to understand the farmers’ motivations, decision making processes and the impact of their decisions on the genetic structure of breeds. In this PhD thesis we explore different social, economic and genetic aspects involved in the conservation of local cattle breeds, i.e. FAnGR, in Europe seeking to contribute to the scientific understanding of this complex issue. We aim to achieve a comprehensive view of the processes involved in the conservation and development of local cattle breeds and have made special efforts in discussing the implications of the research results in this respect. The final outcome of the thesis is to illustrate how quantitative methods can be exploited in designing and establishing sound strategies and programmes for the conservation and development of local livestock breeds. Firstly we explored the public non-market attributes of the total economic value (TEV) of livestock, using the Spanish Alistana-Sanabresa (AS) cattle breed as a case study. Total economic value of any good comprises both use and non-use components, where the latter include option, bequest and existence values. For livestock, the direct use values are mainly stemming from production outputs. Indirect use values relate to the role of livestock as a maintainer of rural culture and landscape. The option value is related to the potential use of livestock, the bequest values relate to the value associated with the inheritance of the resources to future generation and the existence values relate to the utility perceived by people from knowing that specific resources exist. We aimed to determine the relative importance of the non-market components of the TEV of the AS breed, the socio-economic variables that influence how people value the different components of TEV and to assess the implications of the Spanish national conservation strategy for the AS breed. To do so, we used a choice experiment (CE) approach and applied the technique to assess people’s willingness to pay (WTP) for the conservation of AS breed. The use of CE allows the valuation of the individual components of TEV for a given good. We analysed the choice data using a random parameter logit (RPL) model. AS breed was found to have a significant public good value. Its most important values were related to the indirect use value due to the maintenance of Zamorian culture and the existence value (both represent over 80% of its TEV). There were several socioeconomic variables influencing people’s valuation of the public service of the breed. In the case of AS breed, the place of living (city or rural area), having seen animals of the breed, having eaten breed products and the respondents’ attitude towards economic development – environment conflicts do influence people’s WTP for AS conservation. We also found that people do not have a complete picture of all the functions and roles that AS breed as AnGR. Therefore, the actions for increasing awareness of AS should go to that direction. The farmers will need incentives to exploit some of the public goods values and maintain the breed population size at socially desirable levels. One such mechanism could be related to the development of agritourism, which would enhance the private good value and provide an important addition to the conservation and utilisation strategy. However, the farmers need a serious evaluation on how to invest in niche product development or how to improve product quality and brand recognition. Using the understanding on the importance of the public function of local cattle we tried to depict the current diversity regarding technical, economic and social factors found in local cattle farming across Europe. To do so we focused in an international collaborative project on the case of fifteen local cattle breeds in eight European countries. We investigated the variation among the countries to detect the common key elements, which affect the viability of local breeds. We surveyed with interviews a total of 355 farms across the fifteen breeds. We used the planned herd size changes by the farmer as an indicator of breed viability. The questionnaire included several economic, technical and social aspects with potential influence on breeds’ demographic trends. We analysed the data using multivariate statistical techniques, such as discriminat analysis and logistic regression. The factors affecting a local breed’s viability were highly heterogeneous across Europe. In some countries, farmers did not recognise any high social value attached to keeping a local cattle breed. Hence there is a need to develop communication programmes across EU countries making people aware about the diversity and importance of values associated to raising local breeds. The countries were also very variable regarding the importance of local markets and the percentage of farm land owned by the farmers. Despite the country specificities, there were also common factors affecting the breed viability across Europe. The factors were from different grounds, from social, such as the age of the farmer and the social appreciation of their work, to technicalorganizational, such as the farmers’ attitude to collaborating with each other. The heterogeneity found reflects the variation in breeding systems and production environment (in the socioeconomic, technical and ecological sense) present in Europe. Therefore, caution should be taken in implementing common policies at the country level. Variability could also be rather high within countries due to breed specificities. Therefore, the national policies should be flexible to adapt to the specificities. The variables significantly associated with breed viability should be positively incorporated in the conservation strategies, and considered in developing common and/or national policies. The strategy preparation and policy planning should go beyond the provision of a general economic support to compensate farmers for the lower profitability of local breeds. Of particular interest is the observation that the opportunity for farmer collaboration and the appreciation by the society of the cultural, environmental and social role of local cattle farming were positively associated with the breed survival. In addition, farmer's high age is not only a problem of poor generation transfer but it is also a problem because it might lead to a lower attitude to investing in farming activities and to a lower ability to adapt to environment changes. The farmers’ adaptation capability may be a key point for the viability of local breeds. Decision making tools can help to get a comprehensive view on the conservation and development of local breeds. It allows us to use a systematic and structured approach for identifying and prioritizing conservation and development strategies. We used SWOT (Strengths, Weaknesses Opportunities and Threats) analysis for this purpose and recognized that many conservation and development projects rely on farmers. We developed a quantified SWOT method and applied it in the aforementioned collaborative research to a set of thirteen cattle breeds in six European countries. The method has four steps: definition of the system, identification and grouping of the driving factors, quantification of the importance of driving factors and identification and prioritization of the strategies. The factors were determined following a multi-stakeholder approach and grouped with a three level structure. FAnGR expert groups ranked the factors and a quantification process was implemented to identify and prioritize strategies. The structure of the SWOT analysis allowed analyzing the conservation problem from general down to specific perspectives. Joining breed specific analyses into a common SWOT analysis permitted comparison of breed cases across countries. We identified 99 driving factors across breeds. The across breed analysis revealed that irrelevant factors were consistent. There was high heterogeneity among the most relevant factors and strategies. The strategies increased eligibility as they lost specificity. Although the situation was very heterogeneous, the most promising factors and strategies were linked to the positive aspects (Strengths and Opportunities). Therefore, the future of the studied local breed is promising. The results of our analysis also confirmed the high relevance of the cultural value of the breeds. The most important internal factors (strengths and weaknesses) were related farmers and production systems. The most important opportunities were found in developing and marketing new products, while the most relevant threats were found in selling the current conventional products. In this regard, it should be fruitful to work on farmers’ motivation, collaboration, and capacity building. We conclude that European policies should focus on general aspects and be flexible enough to be adapted to the country and breed specificities. As mentioned, farmers have a key role in the conservation and development of a local cattle breed. Therefore, it is very relevant to understand the implications of farmer heterogeneity within a breed for its viability. In the fourth part of the thesis, we developed a general farmer typology to help analyzing the relations between farmer features and farm profiles, herd dynamics and farmers’ decision making. In the analysis we applied and used the sociological framework of economic and cultural capital and studied how the determined farmer types were linked to farm profiles and breeding decisions, among others. The typology was based on measurable socioeconomic factors indicating the economic and cultural capital of farmers. A group of 85 farmers raising the Spanish Avileña-Negra Ibérica (ANI) local cattle breed was used to illustrate and test the procedure. The farmer types were defined by a hierarchical cluster analysis with a set of canonical variables derived from the following five the socioeconomic factors: the formal educational level of the farmer, the year the farmer started keeping the ANI breed, the percentage of the total family income covered by the farm, the percentage of the total farm land owned by the farmer and the farmer’s age. The present ANI farmer types were much more complex than what they were in the past. We found that the farmer types differed in many socioeconomic aspects and in the farms profile. Furthermore, the types also differentiate farmers with respect to decisions about changing the farm size, breeding aims and stated reactions towards hypothetical subsidy variation. We have verified that economic and cultural capitals are not independent and further showed how they are interacting in the different farmer types. The farmers related to the types with high economic, institutionalized and embodied cultural capitals had a higher demand of breeding animals from others farmers of the breed, which may be related to the higher social prestige within the breed. One of the key implications of this finding for the future of the breed is whether or not the prestige of farmers is related to genetic superiority of their animals, what is to say, that it is related with a sound use of tools that farmers have available to make selection decisions. The farmer types differed in the form of collaboration and in the reactions to the hypothetical variation in subsidies. There were farmers with low dependency on subsidies, while most of them are highly dependent on subsidies. Therefore, any drastic change in the subsidy programme might have influence on the development of local breeds. The adaptation of these programme to the farmers’ heterogeneity might increase its efficacy, thus it would be interesting to explore ways of doing it. We conclude highlighting the need to have a variety of policies, which take into account the heterogeneity among the farmers. To finish we dealt with the genetic structure of livestock populations. Farmers’ decisions on the breeding animals and their progeny numbers shape the demographic and genetic structure of the breeds. Nowadays there is a renovated interest in studying the population structure since it can bias the prediction of genomic breeding values and genome wide association studies. We determined the genetic structure of ANI breed using two different methods, a graphical clustering algorithm (GCA) and a Bayesian clustering algorithm (STRUCTURE) were used. We paid particular attention to the influence that the presence of closely related individuals and the genetic differentiation of subpopulations may have on the inferences about the population structure. We first evaluated the performance of the algorithms in simulated populations. Then we inferred the genetic structure of the Spanish cattle breed ANI analysing a data set of 13343 animals (genotyped for 17 microsatellites) from 57 herds. ANI breed is an example of a population with complex relationships. We used the herdbook to study the gene flow, estimation among other things, the contribution of different herds to the genetic composition of the ANI breed. For the simulated scenarios, when FST among subpopulations was sufficiently high, both algorithms consistently inferred the correct structure regardless of the presence of related individuals. However, when the genetic differentiation among subpopulations was low, STRUCTURE identified the family based structure while GCA did not provide any consistent picture. The GCA was a fast and efficient method to infer genetic structure to determine the hidden core structure of a population with complex history and relationships. GCA could also be used to narrow down the number of clusters to be tested by STRUCTURE. Both, STRUCTURE and GCA describe a similar structure for the ANI breed suggesting that the results are robust. ANI population was found to have three genetically differentiated clusters that could correspond to three genetic lineages. These are directly related to the herds with a major contribution to the breed. In addition, ANI breed has also a large pool made of individuals with an admixture of origins. The genetic structure of ANI, assessed by molecular information, shows a stratification that corresponds to the demographic evolution of the breed. It will be of great importance to learn more about the composition of the pool and study how it is related to the existing genetic variability of the breed.

Nutrition and physical activity programs for obesity treatment (PRONAF study). methodological approach of the project

Este trabajo aborda la metodología seguida para llevar a cabo el proyecto de investigación PRONAF (Clinical Trials Gov.: number NCT01116856.) Background: At present, scientific consensus exists on the multifactorial etiopatogenia of obesity. Both professionals and researchers agree that treatment must also have a multifactorial approach, including diet, physical activity, pharmacology and/or surgical treatment. These two last ones should be reserved for those cases of morbid obesities or in case of failure of the previous ones. The aim of the PRONAF study is to determine what type of exercise combined with caloric restriction is the most appropriate to be included in overweigth and obesity intervention programs, and the aim of this paper is to describe the design and the evaluation methods used to carry out the PRONAF study. Methods/design: One-hundred nineteen overweight (46 males) and 120 obese (61 males) subjects aged 18–50 years were randomly assigned to a strength training group, an endurance training group, a combined strength + endurance training group or a diet and physical activity recommendations group. The intervention period was 22 weeks (in all cases 3 times/wk of training for 22 weeks and 2 weeks for pre and post evaluation). All subjects followed a hypocaloric diet (25-30% less energy intake than the daily energy expenditure estimated by accelerometry). 29–34% of the total energy intake came from fat, 14–20% from protein, and 50–55% from carbohydrates. The mayor outcome variables assesed were, biochemical and inflamatory markers, body composition, energy balance, physical fitness, nutritional habits, genetic profile and quality of life. 180 (75.3%) subjects finished the study, with a dropout rate of 24.7%. Dropout reasons included: personal reasons 17 (28.8%), low adherence to exercise 3 (5.1%), low adherence to diet 6 (10.2%), job change 6 (10.2%), and lost interest 27 (45.8%). Discussion: Feasibility of the study has been proven, with a low dropout rate which corresponds to the estimated sample size. Transfer of knowledge is foreseen as a spin-off, in order that overweight and obese subjects can benefit from the results. The aim is to transfer it to sports centres. Effectiveness on individual health-related parameter in order to determine the most effective training programme will be analysed in forthcoming publications.