Structuring of Ranked Models

Prognostic procedures can be based on ranked linear models. Ranked regression type models are designed on the basis of feature vectors combined with set of relations defined on selected pairs of these vectors. Feature vectors are composed of numerical results of measurements on particular objects or events. Ranked relations defined on selected pairs of feature vectors represent additional knowledge and can reflect experts' opinion about considered objects. Ranked models have the form of linear transformations of feature vectors on a line which preserve a given set of relations in the best manner possible. Ranked models can be designed through the minimization of a special type of convex and piecewise linear (CPL) criterion functions. Some sets of ranked relations cannot be well represented by one ranked model. Decomposition of global model into a family of local ranked models could improve representation. A procedures of ranked models decomposition is described in this paper.

A Case-Only Genome-wide Association Study of Gender- and Age-specific Risk Markers for Childhood Leukemia

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.

Insights into Nonpilus Adhesin Functionality and the Molecular Determinants of Nontypeable Haemophilus influenzae Colonization

Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.

After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.

The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.

The (Un)Folding of Multidomain Proteins Through the Lens of Single-molecule Force-spectroscopy and Computer Simulation

Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.

In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.

The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.

Caracterização genotípica de doentes portugueses com patologias associadas ao complexo multienzimático lisossomal: sialidose e galactosialidose

Dissertação de mestrado em Biologi apresentada à Faculdade de Ciências da Universidade do Porto, 2008

KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA

The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.

Flow around a hemisphere-cylinder at high angle of attack and low Reynolds number. Part II: POD and DMD applied to reduced domains

Three-dimensional direct numerical simulations (DNS) have been performed on a finite-size hemispherecylinder model at angle of attack AoA = 20◦ and Reynolds numbers Re = 350 and 1000. Under these conditions, massive separation exists on the nose and lee-side of the cylinder, and at both Reynolds numbers the flow is found to be unsteady. Proper orthogonal decomposition (POD) and dynamic mode decomposition (DMD) are employed in order to study the primary instability that triggers unsteadiness at Re = 350. The dominant coherent flow structures identified at the lower Reynolds number are also found to exist at Re = 1000; the question is then posed whether the flow oscillations and structures found at the two Reynolds numbers are related. POD and DMD computations are performed using different subdomains of the DNS computational domain. Besides reducing the computational cost of the analyses, this also permits to isolate spatially localized oscillatory structures from other, more energetic structures present in the flow. It is found that POD and DMD are in general sensitive to domain truncation and noneducated choices of the subdomain may lead to inconsistent results. Analyses at Re = 350 show that the primary instability is related to the counter rotating vortex pair conforming the three-dimensional afterbody wake, and characterized by the frequency St ≈ 0.11, in line with results in the literature. At Re = 1000, vortex-shedding is present in the wake with an associated broadband spectrum centered around the same frequency. The horn/leeward vortices at the cylinder lee-side, upstream of the cylinder base, also present finite amplitude oscillations at the higher Reynolds number. The spatial structure of these oscillations, described by the POD modes, is easily differentiated from that of the wake oscillations. Additionally, the frequency spectra associated with the lee-side vortices presents well defined peaks, corresponding to St ≈ 0.11 and its few harmonics, as opposed to the broadband spectrum found at the wake.

Molecular analysis of exons 8, 9 and 10 of the fibroblast growth factor receptor 2 (FGFR2) gene in two families with index cases of Apert Syndrome

Introduction: Apert syndrome (AS) is a craniosynostosis condition caused by mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) gene. Clinical features include cutaneous and osseous symmetric syndactily in hands and feet, with variable presentations in bones, brain, skin and other internal organs. Methods: Members of two families with an index case of Apert Syndrome were assessed to describe relevant clinical features and molecular analysis (sequencing and amplification) of exons 8, 9 and 10 of FGFR2 gen. Results: Family 1 consists of the mother, the index case and half -brother who has a cleft lip and palate. In this family we found a single FGFR2 mutation, S252W, in the sequence of exon 8. Although mutations were not found in the study of the patient affected with cleft lip and palate, it is known that these diseases share signaling pathways, allowing suspected alterations in shared genes. In the patient of family 2, we found a sequence variant T78.501A located near the splicing site, which could interfere in this process, and consequently with the protein function.

A case-only genome-wide association study of gender- and age-specific risk markers for childhood leukemia

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. ^ Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5 ) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. ^ The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.^

Ruolo di Glutaredossina-2 nella trasformazione tumorale oncocitaria

Gli oncocitomi sono tumori epiteliali caratterizzati da un accumulo di mitocondri strutturalmente e funzionalmente compromessi, a prognosi generalmente benigna. Le cause genetiche della trasformazione oncocitaria sono tuttora sconosciute; pertanto, lo studio di oncocitomi in contesti familiari sindromici è utile nella ricerca dei determinanti genetici predisponenti il fenotipo. Diversi membri di una famiglia affetta da sindrome dell’iperparatiroidismo con tumore della mandibola (HPT-JT), dovuta ad un'ampia delezione in CDC73, hanno mostrato recidiva di tumori paratiroidei oncocitari. Il sequenziamento dell’esoma ha escluso mutazioni private della famiglia; all'interno della delezione ereditata, tuttavia, sono stati individuati elementi regolatori del gene glutaredossina 2 (GLRX2), codificante un'isoforma mitocondriale deputata alla deglutationilazione proteica reversibile -modificazione modulante l’attività di numerosi target- il cui ruolo nel cancro non è noto. La proteina è risultata assente in tutti i tumori e dimezzata nei tessuti sani dei soggetti. Per indagare se la sua assenza alteri la deglutationilazione proteica predisponendo al fenotipo oncocitario, sono stati generati modelli cellulari TPC1 e HCT116 GLRX2 KO in cui sono stati riscontrati un ridotto tasso proliferativo ed un'alterata glutationilazione proteica, particolarmente in seguito a stress ossidativo. Un esperimento pilota in vivo ha mostrato cellule KO oncocitoidi, con mitocondri morfologicamente alterati, suggerendo che l’alterazione redox innescata dall’assenza di GLRX2 possa indurre una disfunzione metabolica mitocondriale tale da mimare quelle osservate negli oncocitomi. L’analisi proteomica ha individuato diversi target di glutationilazione nei campioni KO identificando proteine del ciclo di Krebs e della catena respiratoria mitocondriale. In particolare, una marcata glutationilazione del complesso della piruvato deidrogenasi (PDHc) è stata correlata ad una ridotta sintesi di ATP dipendente da piruvato. Considerando l'importanza dello stress ossidativo nella fisiopatologia del cancro ed il ruolo del glutatione nella risposta antiossidante, GLRX2 rappresenta un potenziale candidato nella regolazione del metabolismo ossidativo nelle cellule tumorali esposte allo stress e nella modulazione del fenotipo tumorale.

Computational methods for the analysis of protein structure and function

The vast majority of known proteins have not yet been experimentally characterized and little is known about their function. The design and implementation of computational tools can provide insight into the function of proteins based on their sequence, their structure, their evolutionary history and their association with other proteins. Knowledge of the three-dimensional (3D) structure of a protein can lead to a deep understanding of its mode of action and interaction, but currently the structures of <1% of sequences have been experimentally solved. For this reason, it became urgent to develop new methods that are able to computationally extract relevant information from protein sequence and structure. The starting point of my work has been the study of the properties of contacts between protein residues, since they constrain protein folding and characterize different protein structures. Prediction of residue contacts in proteins is an interesting problem whose solution may be useful in protein folding recognition and de novo design. The prediction of these contacts requires the study of the protein inter-residue distances related to the specific type of amino acid pair that are encoded in the so-called contact map. An interesting new way of analyzing those structures came out when network studies were introduced, with pivotal papers demonstrating that protein contact networks also exhibit small-world behavior. In order to highlight constraints for the prediction of protein contact maps and for applications in the field of protein structure prediction and/or reconstruction from experimentally determined contact maps, I studied to which extent the characteristic path length and clustering coefficient of the protein contacts network are values that reveal characteristic features of protein contact maps. Provided that residue contacts are known for a protein sequence, the major features of its 3D structure could be deduced by combining this knowledge with correctly predicted motifs of secondary structure. In the second part of my work I focused on a particular protein structural motif, the coiled-coil, known to mediate a variety of fundamental biological interactions. Coiled-coils are found in a variety of structural forms and in a wide range of proteins including, for example, small units such as leucine zippers that drive the dimerization of many transcription factors or more complex structures such as the family of viral proteins responsible for virus-host membrane fusion. The coiled-coil structural motif is estimated to account for 5-10% of the protein sequences in the various genomes. Given their biological importance, in my work I introduced a Hidden Markov Model (HMM) that exploits the evolutionary information derived from multiple sequence alignments, to predict coiled-coil regions and to discriminate coiled-coil sequences. The results indicate that the new HMM outperforms all the existing programs and can be adopted for the coiled-coil prediction and for large-scale genome annotation. Genome annotation is a key issue in modern computational biology, being the starting point towards the understanding of the complex processes involved in biological networks. The rapid growth in the number of protein sequences and structures available poses new fundamental problems that still deserve an interpretation. Nevertheless, these data are at the basis of the design of new strategies for tackling problems such as the prediction of protein structure and function. Experimental determination of the functions of all these proteins would be a hugely time-consuming and costly task and, in most instances, has not been carried out. As an example, currently, approximately only 20% of annotated proteins in the Homo sapiens genome have been experimentally characterized. A commonly adopted procedure for annotating protein sequences relies on the "inheritance through homology" based on the notion that similar sequences share similar functions and structures. This procedure consists in the assignment of sequences to a specific group of functionally related sequences which had been grouped through clustering techniques. The clustering procedure is based on suitable similarity rules, since predicting protein structure and function from sequence largely depends on the value of sequence identity. However, additional levels of complexity are due to multi-domain proteins, to proteins that share common domains but that do not necessarily share the same function, to the finding that different combinations of shared domains can lead to different biological roles. In the last part of this study I developed and validate a system that contributes to sequence annotation by taking advantage of a validated transfer through inheritance procedure of the molecular functions and of the structural templates. After a cross-genome comparison with the BLAST program, clusters were built on the basis of two stringent constraints on sequence identity and coverage of the alignment. The adopted measure explicity answers to the problem of multi-domain proteins annotation and allows a fine grain division of the whole set of proteomes used, that ensures cluster homogeneity in terms of sequence length. A high level of coverage of structure templates on the length of protein sequences within clusters ensures that multi-domain proteins when present can be templates for sequences of similar length. This annotation procedure includes the possibility of reliably transferring statistically validated functions and structures to sequences considering information available in the present data bases of molecular functions and structures.

Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae

A ribosome association factor (AF) was isolated from the yeast Sacchharomyces cerevisiae. Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-Bromoindolenine (BNPS-skatole). This sequence has a 86% identity to the product of the single-copy S. cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function. Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable. These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process.

The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ‟closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.

Protein binding and signaling properties of RIN1 suggest a unique effector function

Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the “effector domain” of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL–SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.