Survival Prediction in Head and Neck Cancer: Impact of Tumor and Patient Specific Characteristics

Head and neck cancer accounts for almost 5% of all malignant tumors in the Netherlands. The most up‐todate Dutch Cancer Registry (NCR) database from 2009 reported 2878 new patients with an invasive carcinoma of the lip, oral cavity, pharynx and larynx (general incidence 17:100.000). In this thesis we focus on head and neck squamous cell carcinoma (HNSCC). Head and neck squamous cell carcinomas originate from the mucosal lining of the upper aero‐digestive tract. Tobacco and alcohol are irritants to this mucosal lining and therefore form major risk factors for the genesis of malignant epithelial tumors. Other reported etiological factors are malnutrition, viral factors (Epstein Barr virus and Human Papilloma virus), genetic predispositions and occupational hazards

Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome

Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio- generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism’s biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes. IMPORTANCE Studying whole-genome sequences has become an important aspect of biological research. The advent of nextgeneration sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBiogenerated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome sanalyses to facilitate functional studies into an organism’s biology

The 73 S ribosome of Neurospora crassa is the native mitochondrial ribosome

The effect of the inclusion of EDTA and of heparin, in media used in the isolation of mitochondria, on the mitochindrial previous termribosomenext term has been investigated. 1. 1. Mitochondria isolated from previous termNeurospora crassanext term in the presence of EDTA contain only a single type of monomeric previous termribosome, viz. 73next term S. 2. 2. Mitochondria isolated in the presence of Mg2+ contain both 79-S and previous term73next term-S monomeric previous termribosomes.next term The heterogeneity of the previous termribosomesnext term was demonstrated by (a) ultracentrifugation on sucrose gradients, (b) electron microscopy, (c) immunoprecipitation with antibodies against mitochondrial previous term73next term-S and 79-S cytoplasmic previous termribosomes,next term (d) gel electrophoresis of high and low molecular weight RNAs. 3. 3. Inclusion of heparin in all media used for the isolation of mitochondria and previous termribosomesnext term resulted in (a) dissociation of previous term73next term-S mitochondrial previous termribosomesnext term into 50-S and 37-S subunits; (b) stabilization of 79-S cytoplasmic previous termribosomes;next term (c) in the case of mitochondria isolated in the presence of Mg2+ containing both previous term73next term-S and 79-S previous termribosomes,next term heparin causes the selective dissociation of the previous term73next term S monosome to yield previous termribosomesnext term containing only a single monomeric previous termribosomenext term type, viz. 79 S. 4. 4. It is concluded that (a) the 79-S previous termribosomesnext term present in mitochondria isolated in the presence of Mg2+ are contaminating cytoplasmic previous termribosomes,next term (b) the previous term73next term-S previous termribosomesnext term are the real functional mitochondrial previous termribosomes of Neurospora crassanext term

Adaptation and validation of the Dutch version of the nasal obstruction symptom evaluation (NOSE) scale

The nasal obstruction symptom evaluation (NOSE) scale is a validated disease-specific, self-completed questionnaire for the assessment of quality of life related to nasal obstruction. The aim of this study was to validate the Dutch (NL-NOSE) questionnaire. A prospective instrument validation study was performed in a tertiary academic referral center. Guidelines for the cross-cultural adaptation process from the original English language scale into a Dutch language version were followed. Patients undergoing functional septoplasty or septorhinoplasty and asymptomatic controls completed the questionnaire both before and 3 months after surgery to test reliability and validity. Additionally, we explored the possibility to reduce the NOSE scale even further using graded response models. 129 patients and 50 controls were included. Internal consistency (Cronbach’s alpha 0.82) and test–retest reliability (intraclass correlation coefficient 0.89) were good. The instrument showed excellent between-group discrimination (Mann–Whitney U = 85, p < 0.001) and high response sensitivity to change (Wilcoxon rank p < 0.001). The NL-NOSE correlated well with the score on a visual analog scale measuring the subjective sensation of nasal obstruction, with exception of item 4 (trouble sleeping). Item 4 provided the least information to the total sc

Genome sequence and analysis of the tuber crop potato

Potato (Solanum tuberosum L.) is the world’s most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital cro

FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird's-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA](4), a microsatellite that also forms part of the pericentromeres, together with [GA](8), [GATA](4) and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer regio

Prediction of survival with alternative modeling techniques using pseudo values

Background: The use of alternative modeling techniques for predicting patient survival is complicated by the fact that some alternative techniques cannot readily deal with censoring, which is essential for analyzing survival data. In the current study, we aimed to demonstrate that pseudo values enable statistically appropriate analyses of survival outcomes when used in seven alternative modeling techniques. Methods: In this case study, we analyzed survival of 1282 Dutch patients with newly diagnosed Head and Neck Squamous Cell Carcinoma (HNSCC) with conventional Kaplan-Meier and Cox regression analysis. We subsequently calculated pseudo values to reflect the individual survival patterns. We used these pseudo values to compare recursive partitioning (RPART), neural nets (NNET), logistic regression (LR) general linear models (GLM) and three variants of support vector machines (SVM) with respect to dichotomous 60-month survival, and continuous pseudo values at 60 months or estimated survival time. We used the area under the ROC curve (AUC) and the root of the mean squared error (RMSE) to compare the performance of these models using bootstrap validation. Results: Of a total of 1282 patients, 986 patients died during a median follow-up of 66 months (60-month survival: 52% [95% CI: 50%-55%]). The L

Genome bioinformatics of tomato and potato

In the past two decades genome sequencing has developed from a laborious and costly technology employed by large international consortia to a widely used, automated and affordable tool used worldwide by many individual research groups. Genome sequences of many food animals and crop plants have been deciphered and are being exploited for fundamental research and applied to improve their breeding programs. The developments in sequencing technologies have also impacted the associated bioinformatics strategies and tools, both those that are required for data processing, management, and quality control, and those used for interpretation of the data. This thesis focuses on the application of genome sequencing, assembly and annotation to two members of the Solanaceae family, tomato and potato. Potato is the economically most important species within the Solanaceae, and its tubers contribute to dietary intake of starch, protein, antioxidants, and vitamins. Tomato fruits are the second most consumed vegetable after potato, and are a globally important dietary source of lycopene, beta-carotene, vitamin C, and fiber. The chapters in this thesis document the generation, exploitation and interpretation of genomic sequence resources for these two species and shed light on the contents, structure and evolution of their genomes. Chapter 1introduces the concepts of genome sequencing, assembly and annotation, and explains the novel genome sequencing technologies that have been developed in the past decade. These so-called Next Generation Sequencing platforms display considerable variation in chemistry and workflow, and as a consequence the throughput and data quality differs by orders of magnitude between the platforms. The currently available sequencing platforms produce a vast variety of read lengths and facilitate the generation of paired sequences with an approximately fixed distance between them. The choice of sequencing chemistry and platform combined with the type of sequencing template demands specifically adapted bioinformatics for data processing and interpretation. Irrespective of the sequencing and assembly strategy that is chosen, the resulting genome sequence, often represented by a collection of long linear strings of nucleotides, is of limited interest by itself. Interpretation of the genome can only be achieved through sequence annotation – that is, identification and classification of all functional elements in a genome sequence. Once these elements have been annotated, sequence alignments between multiple genomes of related accessions or species can be utilized to reveal the genetic variation on both the nucleotide and the structural level that underlies the difference between these species or accessions. Chapter 2describes BlastIf, a novel software tool that exploits sequence similarity searches with BLAST to provide a straightforward annotation of long nucleotide sequences. Generally, two problems are associated with the alignment of a long nucleotide sequence to a database of short gene or protein sequences: (i) the large number of similar hits that can be generated due to database redundancy; and (ii) the relationships implied between aligned segments within a hit that in fact correspond to distinct elements on the sequence such as genes. BlastIf generates a comprehensible BLAST output for long nucleotide sequences by reducing the number of similar hits while revealing most of the variation present between hits. It is a valuable tool for molecular biologists who wish to get a quick overview of the genetic elements present in a newly sequenced segment of DNA, prior to more elaborate efforts of gene structure prediction and annotation. In Chapter 3 a first genome-wide comparison between the emerging genomic sequence resources of tomato and potato is presented. Large collections of BAC end sequences from both species were annotated through repeat searches, transcript alignments and protein domain identification. In-depth comparisons of the annotated sequences revealed remarkable differences in both gene and repeat content between these closely related genomes. The tomato genome was found to be more repetitive than the potato genome, and substantial differences in the distribution of Gypsy and Copia retrotransposable elements as well as microsatellites were observed between the two genomes. A higher gene content was identified in the potato sequences, and in particular several large gene families including cytochrome P450 mono-oxygenases and serine-threonine protein kinases were significantly overrepresented in potato compared to tomato. Moreover, the cytochrome P450 gene family was found to be expanded in both tomato and potato when compared to Arabidopsis thaliana, suggesting an expanded network of secondary metabolic pathways in the Solanaceae. Together these findings present a first glimpse into the evolution of Solanaceous genomes, both within the family and relative to other plant species. Chapter 4explores the physical and genetic organization of tomato chromosome 6 through integration of BAC sequence analysis, High Information Content Fingerprinting, genetic analysis, and BAC-FISH mapping data. A collection of BACs spanning substantial parts of the short and long arm euchromatin and several dispersed regions of the pericentrometric heterochromatin were sequenced and assembled into several tiling paths spanning approximately 11 Mb. Overall, the cytogenetic order of BACs was in agreement with the order of BACs anchored to the Tomato EXPEN 2000 genetic map, although a few striking discrepancies were observed. The integration of BAC-FISH, sequence and genetic mapping data furthermore provided a clear picture of the borders between eu- and heterochromatin on chromosome 6. Annotation of the BAC sequences revealed that, although the majority of protein-coding genes were located in the euchromatin, the highly repetitive pericentromeric heterochromatin displayed an unexpectedly high gene content. Moreover, the short arm euchromatin was relatively rich in repeats, but the ratio of Gypsy and Copia retrotransposons across the different domains of the chromosome clearly distinguished euchromatin from heterochromatin. The ongoing whole-genome sequencing effort will reveal if these properties are unique for tomato chromosome 6, or a more general property of the tomato genome. Chapter 5presents the potato genome, the first genome sequence of an Asterid. To overcome the problems associated with genome assembly due tothe high level of heterozygosity that is observed in commercial tetraploid potato varieties, a homozygous doubled-monoploid potato clone was exploited to sequence and assemble 86% of the 844 Mb genome. This potato reference genome sequence was complemented with re-sequencing of aheterozygous diploid clone, revealing the form and extent of sequence polymorphism both between different genotypes and within a single heterozygous genotype. Gene presence/absence variants and other potentially deleterious mutations were found to occur frequently in potato and are a likely cause of inbreeding depression. Annotation of the genome was supported by deep transcriptome sequencing of both the doubled-monoploid and the heterozygous potato, resulting in the prediction of more than 39,000 protein coding genes. Transcriptome analysis provided evidence for the contribution of gene family expansion, tissue specific expression, and recruitment of genes to new pathways to the evolution of tuber development. The sequence of the potato genome has provided new insights into Eudicot genome evolution and has provided a solid basis for the elucidation of the evolution of tuberisation. Many traits of interest to plant breeders are quantitative in nature and the potato sequence will simplify both their characterization and deployment to generate novel cultivars. The outstanding challenges in plant genome sequencing are addressed in Chapter 6. The high concentration of repetitive elements and the heterozygosity and polyploidy of many interesting crop plant species currently pose a barrier for the efficient reconstruction of their genome sequences. Nonetheless, the completion of a large number of new genome sequences in recent years and the ongoing advances in sequencing technology provide many excitingopportunities for plant breeding and genome research. Current sequencing platforms are being continuously updated and improved, and novel technologies are being developed and implemented in third-generation sequencing platforms that sequence individual molecules without need for amplification. While these technologies create exciting opportunities for new sequencing applications, they also require robust software tools to process the data produced through them efficiently. The ever increasing amount of available genome sequences creates the need for an intuitive platform for the automated and reproducible interrogation of these data in order to formulate new biologically relevant questions on datasets spanning hundreds or thousands of genome sequences. </p

USING BALD EAGLES TO MONITOR HYDROELECTRIC PROJECTS LISCENSE REQUIREMENTS ALONG THE AU SABLE, MANISTEE AND MUSKEGON RIVER, MICHIGAN

Consumers Energy operated hydroelectric projects located along the Au Sable, Manistee, and Muskegon Rivers underwent environmental studies in the late 1980s and early 1990s as part of the Federal Energy Regulatory Commission relicensing. One of the questions posed during these studies was, would passage of Great Lakes\u27 fishes over barrier dams along these rivers cause detrimental impacts to sensitive wildlife species. Relicensing also required that the operation of all hydroelectric projects on the Au Sable, Manistee, and Muskegon rivers be maintained as run-of-river. Bald eagles (Haliaeetus leucocephalus) were chosen as a biomonitor. This risk assessment included calculating new hazard quotients (HQs) from toxic reference values (TRVs) to determine if it was safe for inland wildlife to be exposed to anadromous fish allowed past barrier dams. A risk assessment was conducted for contaminants of PCBs, DDT, dieldrin, TCDD-EQ and mercury in a fish diet comparing exposure in Great Lakes\u27 accessible regions to interior regions of the Au Sable, Manistee and Muskegon rivers, using fish collected after 1990. The bald eagle population nesting in the study area increased throughout the study period. Mean mercury was greater in fishes in inland than Great Lakes influenced. Mean total PCBs, sum DDT and dieldrin were greater in Great Lakes influenced areas. Total PCBs and sum DDT were greater in Great Lakes influenced nesting areas than inland nesting areas. TCDD-EQ was the limiting factor for bald eagle reproduction on Great Lakes influenced areas with the greatest HQ, which was greater than the adverse population level. My data suggests that if protection of wildlife from environmental contaminants is the management goal, then fish passage should not be allowed past Foote, Tippy and Croton dams. Concentrations of environmental contaminants in nestling bald eagle blood plasma confirm these results. Productivity and success increased on the Manistee and Muskegon Rivers after run-of-river implementation, but there was inconclusive supporting evidence that run-of-river was the factor for the increase