Mutational and molecular analyses of lactococcal bacteriophages TP901-1 and Tuc2009

Bacteriophages belonging to the Siphoviridae family represent viruses with a noncontractile tail that function as extremely efficient bacterium-infecting nanomachines. The Siphoviridae phages TP901-1 and Tuc2009 infect Lactococcus lactis, and both belong to the so-called P335 species. As P335 phages are typically capable of a lytic and lysogenic life cycle, a number of molecular tools are available to analyse their virions. This doctoral thesis describes mutational and molecular analyses of TP901-1 and Tuc2009, with emphasis on the role of their tail-associated structural proteins. Several novel and intriguing findings discovered during the course of this study on the nature of Siphoviridae phages furthers a basic molecular understanding of their virions, and the role of their virion proteins, during the initial stages of infection. While Siphoviridae virions represent complex quaternary structures of multiple proteins and subunits thereof, mutagenic analysis represents an efficient mechanism to discretely characterize the function of individual proteins, and constituent amino acids, in the assembly of the phage structure and their biological function. However, as always, more research is required to delve deeper into the mechanisms by which phages commence infection. This is important to advance our understanding of this intricate process and to facilitate application of such findings to manipulate phage infections. On the one hand, we may want to prevent phages from infecting starter cultures used in the dairy industry, while on the other hand it may be desirable to optimize viral infection for the application of phages as bacterial parasites and therapeutic agents.

Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3 mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 +/- 1.8 mu m in total length, 12.9 +/- 0.8 mu m in body length, 3.4 +/- 0.3 mu m in width, 3.1 +/- 0.1 mu m in thickness and 24.6 +/- 2.2 mu m in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 +/- 0.5 mu m in length and 0.7 +/- 0.1 mu m in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size. (C) 2011 Elsevier B.V. All rights reserved.

Evidence for the conspecificity of Palisada papillosa with P. perforata (Ceramiales, Rhodophyta) from the western and eastern Atlantic Ocean on the basis of morphological and molecular analyses

Morphological and molecular studies were carried out on Palisada papillosa and P. perforata from the Canary Islands (type locality of P. perforata), Mexico and Brazil. The two species have been distinguished by features of their external morphology such as size and degree of compactness of the thalli, presence or absence of arcuate branches, branching pattern and basal system. A detailed morphological comparison between these taxa showed that none of the vegetative anatomical or reproductive characters was sufficient to separate these species. The presence or absence of cortical cells in a palisade-like arrangement, also previously used to. distinguish these species, is not applicable. The species present all characters typical of the genus, and both share production of the first pericentral cell underneath the basal cell of the trichoblast, production of two fertile pericentral cells (the second and the third additional, the first remaining sterile), spermatangial branches produced from one of two laterals on the suprabasal cell of trichoblasts, and the procarpbearing segment with four pericentral cells. Details of the procarp are described for the species for the first time. The phylogenetic position of these species was inferred by analysis of the chloroplast-encoded rbcL gene sequences from 39 taxa, using one other Rhodomelacean taxon and two Ceramiaceae as outgroups. Relationships within the clade formed by P. papillosa and P. perforata have not been resolved due to the low level of genetic variation in their rbcL sequences (0-0.4%). Considering this and the morphological similarities, we conclude that P. papillosa is a taxonomic synonym of P. perforata. The phylogenetic analyses also supported the nomenclatural transfer of two species of Chondrophycus to Palisada, namely, P. patentiramea (Montagne) Cassano, Senties, Gil-Rodriguez & M.T. Fujii comb. nov. and P. thuyoides (Kutzing) Cassano, Senties, Gil-Rodriguez & M.T. Fujii comb. nov.

Multiple nucleolus organizer regions in Leptodactylus mystacinus (Amphibia, Anura) and comments on its systematic position in the L fuscus group based on cytogenetic and molecular analyses

Specimens of Leptodactylus mystacinus from Brazil were karyotyped with conventional and differential staining. The 2n = 22 karyotype is similar to that found for the majority of the Leptodactylus, the karyotypic conservatism also confirmed by the similarity of the replication banding patterns with those previously described. L. mystacinus has a small amount of C-banded heterochromatin, located mainly at the centromeres, although telomeric or interstitial bands have also been noticed. With DA/CMA(3) some chromosome regions showed slightly bright fluorescence, and with DA/DAPI, no particular AT-rich repetitive region was observed. Silver staining showed an extensive inter- and intraindividual variation in the number and position of Ag-positive regions, in 1p, 4p, 8p, 8q, and 11p. Nevertheless, FISH using rDNA probes confirmed only the signals on the short arms of chromosomes 4 and 8 as true NORs. The remaining silver stained regions are probably due to the heterochromatin with some affinity to the Ag-staining. Phylogenetic analysis based on partial cytochrome b sequence revealed that L. mystacinus forms a basal branch, so that the presence of multiple NORs in pairs 4 and 8 in this species indicates an autapomorphy.

Pseudoplatystoma metaense and P. orinocoense (Siluriformes: Pimelodidae) from the Orinoco basin, Venezuela: cytogenetic and molecular analyses

The genus Pseudoplatystoma includes catfish species distributed throughout the fresh waters of South America. These species are important fisheries resources and play a significant ecological role due to their piscivorous and migratory habits. The taxonomy of this genus is still debated: traditionally, only three species have been recognised, but recently, this number was raised to eight. The validity of these eight morphospecies, however, was not confirmed by two subsequent molecular phylogenetic studies, which identified either five or four main clades. In this study, we focused on the two morphospecies restricted to the Orinoco basin, P. metaense and P. orinocoense, which have been assigned to either the same or different clades in previous studies. We carried out cytogenetic analyses to describe their unknown karyotypes and to look for cytotaxonomic markers. We also analysed their mitochondrial sequences in order to assign the sampled specimens to the previously identified molecular clades. The two presumptive species show similar karyotypes (2n=56, 42 biarmed and 14 uniarmed chromosomes) and cytogenetic features in terms of the constitutive heterochromatin distribution and the number and location of minor and major ribosomal genes. Thus, no species-specific chromosome markers could be identified. The analysis of cytochrome b and cytochrome oxidase I mitochondrial genes (carried out by retrieving all the mtDNA Pseudoplatystoma sequences available in GenBank) distributed the sampled specimens into two distinct molecular clades and confirmed the need to re-evaluate, by parallel morphological and molecular analyses, the monophyly of some lineages.

Effects of type I collagen coating on titanium osseointegration: histomorphometric, cellular and molecular analyses

The investigation of titanium (Ti) surface modifications aiming to increase implant osseointegration is one of the most active research areas in dental implantology. This study was carried out to evaluate the benefits of coating Ti with type I collagen on the osseointegration of dental implants. Acid etched Ti implants (AETi), either untreated or coated with type I collagen (ColTi), were placed in dog mandibles for three and eight weeks for histomorphometric, cellular and molecular evaluations of bone tissue response. While the histological aspects were essentially the same with both implants being surrounded by lamellar bone trabeculae, histomorphometric analysis showed more abundant bone formation in ColTi, mainly at three weeks. Cellular evaluation showed that cells harvested from bone fragments in close contact with ColTi display lower proliferative capacity and higher alkaline phosphatase activity, phenotypic features associated with more differentiated osteoblasts. Confirming these findings, molecular analyses showed that ColTi implants up-regulates the expression of a panel of genes well known as osteoblast markers. Our results present a set of evidences that coating AETi with collagen fastens the osseointegration by stimulating bone formation at the cellular and molecular levels, making this combination of morphological and biochemical modification a promising approach to treat Ti surfaces.

Cytogenetic and molecular analyses in troglobitic and epigean species of Pimelodella (Siluriformes: Heptapteridae) from Brazil

Samples from seven different locations of the genus Pimelodella were genetically examined, two caves (exclusively subterranean, upper Tocantins River and Sao Francisco River) and five epigean (from upper Parana River basin). Cytogenetic analyses revealed the same diploid number (2n=46) for all species besides similarities in both number and location of nucleolar organizer regions and C bands. FISH with 5S rDNA probes and CMA(3) staining indicated significant differences among the studied species. Application of PCR-RFLP in ATPase 6 and 8 mitochondrial genes allowed building a minimum evolution phenogram identifying the close evolutionary relationship among groups. Both chromosomal and molecular data were useful to infer the relationships among studied Pimelodella species.

[Molecular analyses for (early) recognition of hematologic diseases - sense and sensibility for molecular analysis: the art of intelligent decision-making]

During the last 10 years several molecular markers have been established as useful tools among the armamentarium of a hematologist. As a consequence, the number of performed hematologic molecular analyses has immensely increased. Often, such tests replace or complement other laboratory methods. Molecular markers can be useful in many ways: they can serve for diagnostics, describe the prognostic profile, predict which types of drugs are indicated, and can be used for the therapeutic monitoring of the patient to indicate an adequate response or predict resistance or relapse of the disease. Many markers fulfill more than one of these aspects. Most important, however, is the right choice of analyses at the right time-points!

Molecular Analyses Define Vα7.2-Jα33+ MAIT Cell Depletion in HIV Infection: A Case-Control Study

Mucosal-associated invariant T (MAIT) cells are an abundant antibacterial innate-like lymphocyte population. There are conflicting reports as to their fate in HIV infection. The objective of this study was to determine whether MAIT cells are truly depleted in HIV infection.In this case-control study of HIV-positive patients and healthy controls, quantitative real-time polymerase chain reaction was used to assess the abundance of messenger RNA (mRNA) and genomic DNA (gDNA) encoding the canonical MAIT cell T cell receptor (Vα7.2-Jα33). Comparison was made with flow cytometry.Significant depletion of both Vα7.2-Jα33 mRNA and gDNA was seen in HIV infection. Depletion of Vα7.2+CD161++ T cells was confirmed by flow cytometry. In HIV infection, the abundance of Vα7.2-Jα33 mRNA correlated most strongly with the frequency of Vα7.2+CD161++ cells. No increase was observed in the frequency of Vα7.2+CD161- cells among CD3+CD4- lymphocytes.MAIT cells are depleted from blood in HIV infection as confirmed by independent assays. Significant accumulation of a CD161- MAIT cell population is unlikely. Molecular approaches represent a suitable alternative to flow cytometry-based assays for tracking of MAIT cells in HIV and other settings.

(Table S1) Molecular analyses and morphological comparisons of deep-sea clams

Large vesicomyid clams are common inhabitants of sulphidic deep-sea habitats such as hydrothermal vents, hydrocarbon seeps and whale-falls. Yet, the species- and genus-level taxonomy of these diverse clams has been unstable due to insufficiencies in sampling and absence of detailed taxonomic studies that would consistently compare molecular and morphological characters. To clarify uncertainties about species-level assignments, we examined DNA sequences from mitochondrial cytochrome-c-oxidase subunit I (COI) in conjunction with morphological characters. New and published COI sequences were used to create a molecular database for 44 unique evolutionary lineages corresponding to species. Overall, the congruence between molecular and morphological characters was good. Several discrepancies due to synonymous species designations were recognized, and acceptable species names were rectified with published COI sequences in cases where morphological specimens were available. We identified seven species with trans-Pacific distributions, and two species with Indo-Pacific distributions. Presently, 27 species have only been documented from one region, which might reflect limited ranges, or insufficient geographical sampling. Vesicomyids exhibit the greatest species diversity along the northwest Pacific ridge systems and in the eastern Pacific, along the western America margin, where depth zonation typically results in segregation of closely related species. The broad distributions of several vesicomyid species suggest that their required chemosynthetic habitats might be more common than previously recognized and occur along most continental margins.

Functional genomic and molecular analyses of transcripts related to glycerol synthesis and trafficking in rainbow smelt (Osmerus mordax) using cold temperature-induced models of glycerol production

The rainbow smelt (Osmerus mordax) is an anadromous teleost that produces type II antifreeze protein (AFP) and accumulates modest urea and high glycerol levels in plasma and tissues as adaptive cryoprotectant mechanisms in sub-zero temperatures. It is known that glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature; however, the precise mechanisms of glycerol synthesis and trafficking in smelt remain to be elucidated. The objective of this thesis was to provide further insight using functional genomic techniques [e.g. suppression subtractive hybridization (SSH) cDNA library construction, microarray analyses] and molecular analyses [e.g. cloning, quantitative reverse transcription - polymerase chain reaction (QPCR)]. Novel molecular mechanisms related to glyceroneogenesis were deciphered by comparing the transcript expression profiles of glycerol (cold temperature) and non-glycerol (warm temperature) accumulating hepatocytes (Chapter 2) and livers from intact smelt (Chapter 3). Briefly, glycerol synthesis can be initiated from both amino acids and carbohydrate; however carbohydrate appears to be the preferred source when it is readily available. In glycerol accumulating hepatocytes, levels of the hepatic glucose transporter (GLUT2) plummeted and transcript levels of a suite of genes (PEPCK, MDH2, AAT2, GDH and AQP9) associated with the mobilization of amino acids to fuel glycerol synthesis were all transiently higher. In contrast, in glycerol accumulating livers from intact smelt, glycerol synthesis was primarily fuelled by glycogen degradation with higher PGM and PFK (glycolysis) transcript levels. Whether initiated from amino acids or carbohydrate, there were common metabolic underpinnings. Increased PDK2 (an inhibitor of PDH) transcript levels would direct pyruvate derived from amino acids and / or DHAP derived from G6P to glycerol as opposed to oxidation via the citric acid cycle. Robust LIPL (triglyceride catabolism) transcript levels would provide free fatty acids that could be oxidized to fuel ATP synthesis. Increased cGPDH (glyceroneogenesis) transcript levels were not required for increased glycerol production, suggesting that regulation is more likely by post-translational modification. Finally, levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were transiently higher. These comparisons also led to the novel discoveries that increased G6Pase (glucose synthesis) and increased GS (glutamine synthesis) transcript levels were part of the low temperature response in smelt. Glucose may provide increased colligative protection against freezing; whereas glutamine could serve to store nitrogen released from amino acid catabolism in a non-toxic form and / or be used to synthesize urea via purine synthesis-uricolysis. Novel key aspects of cryoprotectant osmolyte (glycerol and urea) trafficking were elucidated by cloning and characterizing three aquaglyceroporin (GLP)-encoding genes from smelt at the gene and cDNA levels in Chapter 4. GLPs are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. The highlight was the discovery that AQP10ba transcript levels always increase in posterior kidney only at low temperature. This AQP10b gene paralogue may have evolved to aid in the reabsorption of urea from the proximal tubule. This research has contributed significantly to a general understanding of the cold adaptation response in smelt, and more specifically to the development of a working scenario for the mechanisms involved in glycerol synthesis and trafficking in this species.

Molecular comparison of Scytalidium thermophilum isolates using RAPD and ITS nucleotide sequence analyses

Scytalidium thermophilum plays an important role in determining selectivity of compost produced for growing Agaricus bisporus. The objective of this study was to characterise S. thermophilum isolates by random amplified polymorphic DNA (RAPD) analysis and sequence analysis of internally transcribed spacer (ITS) regions of the rDNA, to assess the genetic variation exhibited by this species complex and to compare this with existing morphological and thermogravimetric data. RAPD analysis of 34 isolates from various parts of the world revealed two distinct groups, which could be separated on the basis of the differences in the banding patterns produced with five random primers. Nucleotide sequence analysis of the ITS region, which was ca 536 bp in length, revealed only very minor variation among S. thermophilum isolates examined. Several nucleotide base changes within this region demonstrated variation. Genetic distance values among type 1 and 2 S. thermophilum isolates, as determined by ITS sequence analysis, varied by a value of 0.005 %. Molecular analyses carried out in the present study would suggest that isolates within this species complex exhibit genetic differences which correlate well with morphological variation and thermogravimetric data previously determined.

A mitochondrial genome phylogeny of termites (Blattodea: Termitoidae) : robust support for interfamilial relationships and molecular synapomorphies define major clades

Despite their ecological significance as decomposers and their evolutionary significance as the most speciose eusocial insect group outside the Hymenoptera, termite (Blattodea: Termitoidae or Isoptera) evolutionary relationships have yet to be well resolved. Previous morphological and molecular analyses strongly conflict at the family level and are marked by poor support for backbone nodes. A mitochondrial (mt) genome phylogeny of termites was produced to test relationships between the recognised termite families, improve nodal support and test the phylogenetic utility of rare genomic changes found in the termite mt genome. Complete mt genomes were sequenced for 7 of the 9 extant termite families with additional representatives of each of the two most speciose families Rhinotermitidae (3 of 7 subfamilies) and Termitidae (3 of 8 subfamilies). The mt genome of the well supported sister group of termites, the subsocial cockroach Cryptocercus, was also sequenced. A highly supported tree of termite relationships was produced by all analytical methods and data treatment approaches, however the relationship of the termites + Cryptocercus clade to other cockroach lineages was highly affected by the strong nucleotide compositional bias found in termites relative to other dictyopterans. The phylogeny supports previously proposed suprafamilial termite lineages, the Euisoptera and Neoisoptera, a later derived Kalotermitidae as sister group of the Neoisoptera and a monophyletic clade of dampwood (Stolotermitidae, Archotermopsidae) and harvester termites (Hodotermitidae). In contrast to previous termite phylogenetic studies, nodal supports were very high for family-level relationships within termites. Two rare genomic changes in the mt genome control region were found to be molecular synapomorphies for major clades. An elongated stem-loop structure defined the clade Polyphagidae + (Cryptocercus + termites), and a further series of compensatory base changes in this stem loop is synapomorphic for the Neoisoptera. The complicated repeat structures first identified in Reticulitermes, composed of short (A-type) and long (B-type repeats) defines the clade Heterotermitinae + Termitidae, while the secondary loss of A-type repeats is synapomorphic for the non-macrotermitine Termitidae.