ABSTRACT. Photodynamic therapy (PDT) is a noninvasive therapeutic method first introduced in the field of dermatology. It is mainly used for the treatment of precancerous and superficial malignant skin tumors. Today PDT finds new applications not only for nononcologic dermatoses but also in the field of other medical specialties such ...

Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of "effector" proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host ...

The organization of eukaryotic genome into chromatin within the nucleus eventually dictates the cell type specific expression pattern of genes. This higher order of chromatin organization is established during development and dynamically maintained throughout the life span. Developmental mechanisms are conserved in bilaterians and hence they have body plan in ...

RNA interference (RNAi) is the process of specific gene silencing by the use of double-stranded RNA (dsRNA). In cultured Drosophila cells, RNAi methodologies are well established and easily executed: dsRNA, when added to the cell culture medium, is efficiently internalized by the cells and, through the activity of endogenous processing ...

The guinea pig pneumonia model mimics Legionnaires' disease as seen in immunocompromised humans, with high untreated fatality rates and as such lends itself to studies of experimental chemotherapy. Guinea pig infection is also used to assess relative virulence of different Legionella bacterial strains, and has also been used to study ...

The elementary level of chromatin fiber, namely the nucleofilament, is known to undergo a hierarchical compaction leading to local chromatin loops, then chromatin domains and ultimately chromosome territories. These successive folding levels rely on the formation of chromatin loops ranging from few kb to some Mb. In addition to a ...

Legionella pneumophila is an intracellular pathogen of free-living protozoa that can also infect alveolar macrophages, L929 fibroblast cells, and HeLa cells. Infection of nonphagocytic cells by L. pneumophila can be used to study invasion mechanisms, compare infectivity of different strains and identify factors important for virulence. Virulent strains of L. ...

Legionella strains are considered biologically inert with respect to traditional identification schemes. Various phenotypic alternatives have been attempted but all have lacked resolution as additional species have been added to what is proving to be a large genus. Only sequence-based schemes have the required resolution to confidently speciate or recognize ...

Disturbances in the general mRNA metabolism have been recognized as a major defect in a growing number of hereditary human diseases. One prominent example of this disease group is Retinitis pigmentosa (RP), characterized by selective loss of photoreceptor cells. RP can be caused by dominant mutations in key factors of ...

Adeno-associated virus (AAV) vectors are in wide use for in vivo gene transfer for the treatment of inherited retinal disease. AAV vectors have been tested in many animal models and have demonstrated efficacy with low toxicity. In this chapter we describe some of the recent methods for small-scale production of ...

Aging is a complex trait and is influenced by multiple factors that are both intrinsic and extrinsic to the organism (Kirkwood et al. 2000; Knight 2000). Efforts to understanding the mechanisms that extend or shorten lifespan have been made since the early twentieth century. Aging is characteristically associated with a ...

Flow cytometry, a valuable technique that employs the principles of light scattering, light excitation, and emission of fluorochrome molecules, can be used to assess the cell cycle position of individual cells based on DNA content. After the permeabilization of cells, the DNA can be stained with a fluorescent dye. Cells ...

p53 levels and activity are controlled in large part through regulated ubiquitination and subsequent destruction by the 26S proteasome. Monoubiquitination of p53 is mediated primarily by the RING-finger E3 ubiquitin ligase MDM2 and impacts p53 activity through modulation of p53 localization and transcription activities. Recently, several E4 ubiquitin ligases (E4s) ...

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins ...

Calcium acts as a prominent second messenger in virtually every cell type and modulates a plethora of cell functions. Thus, Ca(2+) microfluorimetry became a valuable tool to assess information about mechanisms involved in the regulation of the intracellular calcium level in research on living tissues. Here we offer insight into ...

The concept of cancer as a stem cell disease has slowly gained ground over the last decade. A 'stem-like' state essentially necessitates that some cells in the developing tumor express the properties of remaining quiescent, self-renewing and regenerating tumors through establishment of aberrant cellular hierarchies. Alternatively, such capacities may also ...

The development of in vivo retinal fundus imaging in mice has opened a new research horizon, not only in ophthalmic research. The ability to monitor the dynamics of vascular and cellular changes in pathological conditions, such as neovascularization or degeneration, longitudinally without the need to sacrifice the mouse, permits longer ...

Although the field of genetics has grown by leaps and bounds within the last decade due to the completion and availability of the human genome sequence, transcriptional regulation still cannot be explained solely by an individual's DNA sequence. Complex coordination and communication between a plethora of well-conserved chromatin modifying factors ...

Mutant p53 may activate target genes through the interaction of transcription factors or through histone modifications. Chromatin immunoprecipitation (ChIP) is a method commonly used to study these types of protein interactions. In order to generate a list of target genes that may be activated through this mechanism, ChIP sequencing may ...

Those investigators who study the morphology of Legionella and Legionella-infected cells have greatly benefited from the superior resolution afforded by electron microscopy (EM). It can also be said with confidence that EM will continue to reveal as yet to be discovered features of this fascinating intracellular pathogen. In this chapter ...

The recent developments in the field of epigenetics have changed the way the covalent modifications were perceived from mere chemical tags to important biological recruiting platforms as well as decisive factors in the process of transcriptional regulation and gene expression. Over the years, the parallel investigations in the area of ...

Genome packaging is a universal phenomenon from prokaryotes to higher mammals. Genomic constituents and forces have however, travelled a long evolutionary route. Both DNA and protein elements constitute the genome and also aid in its dynamicity. With the evolution of organisms, these have experienced several structural and functional changes. These ...

Legionella pneumophila is the causative agent of the potentially fatal Legionnaires' disease in humans. Mice have proved to be valuable model organisms to study the pathogenesis of this intracellular bacterium, as well as immune responses against it. In this chapter we describe a selection of mouse infection protocols to study ...

Although the study of protozoology has been active for centuries, very few current academic curricula incorporate requirements or even options for coursework on the study of protists; yet, protozoa are becoming widely recognized by investigators as organisms that play a significant role in the evolution, pathogenicity, protection and amplification of ...

Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general (13)C-labeled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the pathways of their utilization can be studied in considerable detail. ...

The introduction of large-scale gene expression profiling studies has greatly increased the need to rapidly obtain high-quality cellular expression patterns of genes found to exhibit differential expression. The use of large-scale nonradioactive RNA in situ hybridization makes this possible, and greatly increases the general usefulness of this data. Here, we ...

DNA replication involves a coordinated progression through S phase, and disruption of these regulated steps may cause gene abnormalities, which may lead to cancer. Different stages of DNA replication can be detected immunofluorescently that would indicate how replication is progressing in a cell population or under specific conditions. We describe ...

The production of outer membrane vesicles (OMVs) is a widespread phenomenon employed by bacteria to secrete cell envelope components into the environment. A contribution of Legionella pneumophila OMVs to the pathogenesis of Legionnaires' disease is likely due to the high number of virulence-related proteins in the vesicles. OMVs are isolated ...

Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (on-chip) is a technique well suited for a comprehensive analysis of transcription factor binding sites, histone modification patterns, and nucleosome occupancy. It can be restricted to a subset of genes or regions but also expanded up to a genome-wide range yielding insight into ...

Caenorhabditis elegans can serve as a simple genetic host to study interactions between Legionellaceae and their hosts, and to examine the contribution of specific gene products to virulence and immunity. C. elegans nematodes have several appealing attributes as a host organism; they are inexpensive, have robust genetic analysis tools, have ...

Currently, several methods are used for the detection of Legionella in clinical samples, and these methods constitute part of the criteria for defining legionellosis cases. Urinary antigen detection is the first-line diagnostic test, although this test is limited to L. pneumophila serogroup 1 (Lp1) (Helbig et al., J Clin Microbiol ...

Transgenic models are invaluable tools for researching retinal degenerative disease mechanisms. However, they are time-consuming and expensive to generate and maintain. We have developed an alternative to transgenic rodent models of retinal degeneration using transgenic Xenopus laevis. We have optimized this system to allow rapid analysis of transgene effects in ...

In higher eukaryotic organisms epigenetic modifications are crucial for proper chromatin folding and thereby proper regulation of gene expression. In the last years the involvement of aberrant epigenetic modifications in inflammatory and autoimmune diseases has been recognized and attracted significant interest. However, the epigenetic mechanisms underlying the different disease phenotypes ...

The adaptation of Legionella pneumophila to the different conditions it encounters in the environment and in the host is governed by a complex regulatory system. Current knowledge of these regulatory networks and the transcriptome responses of L. pneumophila is mainly based on microarray analysis and limited to transcriptional products of ...

Outbreaks of Legionnaires' disease create high levels of public anxiety and media interest and inevitably consume a great deal of public health resources. Investigations should begin as early as possible in order to rapidly identify suspected sources of infection, control the outbreak and prevent further cases occurring. The investigations should ...

The induction of retinal degeneration by light exposure is widely used to study mechanisms of cell death. The advantage of such light-induced lesions over genetically determined degenerations is that light exposures can be manipulated according to the needs of the experimenter. Bright white light exposure can induce a synchronized burst ...

The professional phagocyte Dictyostelium discoideum is a simple eukaryotic microorganism, whose natural habitat is deciduous forest soil and decaying leaves, where the amoebae feed on bacteria and grow as separate, independent, single cells. In the last decade, the organism has been successfully used as a host for several human pathogens, ...

Legionella pneumophila the causative agent of Legionnaires' disease, actively manipulates host cell processes to establish a replication niche inside host cells. The establishment of its replication niche requires a functional Icm/Dot type IV secretion system which translocates about 300 effector proteins into host cells during infection. Many of these effectors ...

Histones are the protein components of chromatin and are important for its organization and compaction. Although core histones are exclusively expressed during S phase of the cell cycle, there exist variants of canonical histones that are expressed throughout the cell cycle. These histone variants are often deposited at defined regions ...

The study of transcriptome responses can provide insight into the regulatory pathways and genetic factors that contribute to a specific phenotype. For bacterial pathogens, it can identify putative new virulence systems and shed light on the mechanisms underlying the regulation of virulence factors. Microarrays have been previously used to study ...

The core histone N-terminal tails dissociate from their binding positions in nucleosomes at moderate salt concentrations, and appear unstructured in the crystal. This suggested that the tails contributed minimally to chromatin structure. However, in vitro studies have shown that the tails were involved in a range of intra- and inter-nucleosomal ...

The p53 tumor suppressor protein plays a central role in mediating the cellular response to a variety of stresses. Activation of p53 signaling will trigger cell cycle arrest or apoptosis in normal cells, depending on such factors as cell type and genetic context. The ability of a cell to circumvent ...

This chapter presents methods for interrogating the involvement of p53 in signaling to apoptosis, autophagy, and senescence. The well-known association of p53 with the stress response to chemotherapy and radiation is the basis for presenting these approaches. The development of quantitative and efficient in vitro assays has enabled researchers to ...

The lipopolysaccharide(LPS) of Legionella spp. is an immuno-dominant antigen and the basis for Legionella pneumophila serogroup classification. The LPS shows a peculiar structure composed of a very hydrophobic lipid A acylated by long chain fatty acids and an O-antigen-specific chain consisting of homopolymeric legionaminic acid. In this chapter we describe ...

The inner blood-retina barrier (iBRB) is essential in restricting the movement of systemic components such as enzymes, anaphylatoxins, or pathogens that could otherwise enter the neural retina and cause extensive damage. The barrier has evolved to confer protection to the delicate microenvironment of the retina, and the tight junctions located ...

RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its ...

The human p53 protein isoforms are expressed in several cell lines and modulate p53 tumor suppressor -activity, mainly through modulation of gene expression (1-4). Thus, identifying the pattern of p53 isoforms expression in cell lines is a key step for future studies of the p53 network (5). At the moment, ...

Legionella pneumophila is an intracellular bacterium that was evolutionarily selected to survive in freshwater environments by infecting free-living unicellular protozoa. Once humans inhale contaminated water droplets, the bacteria reach the pulmonary alveoli where they are phagocytized by resident alveolar macrophages. Depending on host immunity and bacterial virulence genes, the infection ...

Various types of retinal neurons, including amacrine, ganglion, and horizontal cells, expand neurites (dendrites or axons) in horizontal direction and make synaptic or electrical contacts with other cells to integrate the visual information. Many types of ion-channels and receptors are located along these neurites, and these horizontal connections critically contribute ...

Inherited retinal diseases display a very high degree of clinical and genetic heterogeneity, which poses challenges in identifying the underlying defects in known genes and in identifying novel retinal disease genes. Here, we outline the state-of-the-art techniques to find the causative DNA variants, with special attention for next-generation sequencing which ...