An invasive-native mammalian species replacement process captured by camera trap survey random encounter models

Camera traps are used to estimate densities or abundances using capture-recapture and, more recently, random encounter models (REMs). We deploy REMs to describe an invasive-native species replacement process, and to demonstrate their wider application beyond abundance estimation. The Irish hare Lepus timidus hibernicus is a high priority endemic of conservation concern. It is threatened by an expanding population of non-native, European hares L. europaeus, an invasive species of global importance. Camera traps were deployed in thirteen 1 km squares, wherein the ratio of invader to native densities were corroborated by night-driven line transect distance sampling throughout the study area of 1652 km2. Spatial patterns of invasive and native densities between the invader’s core and peripheral ranges, and native allopatry, were comparable between methods. Native densities in the peripheral range were comparable to those in native allopatry using REM, or marginally depressed using Distance Sampling. Numbers of the invader were substantially higher than the native in the core range, irrespective of method, with a 5:1 invader-to-native ratio indicating species replacement. We also describe a post hoc optimization protocol for REM which will inform subsequent (re-)surveys, allowing survey effort (camera hours) to be reduced by up to 57% without compromising the width of confidence intervals associated with density estimates. This approach will form the basis of a more cost-effective means of surveillance and monitoring for both the endemic and invasive species. The European hare undoubtedly represents a significant threat to the endemic Irish hare.

LaGomiCs - Lagomorph Genomics Consortium: An International Collaborative Effort for Sequencing the Genomes of an Entire Mammalian Order

The order Lagomorpha comprises about 90 living species, divided in 2 families: the pikas (Family Ochotonidae), and the rabbits, hares, and jackrabbits (Family Leporidae). Lagomorphs are important economically and scientifically as major human food resources, valued game species, pests of agricultural significance, model laboratory animals, and key elements in food webs. A quarter of the lagomorph species are listed as threatened. They are native to all continents except Antarctica, and occur up to 5000 m above sea level, from the equator to the Arctic, spanning a wide range of environmental conditions. The order has notable taxonomic problems presenting significant difficulties for defining a species due to broad phenotypic variation, overlap of morphological characteristics, and relatively recent speciation events. At present, only the genomes of 2 species, the European rabbit (Oryctolagus cuniculus) and American pika (Ochotona princeps) have been sequenced and assembled. Starting from a paucity of genome information, the main scientific aim of the Lagomorph Genomics Consortium (LaGomiCs), born from a cooperative initiative of the European COST Action “A Collaborative European Network on Rabbit Genome Biology—RGB-Net” and the World Lagomorph Society (WLS), is to provide an international framework for the sequencing of the genome of all extant and selected extinct lagomorphs. Sequencing the genomes of an entire order will provide a large amount of information to address biological problems not only related to lagomorphs but also to all mammals. We present current and planned sequencing programs and outline the final objective of LaGomiCs possible through broad international collaboration.

Characterisation of ZG16p, a unique mammalian lectin from pancreatic zymogen granules

The mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.

Functional analysis of String/CDC25 phosphatase in post-mitotic neurons

Cell cycle and differentiation are two highly coordinated processes during organ development. Recent studies have demonstrated that core cell cycle regulators also play cell cycle-independent functions in post-mitotic neurons, and are essential for the maintenance of neuronal homeostasis. CDC25 phosphatases are well-established CDK activators and their activity is mainly associated to proliferating tissues. The expression and activity of mammalian CDC25s has been reported in adult brains. However, their physiological relevance and the potential substrates in a non-proliferative context have never been addressed. string (stg) encodes the Drosophila CDC25 homolog. Previous studies from our group showed that stg is expressed in photoreceptors (PRs) and in lamina neurons, which are two differentiated cell types that compose the fly visual system. The aims of this work are to uncover the function of stg and to identify its potential neuronal substrates, using the Drosophila visual system as a model. To gain insight into the function of stg in a non-dividing context we used the GAL4/UAS system to promote downregulation of stg in PR-neurons, through the use of an RNAi transgene. The defects caused by stg loss-of-function were evaluated in the developing eye imaginal disc by immunofluorescence, and during adult stages by scanning electron microscopy. This genetic approach was combined with a specific proteomic method, two-dimensional difference gel electrophoresis (2D-DIGE), to identify the potential substrates in PR-cells. Our results showed that stg downregulation in PRs affects the well-patterned retina organization, inducing the loss of apical maintenance of PR-nuclei on the eye disc, and ommatidia disorganization. We also detected an abnormal accumulation of cytoskeletal proteins and a disruption of the axon structure. As a consequence, the projection of PR-axons into the lamina and medulla neuropils of the optic lobe was impaired. Upon stg downregulation, we also detected that PR-cells accumulate Cyclin B. Although the rough eye phenotype observed upon stg downregulation suggests neurodegeneration, we did not detect neuronal death during larval stages, suggesting that it likely occurs during pupal stages or during adulthood. By 2D-DIGE, we identified seven proteins which were differentially expressed upon stg downregulation, and are potential neuronal substrates of Stg. Altogether, our observations suggest that Stg phosphatase plays an essential role in the Drosophila visual system neurons, regulating several cell components and processes in order to ensure their homeostasis.

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

Differential regulations of AQP4 and Kir4.1 by triamcinolone acetonide and dexamethasone in the healthy and inflamed retina.

PURPOSE: Glucocorticoids are used to treat macular edema, although the mechanisms underlying this effect remain largely unknown. The authors have evaluated in the normal and endotoxin-induced uveitis (EIU) rats, the effects of dexamethasone (dex) and triamcinolone acetonide (TA) on potassium channel Kir4.1 and aquaporin-4 (AQP4), the two main retinal Müller glial (RMG) channels controlling retinal fluid movement. METHODS: Clinical as well as relatively low doses of dex and TA were injected in the vitreous of normal rats to evaluate their influence on Kir4.1 and AQP4 expression 24 hours later. The dose-dependent effects of the two glucocorticoids were investigated using rat neuroretinal organotypic cultures. EIU was induced by footpad lipopolysaccharide injection, without or with 100 nM intraocular dex or TA. Glucocorticoid receptor and channel expression levels were measured by quantitative PCR, Western blot, and immunohistochemistry. RESULTS: The authors found that dex and TA exert distinct and specific channel regulations at 24 hours after intravitreous injection. Dex selectively upregulated Kir4.1 (not AQP4) in healthy and inflamed retinas, whereas TA induced AQP4 (not Kir4.1) downregulation in normal retina and upregulation in EIU. The lower concentration (100 nM) efficiently regulated the channels. Moreover, in EIU, an inflammatory condition, the glucocorticoid receptor was downregulated in the retina, which was prevented by intravitreous injections of the low concentration of dex or TA. CONCLUSIONS: The results show that dex and TA are far from being equivalent to modulate RMG channels. Furthermore, the authors suggest that low doses of glucocorticoids may have antiedematous effects on the retina with reduced toxicity.

Cellular source and mechanisms of high transcriptome complexity in the Mammalian testis.

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexity in major mammalian organs have been scarce. Here, using extensive RNA-seq data, we show that transcription of the genome is substantially more widespread in the testis than in other organs across representative mammals. Furthermore, we reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding and long noncoding RNA genes but also poorly conserves intergenic sequences, suggesting that it may not be of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data suggest that this prevalent transcription, which most likely promoted the birth of new genes during evolution, is facilitated by an overall permissive chromatin in these germ cells that results from extensive chromatin remodeling.

Problems of allometric scaling analysis: examples from mammalian reproductive biology.

Biological scaling analyses employing the widely used bivariate allometric model are beset by at least four interacting problems: (1) choice of an appropriate best-fit line with due attention to the influence of outliers; (2) objective recognition of divergent subsets in the data (allometric grades); (3) potential restrictions on statistical independence resulting from phylogenetic inertia; and (4) the need for extreme caution in inferring causation from correlation. A new non-parametric line-fitting technique has been developed that eliminates requirements for normality of distribution, greatly reduces the influence of outliers and permits objective recognition of grade shifts in substantial datasets. This technique is applied in scaling analyses of mammalian gestation periods and of neonatal body mass in primates. These analyses feed into a re-examination, conducted with partial correlation analysis, of the maternal energy hypothesis relating to mammalian brain evolution, which suggests links between body size and brain size in neonates and adults, gestation period and basal metabolic rate. Much has been made of the potential problem of phylogenetic inertia as a confounding factor in scaling analyses. However, this problem may be less severe than suspected earlier because nested analyses of variance conducted on residual variation (rather than on raw values) reveals that there is considerable variance at low taxonomic levels. In fact, limited divergence in body size between closely related species is one of the prime examples of phylogenetic inertia. One common approach to eliminating perceived problems of phylogenetic inertia in allometric analyses has been calculation of 'independent contrast values'. It is demonstrated that the reasoning behind this approach is flawed in several ways. Calculation of contrast values for closely related species of similar body size is, in fact, highly questionable, particularly when there are major deviations from the best-fit line for the scaling relationship under scrutiny.

Tgfbi/Bigh3 silencing activates ERK in mouse retina.

BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.

The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.

Factors affecting DNA uptake by mammalian cells

The ability to introduce DNA and express custom DNA sequences in bacteria opened the door for improvements in a large number of fields including agriculture, pharmacology, medicine, nutrition, etc. The ability to introduce foreign DNA sequences into mammalian cells in an efficient manner would have a large impact on therapeutic applications especially gene therapy. The methods in use today suffer from low efficiencies and sometimes toxicity. In this work a number of factors were evaluated for their effect onONA uptake efficiency. The factors studied included exposure to sublethal concentration of hydrogen peroxide which have been show to lead to destabilisation ofthe lysosomes. These exposures have proven to be very toxic to cells when combined with either the calcium phosphate or the lipofectAMINE® transfection methods. Another factor evaluated was exposure to Electro-Magnetic Fields (EMF). This was fuelled by the fact that EMF have been shown to mediate a number of effects on cell structure and/or physiology. EMF exposure by itself was not sufficient to induce the cells to pick up the DNA, therefore its effect on calcium phosphate and lipofectAMINE® was tested. Although some positive results were obtained, the variability of these results exceeded by far any observed enhancements which discouraged any further work on EMF. Also tested was the possible effect the presence of the cytomegalovirus (CMV) sequence might have on DNA uptake (based on previous results in this lab). It was found that the presence ofCMV in the DNA sequence does not enhance uptake or slow down degradation of the internalised DNA. The final factor tested was the effect of basic amino acids on transfection efficiency. It was found that arginine can enhance DNA uptake by about 170% v/ith calcium phosphate and about 200% with LipofectAMINE®. A model was proposed to explain the effect of arginine as well as the lack of effect from other amino acids.