Memoria para optar al Título Profesional de Ingeniero Forestal

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy, 2017 / Under current and previous global climate change, environments are changing and have changed at a rapid rate. Species with the potential to undergo adaptive radiation are likely to survive environmental change. The genus Rhabdomys is widespread in southern Africa, occurring along the east-west rainfall gradient in South Africa. Rhabdomys may have undergone adaptive radiations in the past, which may have resulted in the current suite of species in various habitats of different aridity. Some Rhabdomys species also occur in sympatry in some locations in South Africa. The aim of my study was to investigate adaptive variation in Rhabdomys by studying the behaviour of 5 populations, representing 3 Rhabdomys species, across South Africa. Using selected taxa, my approach was, firstly, to describe variation in two traits, personality and spatial cogntion, well known for showing environmentally-linked (i.e. adaptive) variation. Secondly, I manipulated the development of exploratory and anxiety behaviour to assess the limits of the adaptive variation (i.e. test the nature of the reaction norm of the characters measured). I first established the taxon-level personality of 4 taxa (2 sympatric) in 5 standard behavioural tests. Generally, the semi-desert living R. pumilio was the boldest together, surprisingly, with R. d. dilectus occurring in grasslands of central South Africa, contradicting previously published results. Comparatively, R. bechuanae from central South Africa and R. dilectus from far north-eastern South Africa, also occurring in grasslands were less bold, even though R. bechuanae is sympatric with R. dilectus in central South Africa. My data indicate adaptive variation at the extreme populations and possibly character displacement in the sympatric populations. In the next chapter, I investigated whether early rearing environment shapes exploratory behaviour and anxiety responses of R. pumilio and R. bechuanae. I predicted that using an interspecies cross-fostering protocol would reveal a gene x environment interaction on behaviour, so that fostered offspring would display an intermediate behaviour phenotype compared to their non-fostered siblings. I showed that a novel rearing environment mostly did not influence the adult behaviour of cross-fostered inidividuals. This indicates genetic constraints on exploratory behaviour and anxiety responses. Next, I tested whether physical rearing environment shapes exploratory behaviour and anxiety responses. I reared semidesert R. pumilio, sympatric R. bechuanae and R. dilectus and allopatric R. bechuanae under either no cover or high cover for 2 generations. The taxa were mostly similar and altering the phyical housing condition did not alter behaviour, but there were small differences between the taxa in exploratory behaviour. In the final experimental chapter, I established whether the environment predicts the spatial cognition in semi-desert R. pumilio, sympatric R. bechuanae and R. dilectus and an allopatric population of R. dilectus from far north-eastern South Africa. The populations showed very similar performance in a modified Barnes maze, indicating a possible phylogenetic constraint on spatial cognition. Overall, my study suggests that there is adaptive variation in personality but not spatial cognition. In contrast to previous studies in the genus, alterations to the social and physical environments failed to separate out genetic and environmental effects (i.e. reaction norm) that would potentially provide the mechanisms for adaptive variation within and between species. The similarity in spatial cognition between taxa and similar responses to environmental modification indicate phylogenetic constraints on traits that were predicted to vary geographically. / XL2018

Des mises au point de nouvelles stratégies d’expansion ex vivo des cellules souches hématopoïétiques (CSH) sont développées depuis quelques années afin de pallier le problème du faible nombre de ces cellules pour le traitement des hémopathies ou de certaines tumeurs solides. Notre équipe avait établi un modèle d’expansion des CSH via leur exposition aux homéoprotéines HOXB4 ou HOXC4. L’étude comparative des transcriptomes de ces cellules a permis l’identification de cibles précoces des facteurs HOXB4/C4 parmi lesquels les gènes codant les régulateurs post-transcriptionnels Pumilio (de la famille PUF). Les facteurs PUF sont impliqués en particulier dans le maintien des cellules souches germinales dans différents modèles animaux, chez les vertébrés ou les invertébrés. Cependant, le rôle des facteurs PUF humains (hPum1 et hPum2) dans les cellules hématopoïétiques humaines n’avait jamais été étudié.Mon travail de thèse exposé ici a consisté, d’une part, en l’étude du profil d’expression des facteurs hPum1 et hPum2 dans différentes lignées hématopoïétiques et au cours de l’hématopoïèse humaine, démontrant une expression plus importante de ces gènes dans les cellules les plus immatures ainsi que dans les progéniteurs dont la prolifération est activée. D’autre part, l’étude fonctionnelle des facteurs hPum1 et hPum2 a mis en évidence leur implication dans l’expansion et la survie des cellules CD34+. L’inhibition spécifique de hPum1 ou de hPum2 in vitro par des shARN, induit une diminution significative du nombre absolu des cellules ainsi qu’une augmentation de leur apoptose. Cela corrèle avec une accumulation des CSH en phase G0-G1 du cycle cellulaire. Par ailleurs, la répression de l’expression de hPum1 ou de hPum2 diminue la reconstitution de l’hématopoïèse in vivo dans des souris immunodéficientes NOD-SCID-γC-/-. L’analyse des ARNm cibles des facteurs Pum par une étude comparative des transcriptomes des CSH transduites ou non par des vecteurs lentiviraux contenant des shARN hPum1 ou hPum2, a permis l’identification de nombreux gènes impliqués dans le contrôle de la croissance, de la survie ou du cycle cellulaire. L’ensemble de nos résultats montre l’indispensable implication des facteurs Pumilio dans le maintien de l’état souche, la prolifération et la survie des CSH humaines. Nous avons démarré des études fonctionnelles dans les cellules leucémiques myéloïdes primaires afin d’évaluer le rôle éventuel des facteurs Pumilio dans la leucémogenèse. Ultérieurement, la caractérisation de hPum1 et hPum2 comme de nouvelles molécules impliquées dans l’expansion des CSH permettra d’envisager leur étude dans la perspective de nouvelles stratégies thérapeutiques. / Ex vivo expansion of hematopoietic stem cells (HSCs) could improve new therapeutic strategies for the treatment of hematopoietic malignancies and solid tumors. Our team had developed an original method to expand human HSCs, consisting in the transfer into these cells of active HOXB4 or HOXC4 homeoproteins. The comparative transcriptomic analysis of CD34+ cells exposed or not to HOXB4 or HOXC4 proteins induced over-expression of Pumilio (PUF) genes. PUF proteins are post-transcriptional regulators of gene expression. They are involved in different biological functions among which the maintenance of stem cells. However, the function of human PUF factors (hPum1 and hPum2) in hematopoietic stem cells has never been investigated. The work that I developed during my thesis first consisted in analyzing the expression of PUF factors in different hematopoietic cell lines and during human hematopoiesis. The results highlighted a high expression of the hPum1 en hPum2 genes in the most immature cells and in the proliferating active progenitors. The study of human PUF factors by inducing their inhibition using specific shRNAs revealed their involvement in proliferation and survival of CD34+ cells. In vitro, inhibition of hPum1 or hPum2 decreases the expansion of human HSCs and increases cell apoptosis. The hPum1 or hPum2 repression also increases the number of HSCs in G0-G1 phase of the cell cycle. Moreover, the inhibition of hPum1 or hPum2 reduces the capacity of human HSCs to reconstitute in vivo hematopoiesis of immunodeficient NOD-SCID-γC-/- mice. The identification of PUF target mRNAs by a comparative transcriptomic analysis of human HSCs infected or not with lentiviral vectors containing hPum1/2 shRNAs, revealed a large number of genes involved in the regulation of cell growth, survival or cell cycle. On the whole, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of human HSCs. Functional studies in primary myeloid leukemic cells are in progress to assess the potential role of the Pum factors in the leukemogenic process. Later on, identification of Pumilio factors as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.

Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and translational regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant negative mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length compared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth.

In skeletal muscle acetylcholinesterase AChE is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Scientists have elucidated many aspects of synaptic AChE structure, function, and localization during the past 80 years. However our understanding of the molecular mechanisms underlying its regulation is incomplete, but it appears to involve both translational and post-translational events as well. We found that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates and that PUM2 binds to the AChE transcripts when immoprecipitation studies were performed. A direct binding between a recombinant PUM2-HD and the Pumilio Binding Site (PBE) in a segment of the AChE 3’UTR was demonstrated by Gel shift assays. Transfecting skeletal muscle cells with shRNAs specific for PUM2 upregulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. We found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific synaptic proteins involved in modulating synaptic transmission, analogous to CNS synapses. PUM2 expression is critically important in many cell types, virtually nothing is known about the regulation of PUM2 expression itself. Analyzing the PUM2 mRNA 3’UTR we found fifteen possible PBEs in the 3 Kb 3’ UTR. We show that PUM2 binds in vivo to its own mRNA. Overexpression of PUM2 in several cell types transfected with a green fluorescent protein (GFP) reporter construct linked to the full length PUM2 3’UTR (GFP-PUM2-3’UTRFL) suppresses GFP expression suggesting that PUM2 downregulates its own expression by binding to its own 3’UTR. Mutations of the first five PBEs yield the expression of the reporter gene indicating that at least one PBE is functional in the autoregulation of PUM2. These observations suggest a novel model for the localized regulation of protein translation through a negative feedback loop. Much is known about PUM2 as a translational regulative protein but little is known about PUM2 cell localization and possible mechanism of translational regulation. In this work we found PUM2 to be highly localized to the cell rough endoplasmic reticulum and that PUM2 is associated with ribosomal RNA. In addition, we found that the GFP protein itself, together with its mRNA and ribosomal RNA (rRNA), were localized in the PUM2 positive complexes when GFP-PUM2-3’UTRFL was transfected into muscle cells. These observations further suggest a mechanism of regulation where translation of the protein occurs but the protein remains associated with the ribonucleoprotein complex, possibly to be transported together with its mRNA to specific domains inside the cell. Thus when needed, more protein is produced in those specific cell regions.

Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and translational regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant negative mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length compared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth.

<p>Much of the regulation of gene expression occurs at the posttranscriptional level, and much of this regulation is controlled and coordinated by RNA binding proteins (RBPs). Many RBPs have multiple mRNA targets, and the proteins encoded by these targets often share functional relationships, forming posttranscriptional RNA operons. These operons often reflect the function of the RBP, thus determination of the genome-wide targets of RBPs allows insight into their functions.</p> <p>The PUF family of RBPs is characterized by the presence of an extremely well conserved RNA binding domain, typically consisting of 8 repeats of an RNA binding motif, with each repeat binding to one RNA base. PUF proteins are proposed to have an ancestral role in self-renewal of stem cells and have been shown to affect a number of developmental processes. Human and other vertebrate genomes contain two canonical PUF genes, Pum1 and Pum2, and at the outset of this study there was very little known about functions or targets of either protein, especially Pum1.</p> <p>In order to identify the genome-wide targets of human Pum1 we used RNA immunoprecipitation followed by microarray, or RIP-Chip, analysis. RIP-Chip allowed us to identify Pum1 target mRNAs in human HeLa cells. We found that there were numerous functional relationships among the proteins encoded by these mRNAs, forming putative RNA operons. Some of these potential operons are progression of cell cycle, cell differentiation and proliferation, and regulation of transcription. We were also able to find a consensus Pum1 binding motif, UGUAHAUA, in the 3' UTRs of Pum1 target mRNAs. </p> <p>The genome-wide targets of PUF proteins from other species have been previously identified, and by comparing the targets of human Pum1 to targets of Drosophila Pumilio and yeast Puf3, both of which bind to the same RNA sequence as Pum1, we determined that there has been evolutionary rewiring of regulation by Puf proteins. While the PUF RNA binding domain and consensus binding sequence have remained almost identical through evolution, the surrounding protein sequence and the mRNAs bound have changed dramatically, indicating that evolutionary rewiring is occurring in a modular fashion. </p> <p>After identifying Pum1 associated mRNAs, we went on the study the function of Pum1. Through Pum1 knockdown assays we found that Pum1 enhances decay of target mRNAs, and that this effect is likely due to Pum1 enhancing deadenylation of these mRNAs. We also showed by immunofluorescence that Pum1 protein has a cytoplasmic granular subcellular localization and upon oxidative stress relocates to stress granules but not processing bodies. We were, however, unable to detect any difference in Pum1 mRNA targeting after stress. We were also unable to detect any changes in progression through cell cycle after Pum1 knockdown. </p> <p>In this study we identified the genome-wide mRNAs associated with Pum1, determined functional relationships among these targets related to the proposed ancestral role of PUF proteins in self-renewal of stem cells, and identified a sequence motif to which Pum1 binds in these mRNAs. We also demonstrated that Pum1 enhances decay of associated mRNAs, and that this effect is likely due to Pum1 enhancing deadenylation of associated mRNAs. These results provide a description of mRNA targets and mechanisms of action of Pum1 proteins, which will provide a strong foundation for future experiments to further explore the functions of the Pum1, especially as they relate to human stem cells.</p> / Dissertation

The striped-mouse, Rhabdomys pumilio, is widely distributed throughout southern Africa within a variety of habitats and rainfall regimes. It is found at sea level in the Eastern and Western Cape regions and at altitudes above 2700 m in the Drakensberg mountains. The attraction of R.pumilio to cultivated land and crops has resulted in extensive damage to plants and cultivated crops. A study of the genetic variation between populations of R.pumilio from different regions of southern Africa was undertaken by protein electrophoresis and randomly amplified polymorphic DNA using the polymerase chain reaction (PCR-RAPD). A cytogenetic study was also undertaken. The mean heterozygosity (H=0.074) for R.pumilio was more than twice that estimated for mammals (H=0.036) while the mean percent polymorphism (P=16.1%) was only slightly higher than the mean percent polymorphism obtained for mammals (P=14.7%). The highest heterozygosities were recorded in the Potchefstroom (H=0 .145) and Zimbabwe (H=0 .118) samples and the lowest mean heterozygosity was recorded in the peninsular Western Cape (H=0. 032). A mean Fst value of 0.459 was obtained, suggesting a high degree of genetic differentiation between the samples of R.pumilio but the negative Fis (-0.01) value emphasized that R.pumilio retained an outbreeding population structure. The similarity coefficient between the samples of R.pumilio using PCR-RAPD's ranged between 0.471 and 0.853 and substantiated the argument for genetic divergence between the samples of R.pumilio. An isolation by distance model for the population genetic structure of R.pumilio was supported by the allozymes (r=0.58, p<0.00l) and PCR-RAPD's (0.75, p<0.00l). Temperature and rainfall also had an influence on the allelic frequency distribution of certain loci of R.pumilio. Rogers (1972) genetic similarity varied between 0.796 and 0.988 while the values for Nei's (1978) unbiased genetic distance varied between 0.000 and 0.189 for the different samples of R.pumilio. Subgrouping of the KwaZulu-Natal samples, the peninsular Western Cape and Eastern Cape samples of R.pumilio was evident with the allozymes. With the PCR-RAPD' s the Zimbabwe sample showed the least similarity to the other samples with a KwaZulu-Natal/Potchefstroom subgroup separating from the less well defined Eastern Cape and Western Cape subgroup. Cytogenetic studies of specimens of R.pumilio from some of the localities in southern Africa revealed a chromosomal number of 2n=48 , while the Potchefstroom and Zimbabwe specimens displayed a chromosomal number of 2n=46. Homology in G-and C-banding was recorded. The allozymes, PCR-RAPD's and chromosomal studies suggested subspecies status for the Zimbabwe population of R.pumilio. The Potchefstroom sample displayed a greater genetic similarity to the remaining South African samples of R.pumilio than the Zimbabwe samples and therefore could not be considered for subspecies status. Although the South African samples of R.pumilio displayed a certain degree of genetic divergence, it was insufficient to warrant subspecies status although evolution in this direction was suggested. / Thesis (Ph.D.)-University of Natal, Durban, 1999.

Memoria para optar al Título Profesional de Ingeniero Forestal / Nothofagus pumilio, conocida vulgarmente como Lenga, es una especie autóctona del sur de Chile y a diferencia del resto de muchas otras locales crece como un bosque monoespecífico. Su madera es estéticamente atractiva y mecánicamente resistente en comparación a otras especies nativas. Este estudio analiza los principales productos fabricados a partir de esta especie, el monto de sus exportaciones y la contribución que la industria hace a la economía local.

Memoria para optar al Titulo Profesional de Ingeniero Forestal